Multiple Molecular Forms of Cholinesterase from Elongated Animals Citation Johnson, Carl Douglas (1977) Multiple Molecular Forms of Cholinesterase from Elongated Animals. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/x6s3-4159. https://resolver.caltech.edu/CaltechTHESIS:12152025-215621387 Abstract Cholinesterase activity in Electrophorus electricus and Caenorhabditis elegans has been fractionated, primarily by velocity sedimentation in sucrose gradients. The electric organs of E. electricus contained five separable forms (7,5 S, 9 S, 12 S, 14 Sand 18 S). Three, so-called native forms (9 S, 14 Sand 18 S) are insoluble in low-ionic strength solutions and they were all shifted to higher sedimentation constants by treatment with bacterial collagenase. The other two (globular) forms are soluble in low-ionic strength solutions and unaffected by collagenase. Trypsin converts the native forms to the 12 S form. Neither collagenase nor trypsin affect the molecular size of the active subunit as determined by SDS-polyacrylamide gel electrophoresis. Four forms of cholinesterase activity were separated from extracts of the soil nematode Caenorhabditis elegans (5 S, 7 S, 11 S, 13 S). The smaller two forms as a pair and the larger two forms as a second pair are kinetically similar (Kms, substrate and inhibitor specificity). There are significant differences between the pairs. A screening procedure using the selective inactivation of the 5 Sand 7 S forms by the anionic detergent sodium deoxycholate has been applied to 89 mutants of C. elegans. One uncoordinated mutant (BC46) apparently devoid of 11 S or 13 S activity was identified. The 5 S and 7 S forms in this mutant are unaltered. The behavioral defect is limited to the body region. Head movements and sensory responses to mechanical, chemical and osmotic stimuli seem unaltered. The mutation is X-linked. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Biology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Russell, Richard L. Thesis Committee: Unknown, Unknown Defense Date: 10 June 1976 Record Number: CaltechTHESIS:12152025-215621387 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:12152025-215621387 DOI: 10.7907/x6s3-4159 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17801 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 16 Dec 2025 17:01 Last Modified: 16 Dec 2025 17:09 Thesis Files PDF - Final Version See Usage Policy. 36MB Repository Staff Only: item control page

MULTIPLE MOLECULAR FORMS OF CHOLINESTERASE FROM ELONGATED ANIMALS Thesis by Carl Douglas Johnson In Partial Fulfillment of the Requirements for the De gree of Doctor of Philosophy California Institute of Technology Pasadena , •California 1977 (Submitt ed June 10, 1976) ii Acknowledgement First I would like to acknowledge my major advisor Dick Russell. He not only initiated many of the studies reported here but also provided direction and ideas as I struggled to extend them. The collaboration of Susan Smith taught me how much more two people working together could accomplish. In pushing this thesis to completion, the worm biochemists - Jim Rand, Ed Hedgecock, Stu Scherer and Maurice Zwass provided motivation and many good times ( also known as long n~ghts) . Other members of the Russell g·roup allowed me to observe some of the frustrations and joys of electrophysiology (Lou Byerly and Dave Weisblat) , electron microscopy (Dave Hall and Randle Ware) and behavioral studies (Joe Cuolotti and Dave.Dusenbery). I would also like to thank the members of the Garfias House Feed Group, especially Welcome Bender who never seemed to tire of hearing about today's experiment and Carole Johnson who never understood today's experiment but was always supporti v e. She also provided an envi r ot1ment in which the relaxed and organic life-style we both preferred could flourish. Finally, I would thank my parents for support and encouragement in my earlier years and dedicate this thesis to the memory of my late uncle Carl A. Peterson. lll Abstract Cholinesterase activity in Electrophorus electricus and Caenorhabditis elegans has been fractionated, primarily by velocity sedimentation in sucrose gradients. The electric organs of E. electricus contained five separable forms (7,5 S, 9 S, 12 S, 14 Sand 1 8 S). Three, so-called native forms (9 S, 14 Sand 18 S) are insoluble in low-ionic strength solutions and they were all shifted to higher sedimentation constants by treatment with bacterial collagenase. The other two (globular) forms are solubl e in low-ionic strength solut ions and unaffected by collagenase. 12 S form. Trypsin converts the native forms to the Neither collagenase nor trypsin affect the molecular size of the active subunit as determined by SDS-polyacrylamide gel electrophoresis. Four forms of cholinesterase activity were separated from extracts of the soil nematode Caenorhabditis elegans (5 S, 7 S, 11 S, 13 S ). The smaller two forms as a pair and the larger two forms as a second pair are kinetically similar (Kms, substrate and inhibitor spec ifi city). There are significant differences between the pairs. A screening procedure using the selective inactivation of the 5 Sand 7 S forms by the anionic detergent sodium deoxycholate has been applied to 89 mutants of C. elegans . One uncoordinated mutant (BC46) apparently devoid of 11 Sor 1 3 S activity was identified. The 5 Sand 7 S iv forms in this mutant are unaltered. The behavioral defect is limited to the body region. Head movements and sensory responses to mechanical, chemical and osmotic stimuli seem unaltered. The mutation is X-linked. V _Table of Contents Title . . . . . i Acknowledgement ll Abstract . . iii , Table of Contents . . . . . , . , . . . . . . General Introduction . . . , . . . , . . . . References . . . I , , . . . , v 1 7 A Rapid, Simple Radiometric Assay for Cholinesterase, Suitable for Multiple Determinations . . . . . 9 Materials & Methods 11 Results . 15 Discussion 17 References II . . . . . . . . . . . . . 18 Separation and Selective Modification of Electrophorus electricus Acetylcholinest e rase by Collagenase . . . . Abstract a f C G O • I e • • • I • • 20 e 21 Introduction . 22 Materials . . 24 Methods . 25 Results . 28 Discussion. 52 Referenc es . 58 vi III Separation and Characterization of Multiple Forms of Cholinesterase Activity from the Nematode Caenorhabditis elegans . . 60 Abstract , . . 61 . .. , Introduction 62 Materials . . . , 63 Methods . . . . . . 65 Results . 72 Discussion . II o • • • • " References . . . . , . . . IV • • • • • • • • . .. 99 10~, Biochemistry and Behavior of a Mutant Nematode Missing Forms of Cholinesterase . 107 Abstract 108 Introduction 109 Materials & Methods 111 Results . . 113 ............ Discussion . References . t t • 0 t 0 141 149 1 General Introduction The suggestion that organisms have enzymes capable of hydrolyzing the ester linkage of acetylcholine is as old as the chemical identification of ACh as a pharmacologically active substance (Dale, 1914). After it had been demonstrated that acetylcholine was a neurotransmitter, it was assumed that in vivo the se cholinesterases func- - -- tioned to terminate the action of the transmitter and this assumption received strong experimental support from biochemical studies showing that the enzyme was concentrated at the synapses of cholinergic motoneurons in vertebrate skeletal muscle (Marnay & Nachmansohn, 1938). Further and more comprehensive studies of the localization of cholinesterase activity has since revealed anomalous localizations in structures containing no cholinergic synapses (e.g. in erythrocytes and plasma). Furthermore, biochemical s tudies of single n eu rons in lobster (Hildebrand, Townsel, & Kravitz, 1974) and in Aplysi a (Giller & Schwartz (1971) have demonstrated little difference in the level of cholinesterase between cholinergic cells and cells using other transmitters, Although a number of functions have been suggested for non-synaptic cholinesterase (e .g. memb rane permeability, Duncan, 1967; axonal conduction, Nachmansohn, 1946) none of the suggestions have received much experimental support and most have been discredited by clear demonstrations 2 of the presume d cholinesterase -d ep end ent function in the absence of the enzyme. Two observa tions suggest that the function of some cholinesterases may not be essential; 1) comparative st udies often show large di fferences in the l eve ls of cholinesterase activity among c los ely related organisms (Hart & Lee , 1966 ; Zagic ek, 1957) and , 2 ) genetic variants have been i denti f ied in which p a rticular cholinesterase activities are eliminated without producing a recognizable phenotype (Rubenstein, et al ., 1970). In many org2Jnsrns cholinesterases occur in several s eparable mol ec ular forms. In studying th e properti es of these forms, one can hope to learn whether they result from the expression of multiple gene s or from the forma-tion of ~ss ociations between a common a ctive subunit and other component s . Forms vri th a common activ e subunit may represent sequential stages in the formation of complexes with specific loc a lizations and fun ctions , t hey may be involved in transport of the enzyme , or th ey may result from degradative processes. If spec ific forms could be shown to be lo calized at synapses, the appearance of th ese forms could provide a bio chem ic a l marker for the events of synapse formation . The studies presented in t h i s thesi s conc ern the multiple fonns of cholinesterase ac tivity in two elongated 3 organisms: the electric eel Electrophorus electricus and the soil nematode Caen orhabdit is elegans . Part I d escribes a r apid , r a diometric liquid extraction assay for cholinesterase activity without which th e large numb er of assays invol ve d in fractionating and characterizing separable f orms ·would not have been pos s ibl e . Ac e tylchol ines terase has been directly solubilized from the e l ectric organs of E . electricus with buffered 1 M NaCl (Silman & Karlin, 1967) and fractionated by veloc ity sedimentat ion into three " native " forms (9 S, 14 Sand 18 S (Massoulie & Rieger, 1969). are insoluble The se forms in low-ionic strength solutions, Two additional "d egraded" forms (7,5 Sand 12 S) are found in autolyzed preparations. Th ese forms can be produced b y proteolytic treatment or by sonication of th e native forms (Massouli e , Rieger & Tsuji, 1970; Massoulie, Rie g er & Bon , 1 9 71). so lution s . They are solubl~ . in low ion ic streng th The ~=inetic prop ert ie s of the f orrns do not diffe rentiate them (Massouli e & Rie g er, 1969 ) and antibodi es prep ared against any form wil l precipitate all of the f orn1S (Ri ege r, et al., 1973b ; Gurari, Silman & Fuchs, 1974). The se cha racteri s tics suggest that all of the forms have th e same active s ubunit . The differences b e tween the "native" and the "de g raded" forms lies in the association of the active subunits with an elongated 4 0 (500 A) tail which has been observed in the electron microscope (Rie ge r et al ., 197Ja). In part II, a so.iubi lity • base d fractionation procedure was used to faci lit ate the separation of all five forms. Treatment with bacterial collagenase selectively shifted the three native forms to faster sedimenting forms . This suggests that the native forms may contain a collagen -like compo nent, presumably in the tail. Caenorhabditis elegans is a small ("-'l mm long ) free living nematode whose self-fertilizing hermaphroditic mode of reproduction greatly facilitates the isolation of recessive mutants (Brenner, 1974). The nervous system of the animal contains only about JOO cells. Parts III and IV concern the separation and characterization of four separable forms of cholinesterase activity in wild type Q. elegans and in an uncoordinated mutant in which two of these forms are missing . The function of cholinesterases in nematodes is unknown. Electrophysiological studies in the large nematode Ascaris lumbricoides have shown 1) tha-t bath application of 5 x 10 -6 IVi ACh produces a measurab l e depolarization of the muscle ce ll membrane and 2 ) foca l application of ACh is effective only when directed towards the re g ion of neuromuscular junctions suggesting that ACh is a transmitter at neuromuscular synapses 5 (del Castillo, de Mello & Morales , 1963). However, the complex anatomy of this region (the musc l e cells send processes which approach the nerve cord and form a complex synctium under which the neuromuscu l ar synapses occur (Ro senbluth , 1965))prevents the study or reproduction with iontophoretic application of the actual release of transmitter which is usually required to firmly establish a particular case of chemical transm is sion . It has also not proved possible to determine vrhich cells release the transm itter. Histochemical staining for cholinesterase has been reported in Ascaris muscle (Lee, 1962). Biochemical studies of nematode cholinesterases have largely concerned themselves with the mode of act ion of anthe l minthic s( Hart & Lee, 1966; Knowl es & Casida, 1966). One result of these studies has been to measure levels of cholinesterase activity in a variety of parasitic nematodes (Hart & Lee, 1966). What is most striking is the variation (from O.J u/gTn to JOO u/gm). Cholinesterase from the nematode Nippostro"!}_gylus brasiliensis (a rat intestinal parasite) has been fractionated by ge l el ectrophoresis into three bands, two fast and one s low (Edwards, et al., 1971). The proportions of these forms are altered in nematodes who survive the host's immune responses (Jones & Ogilvie, 1971). Furthermore rats infected with this par8.site acquire 6 antibodies to nematode cholinesterases . In some cases, these antibodies react only with the two fast bands , in oth ers they react with al l three bands . The l eve l of activity observed in the slow band seems to be related to its antibody. If non-immune rats are g i ven an antibodies f rom an immune rat, the level of the slow band of nematode cholinesterase rises. Alternatively, if immune rats are suppressed by injection of cortisone, the l evel of the s low band fa ll s . Lee has shown that cholin- esterase in this animal is localized in the anterior glands (Lee, 1970). 7 References Brenner, S. (1974) Genetics 77, 71-94, del Castillo, J., de Mello, W.C., & Morales, T. (1963) Arch. Int. Phys. et Biochim. 71, 741-757, Dale, H.H. (1914) J. Pharmacol. Exp. Ther. 6, 147-190, Duncan, C.J. (1967) The Molecular Evolution of Excitable Cells (Pergamon Press, New York) p, 112-124. Edwards, A.J., Burt, J.S. & Ogilvie, B.M. (1971) Parasitol. 62, 339, Giller, E. Jr. & Schwartz, J.H. (1971) J. Neurophysiol. 34, 108-115. Gurari, D., Silman, I. & Fuchs, S. (1974) Eur. J. Biochem. 43, 179-187, Hart, R.J. & Lee , R.M. (196 6) Exp. Parasit . 18, 332-337, Hildebrand, J., Townsel, J. & Kravitz, E. (1974) J. Neurochem. 23, 951-963 , Jones, V.E. & Ogilvie, B. M. (1971) Immunology 22, 119-129. Knowles, C.O. & Casida, J.E. (1966) J. Agr. Food Chem. 14, 566-572, Lee, D.L. (1962) Parasitology 52, 241-260. Lee, D.L. (1970) Tissue & Cell 2, 225-231. Marnay, A. & Na'chmansohn, D. (1938) 92, 37-47, J. Physiol. Land., 8 Massoul ie, J, & Rieger, F. (19 69 ) Eur . J. Biochem . 11, Massouli e , J., Ri eger , F. & Bon , S. (1971) Eur. J. Biochem. 21, 542-551, Mass ouli e , J., Rieger, F. & Tsuji, S. (1970) Eur. J. Biochem. 14, 430 - 439. Nachmansohn, D. (1 946 ) Ann. N.Y. Acad. Sc i. 47, 395-475, Rieger, F., Benda, P., Bauman, A. & Rossier, J. (1973) FEBS Letters 32 , 62-68 Rieger, F., Bon, S., Massoulie, J. & Cartaud, J. (1973a) Eur. J. Bio chem . 34, 539-547, Rosenbluth, J. (1 965 ) J. Cell Biol. 26 , 579-591. Rubinstein, H . I'!i . , Dietz, A .A., Hodges , L .K. , Lubrano , T., Czebotar, V . (1970) J. Clin. Inve st. 49, 479-486. Silman, M.I. & Karlin, A. (1967) Proc. natn. Acad. Sci . U.S.A. 58 , 1664-1 668 . Zajic e k, J. (1 957 ) 1 38 , 1-32, Acta Physiol. Scand . 40 , Suppl. 9 I A Rapid, Simple Radiometric Assay for Cholinesterase, Suitable for Multiple Determinations CARL D. JOHNSON AND RICHARD L. RUSSELL ANALYTICAi. BIOC H E MISTRY 64, 229-238 ( J 975) A Rapid, Simple Radiometric Assay for Cholinesterase, Suitable for Multiple Determinationsl CARL 0. JOHNSON AND RICHARD L. RUSSELL Di1·isio11 of Biology . California Institute of Technology. Pasadrna. Califomia 9 I /09 Received November 12, 1973: accepted October I I, 1974 A rapid and simple radiometric ass ay for cholinesterase, suitable for multiple determin ation s, has been developed. (" H-ace tyl) choline is enzyma ti cally hydrolyzed in a small reaction volume in a scintilla tion vial. The released [3 H)acetate is then extracted into a toluene-based scintillator added directly to the vial , without removing the reaction volume. The extracted [ 3 H)acetate counts cfiiciently , but the unhydrolyzed [ 3 H)acetylcholine remains unextracted in the small aqueous reaction volume, from which its weak ,13-particles of decay do not escape to excite the scintillator. The assay is highly reproducible , quite sensitive, and useful for applications in which multiple sa mples must be quickly assayed. Many techniques for the quantitative assay of cholinesterase have been described and recently reviewed (I). Among these, radioisotopic assays based upon the estimation of [ 3 H] or [ 14 C]acetate produced by hydrolysis of labeled [ 3 H] or [ 14 C]acetylcholine are particularly valuable for their sensitivity and their usefulness over a wide range of substrate and enzyme concentrations. The available raclioisotopic assays for cholinesterase differ, in essence, only in the procedure used to separate the labeled acetate product from unhydrolyzed labeled acetylcholine substrate. Winteringham and Disney (2) measured the acetylcholine remaining following acidification and drying of assay samples to remove " volatilized" (i.e. , protonated) acetate. Reed et al. (3) bound acetylcholine with an ion-exchange resin, centrifuged and measured labeled acetate in an aliquot of the supernatant. Potter (4) selectively extracted acetate from acidified reaction mixtures into an organic solvent system consisting of 5: 1 toluene : isoamyl alcohol. McCaman et al. (5) selectively removed labeled acetylcholine by precipitation as the reinecke salt in 0.33 M HCI, and Wilson et al. (6) removed it by passage over a cation exchange column. In each of these techniques, the sample must be taken through at least 'This work was supported by a Grant from th e U.S. Public He:.ilth Service (NS 09 654) and by a Sloa n Foundation Neuroscie nces Research Grant to R.L.R. 229 C0p ) ri!! ht (0 1975 by ,\ cackm ic Press. Inc. Print ed in the U nitetl States. All righh or re prudu c 1io n in an y form re sa ved. 230 JOHNSON AND RUSSELL one postassay manipulation (usually centrifugation or passage over a column) before counting; consequently these techniques are not convenient for assaying the large numbers of samples which are usually encountered in fractionation schemes to purify cholinesterase or to analyze its molecular properties. In this paper we describe a radioisotopic assay which is convenient for these purposes because it keeps postassay manipulations to an absolute minimum. The assay is based in principle on the liquid-extraction technique of Potter ( 4), but includes a few simple and important modifications. [ 3 H]acetyl-labeled acetylcholine is used as substrate for hydrolysis in a reaction carried out in a scintillation counting vial. After hydrolysis, the reaction mixture is stopped by addition of strong pH 2.5 buffer; this protonates the 3 H-labeled acetate produced, and allows it to be efficiently extracted into a large volume excess of toluene-based scintillation fluid added directly to the scintillation vial. Without further additions, the vial is simply counted; the 3 H-labeled acetate in the organic phase is counted efficiently, but the 3 H-labeled acetylcholine which remains in the small volume of aqueous reaction mixture does not count because the weak /3 particles from the 3 H decay are trapped in the aqueous phase before reaching the scintillator. In short, then, the assay selectively measures enzymatically produced [ 3 H] acetate by using two simple fixed-volume additions to a subsequently counted scintillation vial. MATERIALS AND METHODS Acetylcholinesterase of the electric eel Electrophoms electricus was the ECHP preparation from Worthington Biochemical Corp., or the preparation 461-2 from P-L Biochemicals. [:1H]acetylcholine chloride was the preparation TRA-277, 290 mCi/mmole from Amersham-Searle. [ 3H]sodium acetate was the preparation TRA-12, 500 mCi/mmole from Amersham-Searle. All other chemicals were standard reagent grades. Labeled A cetylcholine Substrate One millicurie of (3H-acetyl)choline chloride was dissolved in 2.0 ml H 2 0 (measured pH 4-5, presumably by equilibration with atmospheric CO 2 ) and stored frozen at -20°C. As received, samples contained 2-5% 1 3 [: H] acetate. For most purposes this level of [ H] acetate could be tolerated as background in the assay, permitting use of the substrate without further purification. When required, the background was reduced to 0.2% by; (1) addition of 0.005 ml glacial acetic acid/ml of 3 3 [ H]acetylcholine; (2) repeated extraction of [ H]acetate with 10 vol of toluene:isoamyl alcohol (IO: I); (3) repeated extraction with 100% toluene to remove isoamyl alcohol from the aqueous phase; and (4) blowing with an air stream to remove residual toluene. The 0.2% RADIOMETRIC CHOLINESTERASE ASSAY 23 I background level achieved in this way was not stable, rising to 2-5% upon storage at - 20°C over a period of a few weeks. Although in vacuo storage of [ 3 H]acetylcholine to provide continuous removal of [ 3 H]acetate was envisaged as a method for stabilizing the low background level, it was not tried. The [:iH]acetylcholine chloride solution was diluted with freshly prepared solutions of unlabeled acetylcholine iodide to provide labeled substrate at appropriate concentrations and specific activities. Assays were routinely performed at two acetylcholine concentrations - 2 x 10- :i M and 5 x 10- 5 M. Substrate was prepared at 5X these concentrations (i.e., 10- 2 M and 2.5 x 10- 4 M), at specific activities of l mCi/mmole and 40 mCi/mmole, respectively, so that 0.020 ml would in either case yield about 10 5 cpm upon complete hydrolysis. (Under the standard assay conditions, 70-75% of the [ 3 H]acetate produced by hydrolysis was extractable and countable, and our counting efficiency for extracted [ 3H]acetate was 45-46%.) Aliquots of substrate sufficient for about 200 assays were prepared and stored at 4°C; under those conditions the background level of extractable label was quite stable for several weeks at 2-5%. Standard Assay Procedure The standard assay mixture contained 0.05 ml of 0.05 M potassium phosphate buffer (pH 7.0), 0.02 ml of acetylcholine substrate at the required concentration (see above), and enzyme plus H~O to give a final volume of 0. I ml. When equal aliquots of many fractions Were to be assayed, the buffer, substrate, and water were premixed and aliquoted to the reaction vessels, and reactions were started shortly thereafter by addition of enzyme from the fractions. The reaction vessels were small (I dram) glass vials (cat. no. 7475, Rochester Scientific Co., Inc., purchased with plastic caps) suitable for later insertion into standard widemouth scintillation vials. Incubations were carried out at room temperature (25 ± l°C) except where indicated otherwise. After a suitable incubation time (IO min was frequently adequate), the reaction was stopped by adding 0.1 ml of stopping mixture (see below), followed immediately by 4.0 ml of scintillation mixture (see below) and the vials were capped, shaken, and inserted into wide-mouth scintillat ion vials for counting. The entire stopping procedure could be accomplished, with a little practice, in 12-15 sec, using automatic pipettors for the stopping and scintillation mixtures, so that a large number of assays could be run simultaneously by using staggered starting and stopping times. When the occasion demanded, we were able to perform up to 300 assays in a total time of 2.5 hr. Counting of the vials (in a Beckman LS-200 scintillation counter) was usually initiated 30 min after stopping the reaction, since 232 JOHNSON AND RUSSELL with this precaution a single vigorous shaking of the vials at the time of stopping provided a complete and stable extraction of acetate and good separation of aqueous and organic phases. Stopping Mixture The purpose of the stopping mixture was twofold, to stop the reaction and to protonate the [ 3 H] acetate so that it would extract efficiently into the scintillation mixture. In the original paper on which this assay is based, Potter (4) accomplished these purposes by adding HCI to a final concentration of 0.1 M. We found that this process led to an acidcatalyzed nonenzymatic hydrolysis of acetylcholine at a rate ( 1.3% per hr) too high for our purposes (see Fig. 1). Consequently we chose instead to add a strong buffer to bring the pH to 2.5, at which point the reaction is stopped, the [:iH]acetate is at least 98% protonated, and acid-catalyzed hydrolysis of residual acetylcholine is approximately I 00-fold less rapid (see Fig. 1). A convenient buffer for this purpose has been chloroacetic acid, which has an appropriate pK and high solubility. In order to facilitate extraction of the [ 3 H]acetate, NaCl was also added to increase the polarity of the aqueous phase. That this enhanced 60 50 ,<) Q 40 X ., sC E' 30 ~ C. "' § 20 0 u 10 Hours of ter extract ion Fie. I. Effects of various stopping mixtures on the stability of acetylcholine after extrac- tion. One hundred microliters of premixed assay buffer, labeled substrate and H,O were aliquoted to each of 15 vials; each vial received I 0'• cpm of ["H ]acclylcholine and had a final acetylcholine concentration of I o - :i M. No enzyme was added. For each of three stopping mixtures, a set of five vials received 0.1 ml of that mixture, plus 4 ml scintillation mixture at time zero. and was counted at various intervals lhreafter lo determine the extent of nonenzymatic hydrolysis of acetylcholine which had occurred. Incubation was at room temperature. The three stopping mixtures were: (-0-) 0.2 M HCI + 2.0 ~I NaCl; (-9-), I M CICH,COOH + 2.0 M NaCl: (-0 -) IM CICH, COOH + 0.5 M NaOH + 2.0 M NaCl. The last mixture was chosen for standard use. 233 RADIOM ET RIC C HOLI NEST ERA SE ASSAY T ABL E I PARTI T IO ;-; I ~G OF [ " H]A CETYLC HOI.I N E AND ["H]AC ETATF. BETWEEN AQUEOUS A;-;D T O L UENE PHASES UNDER V ARIO US CoNornoNS" Radioactively labeled compound I. 2. 3. ["H]aceta te 4. 5. 6. ; • [[' H]acetylcholine . : unh ydroly zed 9 10. 11. 12 . [" H]-a ce tylcholi ne , 13. hydrolyzed 14. 15. 16. Stopping mixture Toluene phase volume (ml) Aqueous ph ase Tolu ene phase Total Standard Standard Standard Stand ard 0.2 M HCI 0.2 M HCI , 2 M Na Cl Standard Standard Standard Standard Standard Standard Standard Stand a rd Standard Standard-NaCl 4 4 JO IO 4 4 4 4 4 4 4 4 4 4 4 4 23.1 24.9 13.7 13.4 33.0 22.2 90.4 97.0 97.0 21.5 22.6 19.6 20.6 23. 1 23 .6 30.2 71.0 72.2 80.4 84.2 71.3 78.5 0.5 0.5 0.5 76.2 71.0 72 .6 71.4 69.2 74 .2 66.8 94. 1 97. I 94.J 97 .6 104.3 100.7 90.9 97.5 97.5 97 .7 93.6 92.2 92.0 92.3 97 .8 97 .0 % of initial cpm recovered in " ["H]acetylchuline chloride or ["HJ sod ium acetate (- 10; cpm in e ither case) was di luted into 0.05 ml of 10- 3 M unlabeled acetylcholine iodide in a ground glass stoppered 15 ml conical centrifuge tube . 0.05 ml of 0.05 M pot ass ium phosphate buffer, pH 7.0, was added and a 10 min in c uba tion period followed. Wh e n acetylcholine was to be hydrolyzed during this pe riod (lin es 10- 16) the tubes rece ived in addition 0.0 I ml of a soluti o n of electric eel chol inesterase (20 unit s"/ml in 0.01 M Tris buffe r, pH 7.4 , containing 0.1 mg.Im! bov in e serum albumin for stabi lity) ; this was a considera ble excess over th e e nzy me required fo r complete h ydrol ysis. After incubation, th e tubes received 0. 1 ml of th e indica ted stopping mixture and 4 ml or IO ml of to luene phase, as indicated; th e stand a rd stopping mixture was I M CICH 2 CO OH . 0.5 ~, NaOH , 2.0 M NaCl, a nd th e toluene phase \\'as 10% isoa my l alcohol in toluene. After th orou gh shaking 11 nd low-s pee d ce ntrifuga tion . to separate the phases, each ph ase was di sso lved for counting in IO ml Bray's dioxa ne-b ase d sc intilla tion fluid (7) to which had bee n add ed co mpl e me nt ary unl a beled aliquots of the opposite phase, ·s o th a t all sam ples were eventually counted in equivalent solutions. * I unit hydrol yzes I µm ole of acetylcholine/min at 25°C. the ext raction can be seen from Tab le I, lines I 0-16. The final stopping mixture, then , contained l M ch loroacetic acid, 0.5 M NaOH, and 2 M NaCl, and was as effective for acetate extraction as Potter's HCI method (Table I, lines l, 2, and 5). Scintillation Mixture The scintillation mixture was designed to provide efficient extraction and count ing of [ 3 H]acetate without significant counting of [ 3 H ]acetylcholine. Following Potter (4) we added isoamyl alcohol in vary ing 234 JOHNSON AND RUSSELL proportions to a standard toluene-based scintillation fluid (0.5% PPO, 0.03% PO POP in toluene), and found that the efficiency of extraction of acetate from an aqueous phase was relatively constant over the range of 10-20% isoamyl alcohol; we chose 10% for reasons of economy . The volume of scintillation mixture to be used was arrived at empirically; as can be seen from Table 1, lines 1-4, increasing the volume from the standard 4 ml up to l O ml provided a slightly higher efficiency of extraction for [ 3 H]acetate, but for normal use this was judged to be an insufficient increase to justify the added expense. With the standard 4 ml of scintillation mixture and the standard stopping mixture, as shown in Table l, lines 7-15, only 0.5% of the unhydrolyzed [ 3 H] acetylcholine is counted, whereas 70-75% of of the [ 3 H]acetate I'eleased by hydrolysis is counted. RESULTS When acetylcholinesterase from a variety of sources was measured with the Standard Assay Procedure described above, the activity measurements were reproducible, linearly dependent on time and on enzyme concentration, and comparable with the values obtained by a commonly used colorimetric assay. These points are demonstrated by the assays of electric eel (Electrophorus electrirns) acetylcholinesterase described below. Reproducibility. Twenty duplicate 15-min measurements were made on a given sample of electric eel acetylcholinesterase at a substrate concentration of 2 x l 0-:3 M. The mean value and standard deviation for these measurements was 15,813 ± 2 I 3 cpm (representing 15.8% hydrolysis of the substrate). Since these samples were counted for 5 min each (av total counts 79,065), the expected standard deviation from statistical counting error alone is ±58 cpm. The additional error is comparable to the nominal error of the automatic pipettors used (± l % ). Linearity H"ith time and enzyme concentration. Figure 2a and 2b show the results of assays of two different amounts of electric eel acetylcholinesterase over the time ranges 0-30 min and 0-3 hr. In each instance, the amount of hydrolysis increased linearly with time. In other experiments this linearity was found to hold for even longer times, provided that hydrolysis did not exceed 30% (30,000 cpm). The nonenzymatic contribution to this hydrolysis was extremely slight (0.23%/hr), as shown in Fig. 2b, and the constancy of the enzymatic hydrolysis rate over long times shows that electric eel acetylcholinesterase was stable under assay conditions. Figure 2c shows that the enzyme activity measured by these experiments was, as expected, directly proportional to the amount of enzyme added. Comparison H"ith a colorimetric assay. The commonly used colorimetric assay of Ellman et al. (8) was used as a standard against which to RADIOMETRIC CHOLINESTERASE ASSAY r 0 9.X 6 (cl ~ ,., I E 10 C 5 .~,,. ·e fr OO 235 IC - ·-to~o ·=0 "O., ~ ~3 E [? ~~2 .C w ~ C ~I rOt:::b •l E 10 E ::1.. OQ fr 0 0 o 2 4 6 8 LO Undilu ted enzyme solu~ io n added, mµ.1 60 120 180 Minutes F1G. 2a and 2b. Time dependence of the assay. A solution of electric eel eel acetylcholinesterase nominall y 200 units*/ml in 0.01 M Tris buffer, pH 7.4, containing 0.1 mg/ml bovine se rum albumin (BSA) for stabi lity , was diluted as indicated into 0.01 M Tris buffer, pH 7.4, 0.1 mg/ml BSA. Dilution for (a) was 5000-fold, for (b) 25,000-fold. Ten microliters of the diluted enzyme were incubated at room temperature for the indicated time s with 0.05 ml 0.005 M potassium phosphate buffer, pH 7.0, 0.02 ml 10- 2 M 3 H- labe lecl acetylcholine substrate, and 0.02 ml H,O. Reactions were stopped with 0. 1 ml of standard stopping mixture and 4 ml scintil lation mixture and counted in a Beckman LS 200-B scintillation counter. (-0 -) Blank, without enzyme: (-e-) reactions, with enzyme. All assays were petformed in duplicate, and both values are plotted: overlap sometimes obscures the second point. 2c -Concent ration dependence of the assay. From the experiments of Figs. 2a and 2b and other experiments of similar design. measured enzyme ac tiviti es were calculated from the slopes of the kinetic curves. according to the following formula: Acetylcholinesterase activity= µ.moles acetylcholine hydrolyzed/min acetv lcholine cone. (in µ.moles/ml) x 0.1 ml = slope (in cpm/min) X - - · - - - - - -- -- - - - - - cpm afte r total hydrolysis The value of a no-enzyme blank was subtracted from all cpm values. and total hydroly sis was accomplished by adding (to a se parate vial) IO µ.I of an electric eel acetylcholi ne sterase solution , 100 units */ml. The calculated act ivi tie s are plotted here against the amount of added enzyme. * I unit hydrol yzes I µ.mole of acetylc holi ne/min at 25°C. compare the radiometric assay. A sample of electric eel acetylcholinesterase was assayed several times with both assays, as shown in Table 2. The comparison of assay values is complicated because: (I) the colorimetric assay uses acetylthiocholine instead of acetylcholine as substrate, and (2) the colorimetric assay is usually performed at the pH optimum of 8.0. Nonetheless the values obtained when both assays are performed at pH 7 .0 are very close, substantiating the validity of the radiometric assay. (The radiometric assay, like the colorimetric one, can be made about 50% more sensitive by raising the pH to 8.0, but thi s sensitivity gain is accompanied in both cases by a much more extensive 1ise in the background due to nonenzymatic hydroly sis. In practice, thi s background ri se constitutes a serious limitation when using the assay to detect low activity level s, and assays at pH 7.0 are preferable.) 236 JOHNSON AND RUSSELL TABLE 2 COMPARISON OF RADIOM ETRI C /\ND CO LORI ~!ETRIC ASSAYS" Activity Assay pH ( X 10' µmole/min/ml) Colorimetric Colorimetric Radiomet1ic Radiometric 8.0 7.0 7.0 7.0 4.32 ± 0.18 (6) 2.84 ± 0.08 (7) 3. 17 ± 0.17 (5) 3.25 ± 0. 14 (9) " Electric eel acetylcholinesterase (P-L Biochemicals lot no. 461-2) was dis solved at 400 units*/ml in 0.01 M sodium phosphate buffer, pH 7.0, 0. 1 M NaCl, and diluted 1000-fold in the same solution + I mg/ml bovine serum albumin (BSA for stability). The colorimetric assay at pH 8.0 was performed by mixing 2.80 ml of 0.1 M sodium phosphate buffer pH 8.0, 0 . 1 ml of 0.06 M acctylthiocholine, 0. JO ml of 0.0 I M 3,3' dithiobis[6-nitrobenzoic acid) (DTNB ; di ssolved in 0.1 M sodium phosphate buffer as described by Ellman [8]) , and 0. 15 ml of the enzyme dilution. The optical density change at 412 nm was followed in a Beckman Acta Ill recording spectrophotometer. The colorimetric assay at pH 7.0 was identical except that the buffer was 2.80 ml of 0.0232 M sodium phosphate buffer. pH 7.0. Measured rates of optical density change were converted to activities usi ng th e conversion factors of Ellman et al. (8). The radiometric assays were pe1formed as described in the text, using 2 x 10- 3 M ace tylcholine substrate, 15 min incubations , and 0.005 ml of the enzyme dilution. These conditions led to 10-11 % hydrolysis of the substrate. To avoid any possible complications from loss of enzyme ac tivity , one group of radiometric assays (line 3) was performed before the colorimetric assays. and a seco nd group (line 4) immediately thereafter; the two groups do not differ sign ificantly. Enzyme activities were calculated for the radiometric assays using the formula of Fig. 2c. All activity values are given as thc mean ± standard deviation, and the number of rcpli ca te assays is included in parentheses. * I unit hydrolyzes I µmole of acetylcholine in I min at 25°C. D SCUSSION In the radiometric cholinesterase assay described above, [ 3 H]acetylcholine is hydrolyzed in a scintillation vial containing a small reaction volume, from which the [ 3 H]acetate produced by ·hydrolysis is extracted into a toluene-based scintillation fluid added directly to the vial. The [:1H]acetate is then measured simply by counting the whole vial without further manipulations. There is no need to remove the unhydrolyzed [ 3H] acetylcholine; it remains uncounted in the small aqueous reaction volume, from which the weak ,B-particles of its 3 H-clecay do not escape to reach the scintillator. (The same principle, viz. selective counting of a 3 H-labeled product by extraction into an organic scintillation fluid, has been used by Roffman, Sanocka, and Troll (9) to assay proteolytic enzymes such as trypsin in a sensitive and convenient fashion.) The assay differs from that of Potter (4) in relatively minor ways. First, it uses 3 H as opposed to 11 C substrate, which allows it to circumvent Potter's final postassay extraction step and to use the scintillation vial as reaction RADIOMETRIC CHOLINESTERASE ASSAY 237 vessel. And second, it uses a buffered stopping solution, which prevents postassay acid-catalyzed hydrolysis and allows stopped samples to be stored for relatively long times before counting. Although these modifications are relatively minor, we have found in practice that the convenience they afford has made a considerable difference in the ease and willingness with which the assay of large numbers of replicate samples is undertaken. The assay depends for its convenience on the availability of (~Hacetyl)-choline chloride; (1 ·1C-acetyl)-choline chloride will not work because the 14 C-decay produces a more energetic /3-particle which too frequently escapes from the aqueous phase and excites the scintillator. In principle the assay should be easily adaptable to the use of other (acyl- 3 H)-choline substrates, but since none of these is currently available commercially, the assay is not currently well suited for studies of substrate specificity. However, it is well suited for inhibitor studies without further modification . Because of its radiometric nature and the relatively high specific radioactivity of available ('3H-acetyl)-choline chloride, the assay [like the assay of Potter (4)] can be made extremely sensitive: we have used it, for example, to measure the acetylcholinesterase activities of individual nematodes of the species C oe11orhahditis clego11s (total wet wt -· I µg). Because it is by nature an endpoint measurement, it is not as well suited as some colorimetric methods for kinetic studies on relatively large amounts of enzyme; however, its sensitivity and convenience make it useful for multiple-time-point kinetic studies on smaller amounts of enzymes not assayable by the less sensitive colorimetric methods. The assay has been in routine use in our laboratory and in some others for several years and has proved accurate and reliable for the measurement of cholinesterase activities from a variety of sources, including rat diaphragm ( I 0) , Tor11edo rnlifomica electric organ ( I I), and, in our own hands , Electro11!wrt1s electricus electric organ, Bungart1.1· 11111/tici11ct11s venom, Drosophila 111ela11ogaster head s, horse serum, bovine erythrocytes, and extracts of the small soil nematode Cacrwrlwbclitis e/ego11s. ACKNOWLEDGMENT We thank Dr. Sydn ~y Brenner for sugge sting so me of th e ideas developed in thi s assay. REFERENCES I. Au gustin sso n. K.-8. ( 1971) i11 l\-kthods of Biochemical Analysis (Glick. D .. ed.). Suppl eme nt volume. pp. 217-"l.73. lntersc ience , New York. 2. Wintcringham. F. P. W. , and Disney. R. W. (1964) Bioclie111. J. 91, 506-514. 3. Reed , D. J .. G o to. K .. and Wang. C. H . ( 1966) A 11ul. !Jioclre111. 16, 59-64. 4. Potter. LT. ( 1967 ) .I. f'lr ,11·111. Ext>. T/1 er. 156, 500-506. 238 JOHNSON AND RUSSELL 5. McCaman. M. W. , Tomey . L. R. , a nd McCaman, R. E. ( 1968) I.if,.· Sci. 7, 233-244. 6. Wil son , S. H. , Schrier. B. K., Farber. J. L. , Thompson, E . .I., Rosenber·g , R. N ., Blume, A. J., and Nirenberg. M . W. ( 1972) J. Biol. Chem. 247, 3159-3169. 7. Bray, G. A. (1960) Ano/. Bioche111. I, 279-285 . 8. Ellman. G. L., Courtney. K. D .. Andres, Jr. Y., and Featherstone, R. M. ( 1961) Bioche111. Plwmwcol. 7, 88-95. 9. Roffman , S. , Sanocka, Y., and Troll , W. (1970)A110/. Biochem. 36, 11-17. 10. Hall , Z . W. (1973)1. Neurobiv l. 4, 343-361. 11. Duguid. J. R., and Raftery , i\l. A. ( 1973) Biochemistry 12, 3593-3597. 20 II Separation and Selective Modification of Electrophorus electricus Ac etylcholinesterases by Collagenase CARL D. JOHNSON, SUSAN P. SMITH and RICHARD L. RUSSELL 21 Abstract The multiple forms of acetylcholinesterase found in the electric organ of the eel Electrophorus e1ectricus have been fractionated by differential solubilization from an ammonium sulfate precipitate, using a column elution procedure ( King, 1972)0 This procedure cleanly separates "native" forms from "degraded" forms, and subsequent sedimentation reveals three native and two degraded forms. All three native forms, in distinction to the degraded ones, are inso.l ubl e at low ionic strength and are shifted to higher sedimentation constants by limit ed collagenase 0 treatmento These results suggest that the long ( 500 A) tail seen previously on the native forms of this enzyme (Dudai, Herzberg & Silman, 1973; Rieger et al., 1973) may contain collagen. 22 Introduction Multiple molecular forms of the enzyme acetylcholinesterase (EC 3.l,lo7) have been observed in extracts from various sources, including the enzyme-rich electric organs of Electrophorus e1ectricus and Torpedo marmorata (Massoulie & Rieger, 1969). In Electrophorus electricus, various conditions for extraction of these forms have been tested, revealing that most of the native activity requires high ionic strength for its extraction (Silman & Karlin, 1967; Massoulie & Rieger, 1969; Dudai & Silman, 1974a), and that the extracted activity is primarily in three "native" forms with sedimentation constants of 9 SJ 14 s, and 18 S (Massoulie & Rieger, 1969)0 Preparations of al.l three native forms show a common electron microscopic structure, consisting of a globular portion attached to an elongated "tail" (Dudai, Herzberg & Silman, 1973; Rieger et alo, 1973), and it has been suggested that the tail might serve to attach the enzyme to the synaptic area. The three forms differ in the size of the globular portion and apparently contain one, two, and three copies, respectively, of a globular unit, which itself appears tetrameric by electron microscopy (Rieger et a .l ., 1973) o Two additional forms of acetylcholinesterase are also found in Electrophorus electricus extracts; in fresh extracts these represent a minority of the total activity, but they become quantitatively more important in aged 23 extracts (where autolysis has had a chance to occur) or in extracts deliberately sonicated or treated with nonspecific proteases (Massoulie, Rieger & Tsuji, 1970; Massoulie, Rieger & Bon, 1971). One of these forms, with a sedimentation constant of about 12 S, is the predominant form in commercially available Electrophorus electricus enzyme (prepared autolytically, Leuzinger et al., 1968). It lacks the characteristic tail of the native forms, and apparently consists exclusively of the globular tetrameric unit (Ri eger et al., 1973). The second form also lacks the tail and apparently consists of a globular dimer (Ri eger et al., 1973 )0 In this paper we describe a solubility-based fractionation procedure which cleanly separates the "native" tailed forms of Electrophorus electricus acetylcholinesterase, as a group, from the "degraded" globular formso This procedure eases the previously difficult task of separating the smallest tailed form (9 S) from the dimeric globular form (7.5 S). The clean separations achieved permit the forms to be separately characterized~ The tailed and globular forms show the solubility differences expected from previous work, and in addition collagenase selectively affects the tailed forms, suggesting a collagen-like component in the tail. 24 Materials The detergents Triton X-100 and Tween 80, the proteins bovine serum albumin (BSA, Fraction V), yeast alcohol dehydrogenase (ADH), K• coli ~ -galactosidase (gal), bovine c atalase (cat), electric eel acetylcholinesterase (AChE), Clostridium welchii phospholipase C, and the chemicals acetylcholine (ACh) iodide, PP0 and P0P0P were purchased from Sigma Chemical Co. Trypsin (TRL0GC) was from Worthington, Celite 545 was from Fisher Chemical Co. Ammonium sulfate (enzyme grade) was from Schwarz-Mann. Chemicals for polyacrylamide gel electrophoresis were obtained from BioRad Laboratories. Renografin (Squibb) was obtained from a local apothecary. [3HJ-acetylcholine was TRA277 from Amersham-Searle, or NET-113 from New England Nuclear. Diisopropyl-1- t 3}[] -fluorophosphate ( [ 3 H] -DFP) was NET-065 from New England Nuclear. Clostridium histolyticum collagenase was obtained from two sources, Worthington Biochemical Corp. (CLSPA) and Advanced Biofactures Corp. (Form III). Buffers were dilutions of phosphate, pH 7o0 (0.05 M KH2P04-KiHP04); borate, pH 8.8 (0.1 M Na 2B4 o 7 , adjusted with HCl); and Tris, pH 7.5 (0ol M tris (hydroxymethyl) amino methane, adjusted with HCl). 25 Methods Cholinesterase assays were performed by a radiometric liquid extraction assay (Johnson & Russell, 1975). Assays were buffer e d at pH 7.0 with 0.025 M phosphate and included 5 X 10- 5 Mor 2 X 10 [ 3 H]-ACh in 100 µ1. -3 . M acetylchol1ne and 0.1 µCorless Assays were started by the addition of enzyme and stopped b y the addition of 100 µl of l M C1CH 2 C00H, 0.5 M Na0H, 2 M NaCl; then 4.0 ml of a standard toluene scintillation fluid (0.5% PPO, 0o03% POP0P) with 10% isoamyl alcohol or butyl alcohol was added, and the vials were capped, shaken vigorously and counted (the released ( 3 H J -acetate extracts into the toluene phase and counts selectively; see Johnson & Russell, 1975). Most sets of assays included several blanks (without enzyme) and several "complete conversions" ( including J.0 µl of 100 u/ml conmiercial AChE preparation to effect complete hydrolysis of t h e [ 3 H J -ACh and thereby det ermine the maximum number of cpm to be expected). With occasional unimportant exceptions, all determinations were within the linear range of the assay(~ 35% conversion), and are expressed as cpm of extracted [ 3 H J -acetate, with blank subtractedo El ec tric eels were obtained from local suppliers. 0 Usually the animals were killed by cooling t o 0 C for 30 min, and the electric organs were then removed, flash0 frozen in liquid nitrogen and stored at -20 C unti1 use. 26 Aliquots of electric organ were homogenized at 5-10 volumes of 10% sucrose , 0.005 .M MgC1 2 o0 c in (H.K. Mitchell, personal communication) for 2 times 1 minute using a Sorvall Omni-Mixer. The homogenate was spun at 20,000 rpm for 30 min in a Sorvall centrifuge. only 5-10% of the activity. The supernatant contains The pellet was extracted by rehomogenizing in 2-5 volumes of 0.02 M borate, pH 8.8; 1 M NaCl. The 20,000 rpm, 30 min supernatant of this extract contains greater than 80% of the activity and is the source of enzyme for subsequent separation of multiple molecular forms. Earlier experiments at pH 7.0 yielded comparable forms of AChE activity, but since higher pH preparations appear to be more stable, most of the experiments reported are at pH 8.8. Acetylcholinesterase activity in the supernatant of the 1 M NaCl extract was fractionated by a procedure which depends on differential solubility in concentrated ammonium sulfate solutions (King, 1972). Saturated ammonium sulfate was added to 20% saturation, and a 20,000 rpm, 30 min supernatant was prepared; 4 ml of washed, de-fined and settled Celite suspension was added per 100 ml volume of supernatant and then saturated ammonium sulfate was added to 50% saturation. After gentle stirring for several hours, the Celite was collected by settling, poured into a column, and eluted with a gradient from 50% to 0% saturated ammonium sulfate in 0.02 M borate, pH 8.8; 1 .M NaCl. 27 The multiple molecular forms were separated by velocity sedimentation in 5-20% sucrose density gradients (Martin & Ames, 1961). A non-ionic detergent and additional protein were added to some gradients to prevent the large losies of activity seen with dilute enzyme; they do not affect sedimentation velocities. When gradients having different numbers of fractions were to be compared, activity was plotted against "fractional position" in the gradient, calculated with fractional position= 1 - Total fractions fraction number - 0.5 0.5 x sample volume 1 - gradient volume+ sample volume For SDS-polyacrylarnide gel electrophoresis, samples were diluted with one volume of 0.02 M (Na) phosphate, pH 7.0, 10% sucrose, 2% SDS, 00005 M dithiothreitol, 0.01% bromopheno l blue and heated for 5 min in boiling water. A linea r g radient slab gel (15 cm x 11 cm x 0.1 cm thick) from 18.5 t o 7.5% polyacrylamide was used for separation. A 3% polyacrylamide stacJdng gel was layered on top of the separation gel and a removable comb was used to form sample wells. The buffers for separation and stacking gels and the running buffer were those of Laemmli (Laemmli, 1970). current of Electrophoresis was carried out with a 20 mA until 30 min after the bromophenol blue marker reached the bottom of the gelo Proteins were fixed 28 and stained in 10% acetic acid, 10% methanol, 0 . 1 % coomassie brilliant blue, then diffusion destained in the same solvent. When several samples were to be com- pared, they were run in adjacent wells of the same gel. Collagenase digestions were performed at room temperature in Tris, pH 7.5. preparations were useds Two commercial collagenase Worthington collagenase (CLSPA, 400 u/mg) was usually used at 10 µ_g/ml. A several hundred-fold excess of BSA was added to inhibit nonspecific proteolytic effects. The Advanced Biofactures collagenase (Form III nominally 2500 u/ml) was usually used at a 100-fold dilution. This preparation is osten- sibly uncontaminated with nonspecific proteases (Miller & Udenfriend, 1970). Results Homogenates of Electrophorus electricus electric organ, when prepared by the high pH, high ionic strength extraction method described in Materials & Methods, contain at least three major forms of soluble acetylcholinesterase which can be separated by velocity sedimentation, as previously described (Massoulie & Rieger, 1969) and as shown in Figure 1. Further resolu- tion of these forms can be accomplished by differential ammonium sulfate solubility; the activity of an extract is precipitated onto a solid Celite support at high 29 Fig. 1. Sedimentation of homogenate. An aliquot of electric organ homogenate was diluted into 20 volumes of 0.02 M borate, pH 8.8; 1 M NaCl; 1% Triton X-100. Fifty µ1 was loaded onto 4.5 ml 5-20% sucrose gradients in 0.02 N borate; 1 M NaCl; 0.1% Tween 80; 1 mg/ml BSA with 0.5 ml 100% Renografin cushions (shaded area). were centrifuged for 3.5 hr at 65,000 rpm. fractions were collected. Gradients 5-drop Ten µl of each fraction was assayed for 5 min with 5 x 10- 5 M ACh. Complete conversion yielded 19.9 x 10 3 cpm$ the blanks gave 1.1 x 10 3 cpm. fraction number. Enzymatic activity is plotted versus Sedimentation is from right to left. JO 4 r0 3 I 0 X 2 E Q_ (.) ~ 0 0 5 10 15 20 Fraction Number Fi g ure 1 25 30 31 ammonium sulfate concentration and then eluted with a gradient of decreasing ammonium sulfate concentration, as described in :Materials & :Methods. As shmm in Figure 2A, the activity elutes as three distinct peaks. Upon subsequent fractionation by velocity sedimentation, these peaks prove to have different compositions, as shown in Figure 2B. The small initial peak (peak 1) contains the same forms, in essentially the same proportions, as the original ext racto Its origin is unclear, and it was not seen in all runs. The intermediate peak (peak 2) contains two forms, whose properties show them to be the globular tetramer and dimer previously identified (se e below). The third, and by far the most prominent peak of the ammonium sulfate elution pattern (p e ak 3) contains three different forms i;those properties show them to be the "native" tailed forms previously identified (see below). Each of the five forms appearing in peaJ<.s 2 and 3 of the ammonium sulfate elution pattern can be isolated and resedimented as a single discrete peak, and the results described below show that each is a distinct entity. Accordingly, and to avoid confusion with previous nomenclatur e , thes e forms are subsequently referr e d to by the Roman numeral designations I-V, as indicated in Figure 2. When the separated forms I-V are isolated and 32 Fig. 2. Fractionation of AChE on Celite-ammonium sulfate columns. From 11,5 g of electric organ tissue a 1 M NaCl extract was prep a red and fractionated on a Celite-ammonium sulfate column, as described in Materials and Methods. A 50 ml volume of extract was precipitated onto 2 ml of settled Celite, poured into a 1 cm diameter column and eluted with 100 ml of a 50%-0% ammonium sulfate gradient. A. Five µl of each fraction was assayed for 30 sec with 2 x 10- 3 M ACho Complete conversion gave 110.6 x 10 3 cpm, and blanks gave 3.3 x 10 3 cpm. Three sets of fractions, representing the 1st (fractions 19-21), 2nd (fractions 24-27) and 3rd (fractions 31-45) peaks were separately pooled and reprecipitated by addition of saturated ammonium sulfate to 50% saturation. The resulting pellets were redissolved in 0.02 M borat e , pH 8.8; 1 M NaCl. B. Aliquots of peaks 1, 2, and 3 were loaded onto 5 ml 5-20% sucrose gradients in 0.02 M borate, pH 8.8; 1 M NaCl; 0.1% Tween BO. 65,000 rpm for 4.5 hr. Gradients were centrifuged at Five-drop fractions were collected and 10 µ1 of each fraction was assayed for 30 sec with 2 x 10- 3 M ACh. Complete conversion yielded 112.3 x 10 3 cpm; the blank gave 3.4 x 10 3 cpm. plotted against fractional position. Enzymatic activity is .r I I 20 1 CD ® 30 1 40 I @ Fraction Number ~~ 0 -t~~W~S/$4' 10 0 LJ + 11 I ~ I I 10~ 20 ,,.\ I . u 0.. 0 5 1.0 I I I x 10 I E Figure 2 50 \ -'\e _f 0 0 20 I r0 I 10 30~ 8 20 \ I ~I' \ I b 40~ 30~ t 50~ i I A 60'"1 70-1 0.6 ·" 0.4 I CD @ 0.2 Fractional Position 0.8 ,ff ,,;'J) m r \ I\ IL r~ 0 \..,J \..,J J4 resedimented without the 1 M NaCl used for their extraction, the globular forms IV and V remain soluble at low ionic strength and again resediment as discrete uniform peaks, whereas the tailed forms I, II, and III precipitate and therefore disappear from the gradient ( Figure 3). similar results have been reported previously for forms II, III and V; however, the situation for forms I and IV has been more confusing, due to the fact that they have not been reliably resolved. In particular , the presence of significant amounts of form IV in preparations presumed to consist mostly of the native form I has led to the erroneous conclusion that form I is soluble at low ionic strength (Massoulie & Rieger, 1969; Dudai & Silman, 1974a). In view of this precedent, a careful solubility study of form I was undertaken, as shown in Figure 4; form I clearly shows the low-ionic-strength insolubility characteristic of forms II and III. Previous electron microscopic studies of forms I, () ' II and III have shown the presence of a ,-..,500 A tail in addition to globular subunits (Dudai et al., 1973; Rieger et al., 1973). The possibility that these tails contain collagen is suggested by the observation that a large form of acetylcholinesterase (perhaps homologous with form II or III) can be selectively removed from rat diaphragm endplates with collagenase (Hall, 1973). Accordingly the effects of collagenase on forms I-V 35 Fig. 3. Se diment a tion of separated forms I, II, III in 1 M NaCl and in no Na Cl. Separated forms I, II, III were diluted into 0.05 M phosphate, pH 7.0 with ( A) or without (B) 1 M NaCl. One hundred-µl aliquots were loaded onto 5.2 ml 5-20% sucrose gradients in the same buffer. Gradients we re centrifuged at 65,000 rpm for either 4.5 hr (A) or 4.0 hr (B). Fractions were collected and 10 ul (A) or 20 µl (B) of each was assayed for 15 min with 2 x 10- 3 M ACh. ,., 100 x 10 lo3 x 10 3 position. 3 cpm. cpm (B)o Complete conversion yielded Blank was 1.6 x 10 3 cpm (A), or Activity is plotted against fractional 40- Il ; ;;> 1.0 0- 20 40 O 0.6 , 0.4 0.2 \c<ll---M'»,p I \ Fractional Position 0.8 ,r,,. ,..,-v m ~ ~ ._.,.,di ~ 20 ~ I 10-1 40 "'~ Figure J 0 0-t= 1.0 20 40 O ._L, 0.6 0.4 0.2 0 ~ ~2--ri;r a •~.,,,'3'-r--:-3 f\"~ Fractional Position ' 0.8 m "'"""""«f~¾-?<•i-..~,,.,mlhf"'" ~ 20 4 Il ~ .,- I 8: no NaCl 0 it?cf?~~:a'i!m11'sr4'cof-!fH'~~ 10 20 ~ § -I o ~~~~~-r-~~~~ A: I M NaCl 207 °' \..;) 37 Fig. 4. Sedimentation of form I as afunction of NaCl concentration. AChE form I, prepared in 0.05 :M phosphate, pH 7.0; 1 M NaCl, was diluted with 1 volume of 0.05 M phosphate; 0.01% Triton X-100. One or two hundred-µl aliquots were loaded onto nine different 5-20% sucrose gradients containing NaCl concentrations from 0.1 M to 1 M. After centrifugation at 65,000 rpm for 4.5 hr, fractions were collected. Twenty µl of each fraction was assayed for 20 min with 2 x 10 - 3 M ACh. In the main figure the percent of activity recovered in the peak (~--@) and that recovered in the entire gradient (x--x) is plotted as a function of NaCl concentration in the gradient. In the insert, the fractional position of the activity peak is plotted as a function of NaCl concentration; no peak occurs below 0.2 M NaCl. JS 100 / X -- -- - - - - x x. . . x xI 80 ~------ >-. L (1) Ii C 0.7 8 60 /4 :~ 0::: x > (1) -+- ~ 40 /, u L (1) Q_ 20 0.6 ft ~ 0.5 0.4 C 0 IX OO3 X LL 0 . 2 - - - - - - - - ~ • 0.5 0 1.0 [NaCl], M o----~---r---..----.-----.. 0 0 .2 0 .4 0.6 [NaCl] , M Fi g ure 4 0.8 1.0 39 were investigated. Two different collagenase prepara- tions were used at low concentration, and high levels of BSA were added to try to circumvent the effects of any contaminating nonspecific proteases. When careful sedimentation analysis was carried out, using gradients with sedimentation markers, small but reproducible effects of collagenase tr e atment were seen, as shown in Figure 5. Forms I, II, and III were all shifted to slightly higher sedimentation constants, while forms IV and V were unaffected. To control for the possibility that differential collagenase effects on the forms might result from unintended variations in the separate digestions used, a mixture of forms II, III and V was treated with collagenase; as shown in Figure 6, forms II and III were shifted in the same digestion which left form V unchanged. A statistical analysis of a larger series of similar experiments, pr esented in Table 1, serves to verify the differential nature of the collagenase effect. Although collagenase selectively affected the (tailed) forms I, II, and III, it did not alter their char a cteristic low-ionic-strength insolubility (at least for most of the affected molecules) as shown in Figure 7. A small fraction of collagenase-treated forms II-III molecules were rendered soluble in low ionic strength (Figure 7); these molecules sediment with even great.er 40 Fig. 5. Effect of collagenase on sedimentation of separated forms of AChE. Separated AChE forms I, II, III, IV, and v were diluted 10-20X into 100 µl o f 0.1 M Tris, pH 7.5. Ten µl of 25 mg/ml BSA was added to each incuba- tion. Control incubations (-o-) received an additi onal 10 µl of Ool M Tris, pH 7.5. Collagenase incubations (-o-) received 10 µ1 of 100 pg/ml collagenase (Worthington CLSPA). After 1 hr incubation at room temperature, 100-µl aliquots were loaded onto 5.0 ml 5-2CPJo sucrose gradients in 0.02 M borate, pH 8.8; NaCl; 0.1% Tween 80; 1 mg/ml BSA. ADH were added as markers. 1 M ~-Galactosidase and Gradients were centrifuged for 5 hr (A) or for 7 hr (B) at 65,000 rpm. Five-drop fractions were collected and 10 µl of each was assayed with 2 x 10 (form I) or 5.5 x 10 3 -3 M ACh. ~20 x Complete conversion yielded io 3 cpm (forms II-V). cpm (form I) or 1.1 x 10 3 100 x 10 Blanks were cpm (forms II-V). Panel A shows the results for forms II, III, and V; panel B shows the results for forms I and IV, including a small amount of contaminating form II. For clarity, only the regions of the activity peaks are p lotted. Slight variations in the positions of the markers have b een compensated by shifting the fractional position scales 3 41 (by 0.025 or les s ) so that the most relevant marker (gal for A, ADH for B) has the same fractional position in all gradients. arrows, gradients. t. Marker positions are indicated by for control grad i ents, for collagenase gal ADH JI 'l t t 8 4 i t 6 3 ]IT }L r<) I t A II II 1\ 4 I I I 2 E I 0.. u t m 0 X 'l t III -sl. t }L 9 2 / 0 d I I (r.lY 0 1.0 0.8 0 .9 0.6 · 0.5 0 .7 0.4 ADH r0 I I N 'lt N 40 8 t 30 8 N f!:\ 6 I I 0 X ?Q 20 4 0.. I I I I E u I I 10 II 2 p--0.. er 0 0 ,cl I f p-0-~ . -Cf I 1.0 0 .9 0 .8 0.7 0 .6 0.5 Fractional Position 0.4 0 .3 4J Figo 6. Effect of collagenase on sedimentation of mixed forms II, III and V. Ten µl of an AChE preparation containing significant amounts of forms II, III, and V was diluted with 10 µl of 0.1 M Tris, pH 7.5; 10 mg/ml BSA and 10 µl of purified collagenase solution (Adv ance Biofactures Corp.; collagenase form III diluted 1:100 in (-o-) or 10 µl of additional Tris 0.1 M Tris, pH 7o5) buffer (---o---). After 14.5 hr incubation at room temperature, 5-µl aliquots were loaded onto 5.0 ml, 5-20% sucrose gradients in 0.02 M borate, pH 8.8; l M NaCl; Oo1% Tween 80; l mg/ml BSA. Gradients were cen- trifuged for 4.5 hr at 65,000 rpm. Five-drop fractions were collected and 2 µl of each fraction was assayed for 3 min with 2 x 10 22.2 x 10 3 cpm. 3 M ACho Complete conversion yielded The blank was 0.5 x 10 marker is indicated as in Fig. S. 3 cpm. The gal t 6 r<) I 0 5 4 m A I \ I II R ? Q .fl I I ~ 0.6 0.4 0.2 Fractional Position Fi gure 6 0 Legend for Table 1 Single forms of AChE or mixes of roughly equivalent amounts of several forms were incubated with or without collagenase, as in Figure 5. Aliquots of control and collagenase incubations were loaded onto 5.0 ml 5-20% sucrose gradients in 0.02 M borate, pH 8.8; 1 M NaCl; 0.1% Tween 80; 1 mg/ml BSA. ~-Galactosidase, catalase, and alcohol dehydrogenase were included as markers in most 6f the gradients. A total of 33 gradients were run for part A of the table, and 21 gradients for part B. After centrifugation, gradients were collected and assayed for AchE activity. The position of the activity peak was estimated to the nearest 0ol fraction (independent estimates by different persons rarely differed by more than 0.1 fraction) and converted to a fractional positiono Fractional positions in gradients not run for 5 hr were propor tionally corrected, and the resulting values for -each peak were used to calculate the mean fractional position and standard deviation reported in the table. The number of gradients used to calculate particular values is indicated in parentheses. Gradients with and without collagenase were tabulated separately , both for AChE activity peaks and for markerso Ao Collagenase incubations using Worthington ( CLSPA ) enzyme at 10 pg/ml (nominally 400 u/mg ) for 1-25 hr. The lower portion includes data from one set of gradients with no NaCl, run for 4 hr (fractional position not corrected to 5 hr). Bo Corp. Collagenase incubations using Advanced Biofactures (ABC) (Form III) enzyme. tion is nominally 2500 u/ml. This collagenase preparaIt was used at 1-1000 u/ml for 1-12 hr. Variation in incubation time was without effect in the range tested, except when enzyme concentrations >100 units/ml were used; in this case some degradation to form V was seeno Table 1. Eff e ct of Collagena se on the Sediment a tion Velocity of AChE Forms A. Worthington c oll a gena se 1 M NaCl Control Collagenase I II III IV . 40 + - .02 060 -+ .02 - .01 .76 + .34 + - . 01 (6) (6) (6) (4) .65 + - .02 083 + - .01 - .01 033 + (8) (8) (2) 044 ± .01 (9) no NaCl .33 Control (1) .33 -+ .01 Collagenase (2) B. ABC collagena se 1 M NaCl II I Control .60 + - .02 III . 77 (8) (9) Collagenase . 67 ± .04 (15) ± . 02 .84 ± .03 (15) IV 48 Table 1. Effect of Collagenase on the Sediment at ion Velocity of AChE Forms A. ( cont.) Worthington collagenase 1 N NaCl Control Collagenase V -gal cat ADH .52 + .01 - . 6 8 -+ .01 .48 + - .01 - .02 .33 + (3) (8) (4) ( 9) .52 + - .01 .67 + - .01 .48 + - .02 .33 + - .01 (4) ( 9) (6) (12) .51 .69 .so .33 (1) (1) (1) (1) .so +- .02 .68 -+ .01 .so :!:: 0 .33 :!:: 0 (2) ( 2) ( 2) ( 2) no NaCl Control Collagenase B. ABC collagena se 1 M NaCl Control Collagenase V -gal .49 + - .02 .69 + - .03 (7) (5) 049 + - .01 - .03 .67 + (15) (13) Figo 7c Effects of collagenase, phospholipase C, and trypsin on sedimentation of forms II and III. AChE form II (10 µl) and form III (7.5 pl) were mixed and diluted to 50 pl with 0,1 M Tris, pH 7.5. Ten µ1 aliquots were diluted into 100 µl of 0.05 M Tris, pH 7.5; 0.05 M NaCl; 0.005 M Cac1 . 2 One diluted aliquot received 10 µl of 0.1 M Tris and served as a control. Three other aliquots received either 10 µl of collagenase (Worthington, 100 µg/ml), 10 µl of phospholipase C (100 µg/ml) or 10 µl of trypsin (10 JJ.g/ml), all freshly prepared. After 3 hr incubation at room temperature 10 µ1 aliquots of each were loaded onto 5 ml, 5-20% sucrose gradients in 0.02 M borate, pH 8.8, 0,1% Tween 80 and 1 mg/ml BSA, and 'r -galactosidase was added as a marker ( t ) o 1 M NaCl was included in the gradients of set A but not in those of set B. Set A gradients were centrifuged at 65,000 rpm for 4.5 hr, set B for 3,0 hr. fractions were collectedo Five-drop Ten-µl portions from the first 20 fractions of each gradient were assayed for 30 min with 3 2 x 10- M ACh. Complete conversion yielded 113.7 x 10 3 cpm; the blank was 2.2 x 10 3 cpm, except that for the gradients after trypsin treatment, complete conversion yielded 22.7 x 10 3 cpm with a blank of 1.4 x 10 3 cpm. I 0 I 1.0 5 I ~I 0.8 0.6 0.4 0.2 Fractional Position ~.~~~~~ 7 Q ::::'4'":9:-:,')'t' 0 Trypsin 0.8 0.6 0.4 0.2 Fractional Position ~,il?r:$·1r!l!lx:~•!tr£>--ti'Q.,._.,Tt:" 1.0 -I Figure 7 0 I 5 10- 10 Trypsin 15 o• , • , 10-1 ~ u 15 10 7 0-...,_p~ 0 I 20 Phospholipose C 0--1----.-----.----.---,---~ X 20 u X Col logenose ~ 10 20 · Control a: no NaCl 0 r0 Phospholipose C Collage nose 30 E 30 Q_ 10 20 30 0 I O 1 '0-4)~>-0-/4:~~----- 20 30 Or-"711\--r-~~~-r--g_ 30 0 r0 I Control o--~~--.---.------ 10 20 30 A: I M NaCl 0 V\ velocities th an the rest (compare with marker) suggesting that they may have b een more extensively modified. The same collagenase effects were seen over a wide range of collagenase concentrations (from 0.04 u/ml to 40 u/ml), but not with other proteases ·or phospholipase C (Figure 7). However at very high collagenase concentrations or with incubations at higher temperatures, a conversion of forms II and III to form V was observed. This conversion is similar to that reported earlier for collagenase (Dudai & Silman, 1973, 1974a) and for trypsin and chymotrypsin (:Hassoulie & Ringer, 1969; Nassoulie et al., 1970). Whether it represents the effect of contaminating nonspecific proteases or a more complete digestion by collagenase is unclear. (From experiments with trypsin under identical incubation conditions, we calculate that contamination of the collagenase preparation with as little as 0.01% trypsin or related nonspecific pro te ase could produce the observed conversion to form V.) Previous studies of native acetylcholinesterase forms II and III, using SDS-polyacrylamide gels, have shown one major subunit of ~ 82,000 molecular weight; after prolonged autolysis or deliberate trypsin digestion to produce form V, this subunit shows varying extents of cleavage, yielding a ,.., 59,000 molecular weight fragment with a DFP 1.abeled site and a ,.; 25,000 molecular weight 52 fragment without (Dudai & Silman, 1974b). The results of Figure 8 confirm and extend these observations. Our preparations of the native forms II and III contain primarily the native subunit, but also significant amounts of the cleaved, active-site-containing fragment, indicating that cleavage need not alter the sedimentation properties of these native forms, In addition, virtually the same ratio of native subunit to cleaved fragment is seen after collagenase or trypsin digestions which do produce sedimentation changes, indicating that these enzymes do not (under these conditions) attack the active subunito Instead, they must attack a different component of the native forms, one which is covalently unlinJ(ed to the active subunit but is particularly important in determining sedimentation properties. Discussion The ammonium sulfate fractionation procedure described above has served to separate Electrophorus e1ectricus acetylcholinesterases -into two discrete classes with distinct properties. Members of the first class (forms I, II, and III) are insoluble in low ionic strength and sediment slightly more rapidly after collagenase treatment. Previous studies indicate that these forms 53 Fig. 8. Effects of collagenase and trypsin on SDS- polyacrylamide gel electrophoresis of form III. AChE form III was incubated with 2 x 10-S M [ 3 H]~DFP at room temperature for 20 min. Unbound DFP was removed by gel filtration on Sephadex GSO. One hundred-µl aliquots of the labeled AChE were incubated with digestive enzymes. The control received 10 µl of 0.1 M Tris; the collagenase incubation received 10 µl of 100 µg/ml collagenase (Worthington CLSPA); the trypsin incubation received 10 µl of 1 µg/ml trypsin. After incubation at room temperature for 2 hrs. 20 µl aliquots were boiled in SDS and dithiothreitol. Twenty µl aliquots of the denatured, reduced enzyme were loaded onto a polyacrylamide gel and electrophoresed. After fixation and staining for protein, the gel was sliced with an array of razor blades and the distribution of[ 3 H]was determined by liquid scinti l lation counting. against slice nurr~ero Cpm ot[ 3 H]are plotted The position of protein stain corresponding to marker BSA (66,600 dalton) is indicated with an arrow. 54 ~ lt I 0 )... 0 u (/) ~~ ~ 0 C t Cl) C t f C Cl) (/) 0.. >, "- .._ cri 0 0 <..O 0 u 0 lf) L (1) 0 ~ ..a E CX) z H Cl) •rl :J 0 t0 0 (\J 0 \ \'- - -....i---,....--.:..1~, , -1--1----.--1'0 N O t0 N z-01 x wdJ \--1- - - - - - , - - - - - 4 - 0 t0 (\J u (f) (1) :::$ bJl li-i 55 (> possess a 500 A tail (Dudai et al., 1973; Rieger et al., 1973). Members of the second class (forms IV and V) are soluble in low ionic strength and are unaf f e cted by collagena se treatment. Previous studies indicate that these forms l ack a tail (Dudai et al., 1973; Rieger et al., 1973)0 Because forms I (9 S) and IV (7.5 S) are similar in size, they have been unr esolved in many previous studies, and the reported properties of the "peak A" in which they have been jointly included have sometimes been confusing (Massoulie & Rieger, 1969; Dudai & Silman, 1974a~ the ammonium sulfate procedure provides a convenient method for separating these forms. Interestingly, the order of elution in the ammonium sulfate fractionation procedur e sugges t s that the 500 A tail, in addition to causing precipit at ion at low ionic strength, also promotes "salting out" at high ammonium sulfate concentrations. The observed ef f e ct s of collagenase on forms I, II, and III differ from those previously reported. In previous studies, using high collagenase concentrations, the effects of collagenase treatment have varied; in one case the effects resembled those of nonspecific protease digestion in that the native forms II and III were partially or wholly converted to a form with the lowionic-strength solubility and the sedimentation constant of the globular form V (Dudai & Silman, 1973, 1974a). 56 In a second case, collagenase was reported to make the native forms low-ionic-strength soluble without marlrndly altering their sedimentation properties (Ri eger, Bon & Massoulie, 1973). We confirm the occurrence of the former effect at high collagenase concentration; however, we do not see either effect at low concentrations, which nonetheless produce sedimentation shifts of forms I, II, and III. Accordingly we conclude that the sedimentation shifts are more likel y to be the result of specific collagenase action. Only one previous report of similar sedimentation shifts is kno1m to us; this occurred after treatment with a "phospholipase C" preparation from Clostridium perfringens (Ri eger, Bon & Massoulie, 1973). However, in the same report a Bacillus cereus phospholipase C preparation was shmm to have no effect , and we also find a Clostridium welchii phospholipase C preparation to be ineffective, Not ing that Clostridium p erfringens .1-s a rich source of collagenase, we prefer to believe that the previously reported shifts with the Qo perfringens preparation may have been due to low level collagenase contamination. The direction of the collagenase-induced shifts is consistent with the notion that collagenase is removing 0 • part of the 500 A tail. From the relative sedimentation rates of form I ( 9 S, one tetramer plus tail) and form V (12 S , one tetramer only), it appears that the tail 57 significantly slows sedimentation and that it s removal should then generate a sedimentation rate increase. However, the removal does not seem , under these collagenase treatment conditions, to be complete, since ( a ) the sedimentation rate of collagenase-modified form I is still not as high as that of form V, (10 S) (b) most of the collagenase-modified molecules remain low-ionic-strength insoluble, suggesting that some fragment of the tail is retained, and (c) a small fraction of the collagenasemodified molecules jointly acquire low-ionic-strength solubility and an even higher sedimentation rate, as if they had been deprived of an additional portion of the tail in a second, slower collagenase-dependent step. The use of electron microscopy to check the inference that collagenase removes the tail and to determine the extent of tail removal should prove interesting, and together with clcemical studies of normal and col l agenase modif ied forms should serve to determine whether the tail is indeed composed of collageno Whatever the nature of the tail, it is most probably made of subunits which are not covalently linked to the active subunitso Whether this might be accomplished by post-synthes i s, non- covalent association between the two subunit types, or by cleavage to produce the two subunits types from a larger precursor, is uncertaino 58 However, the former alternative makes it easier to understand the association of variable numbers of active subunit tetramers with a tail, and is particularly appealing if the tail is indeed a collagen-like component which serves to fix the enzyme to an extracellular, basement membrane site. References Dudai, Y., Herzberg, M., & Silman, I. (1973) Proc. natn. Acad. Sci., U.S.A. 70, 2473-2476. Dudai, Y. & Silman, I. (1973) FEBS Lett. 30, 49-52. Dudai, Y. & Silman, I. (1974a) J. Neurochem. 23, (1974b) Biochem. Biophys. Res. 1177-1187. Dudai, Y. & Silman, Io Commun. 59, 117-124. Hall, z. (1973) J. Neurobiol. 4, 343-361. Johnson, C.D. & Ru ssell, R.L. (1975) Anal. Biochem. 64, 229-238. King, T.P. (1972) Laemmli, U .L. Biochemistry 11, 367-371. (1970) Nature, Land. 227, 680-685. Leuzinger, W., Ba ker, A. & Cauvin, E. (1968) Proc. Natl. Acad. Sci., U.S.A. 59, 620-623. :Martin, R .G. & Ames, B .N. 1372-1379. (1961) J. Biol. Chem. 236, 59 Massoulie, J. & Rieger, F. (1969) bur. J. Biochem. 11, 441-445. Massoulie, J., Rieg er , Fo & Tsuji, s. (1970) Eur. J. • Biochemo 14, 430-439. Massoulie, J., Rieger , F. & Bon, s. (1971) Euro J. Biochemo 21, 542-551. Miller, R.L. & Udenfriend, S. (1970) Arch. Biochem. Biophys. 139, 104-113. Rieger, Fo, Bon, s. & Massoulie, J. (1973) FEBS Lett. 36, 12-16. Rieger, F., Bon, s., Massoulie, J. & Cartaud, J. ( 1973) Euro J. Biochem. 34, 539-547. Silman , H.I. & Karlin, A. U.S.A. 58, 1664-1668. (1967) Proc. Natl, Acad . Sci. 60 III Separation and Characterization of Multiple Forms of Cholinesterase Activity from the Nematode Caenorhabditis elegans 61 Abstract The nematod e Caenorhabditis elegans has four forms of cholinestera se activity separable by velocity sedimentation in sucrose gradi e nts: form I form III (11 S) and form IV (13 S). (5 S), form II (7 S), Most of form I was released by rep eated extraction with 0.05 M borate pH 8.8. A portion of forms II, III and IV was released with buffer extraction a lone, but a substantial amount of the larger forms was released only after treatment with the anionic detergent sodium deoxycholate (DOC). Forms I and II as a pair and forms III and IV as another pair share similar kinetic properties (i.e. Km, acylthiocholine substrate specificity, inhibitor specificity). Form II was more stable to thermal inactivation at 45°c than form I whereas forms III and IV were only slightly inactivated at 45°c and showed an identical timecourse of inactivation at 45°c and at 56°c. Forms I and II were inactivated by 0.5% DOC wher e as forms III and IV were more stable in DOC but were suppressed by 1% Tween 800 62 Introduction Fractionation of solubilized cholinesterase activity from electric organs (Massouli e & Rieger, 1969; Dudai & Silman, 1974), from muscle (Hall, 1973; Wilson, Mett ler & Asmundson, 1969), from vertebrate brain (Wenthold, Mahler & Moore, 1974; PD & Ellman, 1969; Lim, Davis & Agranoff, 1971; Chan, et al., 1972) and from a variety of invertebrates (Baites, Don & Masters, 1972; Hildebrand, Townsel & Kravitz, 1974; Dudai, in press) has consistently revealed multiple forms of different sizes. Studies of the kinetic properties of the separated forms has demonstrated differences in Km (Miller, et al., 1973) in substrate inhibition (Hall, 1973) and in inhibitor sensitivity (Chan, et a~., 1972) but the overall impr ess ion is of close similarity of kinetic properties (Massoulie & Rieger, 1969; Hall, 1973; Chan, et al., 1972). This suggests that the size differ e nces result from specific associations of a common active-site-containing subunit with other components, either self (forming multimers) or non-self. In contrast, the biochemical characterization of cholinesterases in vertebrates has demonstrated such striking kinetic differences between ("true") acetylcholinesterase (EC 3.l.lo7) and ("pseudo") cholinesterase (EC 3.1.108) that the two activities are treated as separate enzymes and a variety of inhibitors and substrates 6J are routinely used to allow selective determinations of either enzyme without requiring prior separation (Bayliss & Todrick, 1956; Augustinsson, 1963). Pr e liminary studies of the n e matode Caenorhabditis elegans (R.L. Russell, unpublished) had revealed multiple forms of ChE activity separable by velocity sedimentation. In the hope of applying genetic analysis (for which this animal is well suited (Dougherty & Calhoun, 1948; Nigon, 1949; Brenner, 1974)) to the problem of the interrelationship of the multiple forms, we have further characterized the separable forms of Q. elegans cholinesterase activity. This part then, describes the solubilization and properties (Kms, substrate and inhibitor specificities, thermal inactivation and detergent effects) of four separable forms of nematode cholinesterase. :dateria1s The detergents Tween 80 and sodium deoxycholate and the proteins bovine serum albumin (BSA, Fraction V), E. coli ~ -galactosidase, bovine catalase and electric eel acetylcho1inesterase were from Sigma Chemical Co., St. Louis, Mo. Renografin (3,5-diacetarnido-2,4,6,-triiodobenzoic acid methy1g1ucarnine salt) from Squibb, Inc., Princeton N.J. was obtained from a local source. Acetylcholine 64 iodide, eserine sulphate, neostigmine bromide (( m-hydroxyphenyl) trimethylammonium bromide dimethylcarbamate), isoOMPA (tetraisopropylpyrophosphoramide ), DFP (dii sopropylfluorophosphate), acetylthiocholine iodide and butyrylthiocholine iodide were from Sigma. Temik ( 2-methyl-2- (methylthio) propionaldehyde 0-(methylcarbamoyl) oxime) was from Union Carbide Corp., Salinas, Ca. Tensilon (ethyl-m-hydroxy- phenyl)dimethylammonium bromide) was from Hoffman-LaRoche, Inc., Nutley, N .J. BW28L~C51 (1 :5-bis-(4-allyldimethylammoniumphenyl) pentane-3-one diiodide) was from Wellcome Research Laboratori es, Beckenham, England. TEA (tetraethylammonium chloride) and hemicolinium-3 (2, 2 '-(4,4'-biphenylene)-bis (2-hydroxy-4,4'-dimethylmorpholinium bromide)) were from Eastman Kodak Co., Rochester, N.Y. Propionyl thiocholine iodide was from K & K Laboratories Inc., Plainview, N.Y. Paraoxon was a gift from the Department of Nematology, University of Calif., Riverside. Acetyl-~ -methyl- thiocholine iodide and propionyl-~ -m ethyl-thiocholine iodid e were from U.S. Biochemical Corp., Cleveland, Ohio. 3,3'-Dithio (6-nitrob enzoic acid) (DTNB ) was from Eastman. (3H}-acetylcholine was TRA277 from Arnersham-Searle Corp., Arlington Heights, Ill or NET-113 from New England Nuclear, Boston, Mass . Ca. Aquacide II was from Calbiochem, La Jolla, Sepharose 6B was from Pharmacia Chemicals, Inc., Pisc ataway , N.J. grade. Oth er chemicals were standard reagent Borate buffers were dilutions of 0.1 M Na 2B40 7 65 adjusted to pH 8.8 with HCl. Methods Nematodes (Caenorhabditis elegans, Bristol variety, N2 strain) were maintained in the laboratory on agar surfaces ( NGMI·/I media - Table 1) seeded with a lawn of Escherichia coli (OP-50 strain) (Brenner, 1974). The larger number of animals used for biochemical studies were grown in liquid cultures (Figure 1). For food, E. coli (NA-22 strain) was grown to stationary phase at 23°c on M9 media with 0.8% glucose as a carbon source (Table 1). The bacteria were collected by centrifugation and resuspended in S media (Table 1) at up to JO grn/1. An inoculum of nematodes was added and the culture was aerat~ with several spargers (for 10 1 in a 5 gallon carboy) or by the action of a reciprocal shaker (for 500 ml in a 2 1 Erlenmeyer flask). (The shaker has a throw of about L inches and the frequency was adjusted to about 2 cycles/second to produce a sloshing of the media which was sufficient for rapid growth.) large inocula Relatively (0,5-5% of the final yield) were used to produce rapid growth yielding healthier nematodes and avoiding the danger of contamination. Carboy cultures were often seeded with the nematodes harvested from cultures grovm on the shaker. 66 Table 1. Media S media NGMM for 4 1 for 1 1 agar 68 gm NaCl 12 gm 'peptone 10 gm H o to 4 1 2 K7HPo 4 KH 2Po 4 NaCl 5.85 gm mg/ml si tosterol in Tween 80 1 ml H2 o to 1 1 autoclave, cool add, presterilized 1. Trace Metals 10 ml 0.69 gm Feso "7H o 4 2 0.20 gm Mnc1 °4H o 2 2 0.29 gm Znc1 °7H 0 2 2 1.86 gm Na EDTA 2 H o to 1 .1 3 • :•10 autoclave, cool add, presteriliz e d 1 . 1 0 mg/ml cholesterol i n ethanol 4 ml 2. 0.5 M CaC1 8 ml 2 _ 3. 1. 0 M Ng SO 4 ml 4. 1 . 0 M (K ) P8 - 100 ml 4 ( pH 6.0 ) 35. 6 gm K IvIPO 2 4 108.3 gm KH Po 2 4 H o to 1 1 2 2a M9 for 1 1 NaC l NH C1 4 Na HPo 2 4 KH Po 2 4 .MgS0 4 H o to 2 1 gm 6 gm 3. 5 g-m 1 gm 6 gm 3 gm 0al gm 900 ml autoclave, cool add, presterilized 1. 8% (w/v) glucose 100 ml 4, 2 l M (K)citrate 10 ml (pH6.0) 20.0 gm citric acid•H o 2 293a5 gm ( K) citrate•H o 2 H2 o to 1 1 3 1 M Ngso 3 ml 4 D.5 M Cacl 6 ml 2 *or use 10 mg/ml cholesterol in ethanol ( add after autoclaving) f or bubbling cultures - Fig. 1. Growth of Caenorhabditis elegans in liquid culture o A culture of ~o coli was grown to stationary phase at room temperature and the bacteria were collected by centrifugation and resuspended in half the volume of S mediao The nematodes (N2) from several 9 cm petri plates were added to 500 ml of resuspended bacteria in a 2 1 Erlenmeyer flask and incubated at 20°c on a reciprocal shakero Aliquots were removed for counting of bacteria (Petroff-Hauser slide) and of nematodes (Scott slide). During exponential growth, the nematode doubling time was about 14 hours. 68 +(/) (1) > ~ 0 . ..s::::. . X X X X -~ x-L____x✓ E E "-.. (/) '0 (1) )._ (1) -1- 0 0 10 9 X ..0 \ 2 3 Time (days) Fi gure 1 4 5 10 3 -0 0 +0 E Q) C Cultures were harvested by low speed centrifugation or by settling at 4°C. After several washes with cold distill ed water, the nematodes were separated from debris and crystalline material which forms in the cultures by flotation in 35% sucrose (Sulston & Brenner, 1974). After further washes to remove the sucrose , the animals were pelleted in a Sorvall centrifuge and then used immediately or frozen in liquid nitrogen and stored either under liquid nitrogen or at -20°c. Homogenization was by freeze-powdering in liquid nitrogen. Usually, a small amount of water was added to allow the pelleted animals to be pipetted and they were then added dropwise to liquid nitrogen in a cold mortar. The frozen droplets were ground to a fine powder with a cold pestle and the powder was removed with a spatula and tha·w ed. 0-4°C. Subsequent fractionation was performed at Pellets were resuspended by re-freeze-powdering or with a Dounce glass homogenizer. The multiple molecular forms of cholinesterase were displayed by zone vel oci ty sedimentation through sucrose gradients ( rfartin & Ames, 1961) using either a SW 27 or a SW 65 rotor in a Beckman L2-65B ultracentrifuge. SW 27 gradients were collected by lowering a thin capillary pipette to the bottom of the tube and pumping the contents out with a peristaltic pump. SW 65 gradients were collected by drop after puncturing the bottom of the tube 70 with a rigidly held needle, ~ -galactosidase Catalase (11.3 S) and (15.9 S), included in some gradients as markers, were assayed by spectrophotometric assays (Massoulie & Rieger, 1969; Baudhuin, et al. , 1964) . For biochemical characterization, and to permit rerunning of separated forms, the appropriate fractions of sucrose gradients were dialyzed against 0,05 M borate, pH 8.8 buffer and concentrated with Aquacide II. Sucrose was removed from larger fractions by pressure filtration in a Diaflo apparatus using a PM30 membrane, then further concentrated in a dialysis bag with Aquacide II. Separated, concentrated forms were further dialyzed against buffer 0 then stored at -20 C. Assays for cholinesterase were with a radiometric liquid extraction assay (Johnson & Russell, 1975), Assays included, in 100 µl, 0,025 M (K)Po =, pH 7,0 and 0.02-0.1 4 3 µcurie [ HJ-acetylcholine. Cold ACh was usually included . -5 to give 5.0 x 10 M ACh. Assays were started by the addition of enzyme or substrate and stopped with 100 µl of 1 M ClCH COOH, 0.5 M NaOH, 2 M NaCl; four mls of scintilla2 tion fluid (0.5% PPO, 0.03% POPOP in toulene-10% n-butanol) was added, the reaction-scintillation vial was capped, vigorously shaken and counted, The labelled product 3 ( [ HJ-acetic acid) extracts into the toluene phase and is selectively counted. Each set of assays included several blanks and complete conversions (effected with 71 10 pl of lOOu/rnl e l ec tric eel AChE) as controls. The Km for acety lcholine was determined by assays containing variable amounts of cold ACh and a const a nt amount of [ 3 H]-ACh . Buffer and substrates were pr e mixed and 100 µl was used for duplicate assays of each enzyme being tested a nd for triplicate blanks and complet e conver s ions. 0 Enzymes were prepar ed at 4 C in 0.05 M borate, 1 mg/ml BSA , a t a concentration that produced about 30% conversion at the lowest concentration of ACh and were then allowed to equilibrate to room temperature for 10 minutes before beginning the assay. Specificity toward acylthiocholine substrates was determine d with the spectrophotometric assay of Ellman (Ellma n, et al., 1961) modified so as to approximate the conditions of the radiometric assay . Each assay contained 0,86ml 0.025 M (K)Po =, pH 7.0, 0.1 ml 0.0033 N DTNB and 4 0.02 ml 0.05 M acylthiocholine iodide (final concentration 10 - 3 M). The assay was started b y a ddition of enzyme in 0.02 ml. The change in OD 412 was followed on an Acta III recording spectrophotometer with a blank (no enzyme) in the r eference cell. The effects of inhibitors were assessed by radiometric assay at 3 x 10 -6 M ACh (no cold ACh a dded) . Inhibitors were pr e pared fresh and premixe d with buffer and substrate; 100 µl was used for duplicate assays of each enzyme b e ing t ested a nd for tr iplicate blanks and 72 complete conversions. Complete conversion was achieved with 10 µl of 2 M Na0H and neutralized with 10 µl of 1 M H so before stopping with the usual solutions. 2 4 Thermal inactivations were performed by diluting enzyme at least 20X-fold into assay buffer preheated to the inactivation temperature. Aliquots were removed at specific times thereafter and transferred to assay vials on ice. After all time points were taken, the vials were warmed to room temperature and the a~rnay was started by the addition of substrate. Results When nematodes were homogenized by freeze-powdering in liquid nitrogen and thawed into 5 volumes of distilled water, only a small amount (<5%) of the cholinesterase activity measurable in the homogenate was solubilized. For preparative studies, the diluted homogenate was usually centrifuged at 20,000 rpm for 30 min and cholinesterase was extracted from the resulting pellet by resuspension in an extraction buffer. Treatment of the homogenate for 2 hours at 4°c with 0.05 M borate, pH 8.8 released 47% of the measurable activity to a low speed supernatant. Analysis of this supernatant by velocity sedimentation (Figure 2) revealed two peaks of solubilized activity at 5 Sand at 12 S. In addition, a substantial portion of the activity was not solubilized and floated on a cushion 73 Figo 2. Fr actionation of Cholinesterase by velocity sedimentationo Fresh worm homogenate was dilut e d into 10 volumes of 0.05 M borate, pH 80 8 • • . After two hours stirring at 40 C, a 1500 g, 5 min supernatant was prepared (cont aining 47% of the activity of the homogenate) and 1 ml was loaded unto 34 ml 5-20% sucrose gradients (same buffer) with 4 ml cushions of Renografin. The gradient was centrifuged for 25 hours at 27,000 rpm. Fractions were collected and 10 µl of each was assayed for 90 min with 5 x 10- 5 M ACh. The 3 3 blank of [ HJ -ACh was O. 3 x 10 cpm; complete conversion was l8o0 x 10 3 cpm. The position of 0-galactosidase from E.coli (15.9 S) is indicated by the arrow. r0 I 2 0 X E 0. -I 0 0 ~~'4------.--------,-----,..----..---- 0 5 10 15 Fraction Number F igure 2 20 75 of high density Renografin. (When a cushion was not used, this fraction pelleted and was not recovered.) Extraction with high ionic strength solutions (0.05 M borate, pH 8.8; 1 M NaCl) did not appreciably enhance the amount of activity released and no additional sedimentation peaks were revealed (Figure 3). Repeated extraction of high speed pellets with 0.05 M borate, pH 8.8, over several days following homogenization (Figure 4A) released about half of their total activity to supernatants. Much of the released activity in the first few supernatants sedimented at about 5 S. The 5 Speak decreased in the later extractions to reveal an additional peak at about 7 S. In addition, all of the supernatants contained material sedimenting as a broad peak from 11 S to 13 S. Various treatments were tried in attempts to release the activity which remained in the pellet. Only treatment with the anionic detergent sodium deoxycholate(DOC) released most of the activity ( > 85% in two washes) . The / activity extracted from the pellet by DOC sedimented as a 7 Speak and as a broad 11-13 Speak (Figure 4B). Only small amounts of activity sedimented at the 5 S position. When gradient fractions were dialyzed and refractionated by velocity sedimentation (Figures 5 and 6) sharp peaks were observed at four separate positions corresponding to four separable forms: form I + (5.3-0.3 S), form II Fig. 3o Velocity sedimentation after extraction with 1 M NaCl. Fresh worm homogenate was diluted six fold into 0.05 M b orate, pH 8. 8 ; 1 M NaClo . . After 2 h ours stirring at 40 C, a 1500 g, 5 min supernatant was prepared and 1 ml was loaded unto 38 ml 5-20% sucrose gradients. The gradients were centrifuged at 27,000 rpm for 16 hours. Fractions were collected and 10 µl of each was assayed with 5.0 x 10 -5 M ACh for 45 min. . The blank was 0.2 x 10 complete conversion was 17.6 x 10 3 cpmo 3 cpm; 77 r<) 6 I 0 X 4 E 0.. u 2 5 10 l5 Fraction Number Fi g ure J 20 Fig. 4 . Successive extractions with 0.05 M borate, pH 8.8. About 20 gm of fresh nematodes (N2 ) were homogenized by freeze powdering. Without dilution, a 20,000 rpm, 30 min supernatant was pr epa red (yield 6 ml-used for studies of soluble enzymes). The pellet was resuspended in dis- tilled water and sedimented at 20,000 rpm for 30 min. The two S20K supernatants were respun at 40,000 rpm for 30 min to give S40K-l and S40K-2. rrhe combined pellet was re- peatedly resuspended by Dounce homogenizer in 10-20 ml of 0.05 M borate, pH 8.8 as a series of nine 40,000 rpm, 30 min supernatants (S40K-3 thru S40K-ll) were prepared over 4 days. A. Total S40K supernatant activity (1 u = 1 mole ACh hydrolyzed/min at 5 x 10- 5 M ACh ) is plotted against the time of supernatant p reparation. The dashed lines above S40K-5 and S40K-9 represent activity in the resuspended pellet. B. Velocity sedimentation of the S40K supernatants at 65,000 rpm for 6.5 hrs (5 ml 5-20% sucrose gradients in Oo05 M borate, pH 808)0 Slight differences in sample size, length of run and number of fractions were compensated for by plotting activity versus fractional position. Activity assays were with 5.0 x 10 [ 3 H]- ACh. -5 M ACh and 10 5 cpm of The measured cpms (blank subtracted) were corrected to a ;i-o µl, 10 min assay and multiplied by the total volume of each S40K so that the plotted activity was 79 proportional to total activity of the fraction. The final gradient represents activity solubilized by treatment of P40K-9 with 0.5% DOC. u ..c ~ +- 2 3 4 11 Figure ~- u [ X 9 0 l Ti me ( days) 0 ~-~-cl 1.0 I I I ,~- (DOC) pellet I 0.4 0.2 I I Fractional Position 0.8 0.6 I >h I I 0 ~ ~ ~ l > - ·•- t ! - -, - ~ I 1 I~~ lrllgl 3 1 ~ I 2-f-Y '..-.---,---1 0 I fglO I I 0 LO I 15-V 51: ri I I I 1 ~ I I I I I I I A 6 I 10~ 15CJ) 0 ➔- -0 20 25 18 B OJ 0 81 Fig. 5. Velocity sedimentation of separated peaks. The peak. fractions from sucrose gradients were pooled, dialyzed and concentrated. Aliquots were refractionated by velocity sedimentation at 65,000 rpm for A. B. 9 hours and Co 6.5 hours. 12 hours, Samples were Ao 5 S peak. of the first pH 808 extract (S40K-3, Figure 4); B. 7 Speak from a fractionation of later pH 8.8 extracts (pooled S40K-5 thru S40K-9, Figure 4) and Co ll-13 Speak from the first pH 8.8 extract (S40K-3, Figure 4). The peaks are labelled I-IV representing the four separated forms shmm completely separated in Figure 6 o The arrow marJrn a comparable position in the three gradients. Assay blanks were 0.5 x 10 3 , 0.6 x 10 3 , 3 and 3.7 x 10 3 cpm and complete conversions were 18.2 x 10, 16.4 x 10 3 and 96o5 x 10 respectively. 3 cpm for panels A, Band C 82 A 6 4 . . ~ · J 2 o ~ TI • B r<) I 0 4 X ~ 2 0 12 C 8 . ' ,. -. ' ~ ~ ·.,: 0 - - i - - - - , - - - , . - - - - - , - - : : : :••·3· . ~~ I ~~ l.O 0.8 0 .6 0.4 0.2 Fractiona l Position Fi g ure ,; 0 83 Fig. 6. Rerun of separated forms. A one hundred µl aliquot of separated forms I-IV were loaded unto a sucrose gradient in Oo05 M borate, pH 808. After sedimentation for 7 hours at 65 , 000 rpm, fractions were collected and assayed with 5.0 x l □The arrow indicates the position of bovine catalase ( 11.3 S ) . 5 M ACho 84 20 10 0 v ]&> /. Q&) Q , ;j " \0 ? ,,,.,., 8 t0 I 0 4 w X E 0. {_) 1.0 0.8 0.6 0.4 Fractional Position Fi g ure 6 0.2 0 (7.1±0.4 s), form III (11.4±0.5 S) and form IV (13.o±o.6 S). A number of cholinesterases have been reported to be "elongated" based upon a comparison of velocity sedimentation and gel filtration data (Massoulie, Rieger & Bon, 1971; Dudai, in press); by comparison to a globular protein of equivalent molecular weight, elongation leads to slower sedimentation (suggesting a lower molecular weight) but earlier elution from a gel filtration column (sugge sting a higher molecular weight). None of the worm cholinester- ases appear to be substantially elongated by this comparison (Table 2). The molecular weights of the forms, calculated from sedimentation coefficient are: form I (82,000±5000), form II (130,000±10,000), form III (2js,000±1s,OOO) and form IV (343,000±22,000). Since multiple forms could be produced by degradative processes during the relatively lengthy extractions, attempts were m:3.de to directly solubil i ze all of the cholinesterase 2ct ivity of the homogenateo When aliquots of variously extracted homogenate were sedimented on gradients with Renografin cushions, it was found that a 30 minute treatment with 0.5% DOC completely eliminated activity on the cushiono Solubilization with DOC was complicated by a rapid loss of activity sedimenting at 5 S and 7 Sand maximal recovery of the solubilized activity was achieved by short (1 min) treatments with 0.2% DOC 86 Table 2. Average Sedimentation Constants and Gel Filtration of Separated Froms Velocity Sedimentation s Form estimated ➔i" MW ( x10-3daltons } Gel Filtration Kav estimated* ( 6B) MW (xl0- 3 daltons ) I 503 -+0.3 (n-=7 ) s2±s 0.53 95 II + 7 .1-0.4 ( n=S ) 130±10 0.48 135 III 11.4....'..0 .5 ( n=6 } 275±15 0 .34 340 IV 130±0.6 (n=6) 343 ±22 0.32 390 7. 4 -+0.4 ( n:=3 ) 1 35±10 0. 41 220 3 05 0.32 390 .L Drosophila melanogaster Electric eel (V) 12,8 Kav determined relative to blue dextran ( Kav = 0.0 ) and tryptophan e (Kav = 1.0) on a column ( 1 cm dia x 34 cm ) of Sepharose 6B ( run in 0.02 M borate, pH 8.8; 0.1% Tween 80 ) . The marker positions were determined by continuous monitoring of the 0D 280 spectrophotometer. of the eluate with a LKB Unicord II *Molecular we i ghts were es tim a t ed f or se d i mentati on fr om plots of lit e rature s! 0 and IiW da ta . For the ge l f iltra- tion d a ta, MW was estimated off th e chart in the Pharmaci a Ch em i cal Co, p amp h let "S e p h 3. ros e 11 • 87 followed as quic1ny as pos s ible by sedimentation into gradients without DOC and containing 0.1% Tween 80 (Figure 7). The distribution of activity in such gradients was smeared, but consi stent with the presence of all of the separable forms in vivo. Forms I and II have been reliably recovered from reruns of DOC extract gradient fr ac tions. The separation of the larger forms into only forms III and IV was usually less clear and there exists the possibility of additional minor heterogeneity here. Biochemical characterization of the four separated forms is described in Figures 8 and 9 and T ab les 3, 4 and 5. Kms Forms I and II as a class have similar Kms which are significantly different from those of forms III and IV (Figure 8). Electric eel AChE and horse serum cholinester~ ase had Kms of 1.0 x 10 experiment. -5 Mand 32.3 x 10 -5 Min the same (The radiometric assay is not we ll suited for use at the higher concentrations of ACh necessary to assess substrat e inhibition and the precise determination of the extent of substrate inhibition for all of the worm forms has not b een completed. An experiment shown in Part IV (Figur e 7) demonst r ates the substrate inhibition of forms I and III. ) Substrate specificity Tabl e 3 pr esents the substrate specificity for a 88 Fig. 7o Velocity sedimentation after extraction with DOC. One ml of frozen nematodes were freeze powdered and extracted with 0.8 ml of 0.1 M borate, pH 8.8 and 0.03 ml of 4°c. 10% DOC (final 0.17% DOC) at After 2 min, a 1500 g, 1 min supernatant was prepared and 100 µl aliquots were loaded unto 5o0 ml 5-20% sucrose gradients in 0.05 M borate, pH 8.8, 0.1% Tween 80. After centrifugation (65,000 rpm, 4.5 hours), fractions were collected and 10 µl of each was assayed for 10 min with 5.0 x 10- 5 M ACh. The blank was 1.1 x 10 32.0 x 10 10 -5 3 cpm. 3 cpm; complete conversion was Alternate fractions were reassayed with M eserine - - 0 - . 8 t0 I 6 0 .x E 4 0. u 2 Q -l-C)---.0-:~=:e-.-o.~~--.------~.:___~~-0----0 0 15 20 Frachon Number Figure 7 25 90 Fig. 8. Km for acetylcholine . The separated forms I-IV were assayed with 10 con4 centrations of ACh ranging from 2 x 10- 6 to 10- M. Velocity (Vin u/ml) was plotted against V/S, an EadieHofstee ploto The Km (-slope) was calculated with the linear regression program on an HP65 calculator. • ' stand ard d eviations of the slopes wer e : form I 5 5 form II± 0.5 x 10- ± O. l X 10- 5 M. M, form III± 0.1 x 10- The ± 0 .6 x 10 - 5 M, M, form IV 91 I 80- TI Km == 9. 6 x Io- 5 M Km=7.2 X I0- 5 M 60 0- l N Km= I. 9 x I o- 5 M 0 0.5 1.5 0 0.5 2 V/Sx10- _ Fi g ure 8 1.0 92 Table 3. Acyl-thiocholine Substrate Specific i ty of Separated Forms Activity of Subs t rate electric eel AChE horse serum ChE nematode ChE forms I II III IV acetyl-thio choline 100 100 100 100 100 10 0 acety1- Q, methyl-thi o choline 68 37 45 47 66 78 p r opionylt h iochol i ne 61 18 9 54 59 104 103 p rop i onyl -\' methyl thiocholine 36 92 35 34 94 102 Oo3 181 26 25 64 71 I butyrylthi ocholine I Hy drolysis of acyl-thiocholine substrates was followed spectrophotometrically as described in methods. Substrates were at 1 mJv1. All of the enzymes had about the same activity with acetylthiocholine as substrate . ( b. OD 412 /min ) recorded as percent of the act i vity measured wi th acetylthiocholine. Activity 9J number of acylthiocholine compounds. The "true" acetyl- cholinesterase from the·electric eel and the "pseudot' cholinesterase from horse serum are included for comparison. All of the nematode enzymes are intermediate i n specificity between the two vertebrate enzymes. Comparison of the four separable forms of worm ChE again reveals a pairing of forms I and II and of forms III and IV. In general the rate of hydrolysis with forms III and IV was less effected by lengthening the acyl group than was that of forms I and II. Inhibitor specificity The inhibition of electric eel AChE, horse serum ChE and the four separated forms of worm cholinesterase with nine cholinesterase inhibitors (Table 4) divided the four worm forms into the same two classes seen previously in Km and substrate specificity data. The inhibitors were chosen to inclu de several covalent carbamate and organophosphate inhibi to rs as well as reversible inhibitors. Two of the inhibitors are 5 x 10- 4 M TEA, commonly used to block potassi um-channels (Kd=4 x 10- 4 M, Hille, 1967), and 5 x 10- 6 M hemicolinium-3, used to block choline uptake (at 2 x 10- 5 M, Birks & MacIntosh, 1961); both of these compounds are effective cholinesterase inhibitors. The nematode enzymes were only 5-11% inhibited by 2 x 10- 3 M iso0}1PA, the specific inhibitor of ''pseudo" Table 4. Inhibitor Specificity of Separated Form % Inhibition of Inhibitor electric eel AChE horse serum ChE 78 nematode ChE form I II III IV 22 25 19 73 76 89 71 31 20 56 59 10 98 29 22 58 63 paraoxon (l0- 6 N) 44 87 20 22 15 15 iso-01'1PA (2 X 10- 3N) 23 100 5 7 9 11 tensilon (2 X 10- 6 M) 96 0 34 30 61 59 BW284C51_ 6 (2 X 10 M) 99 21 45 48 64 69 TEA 91 26 68 66 76 75 37 86 35 32 70 73 neostigmi9e ( 2 X 10- N) _ (5 X 10 4 :M) hemicolin~um-3 (5 X 10- M) Inhibition of activity in 15 min assays without preincubation of enzyme and inhibitor. Percent inhibition was calculated by comparison with parallel assays without inhibitor. 95 cholinester a se. 2 x 10- 6 M Bl'l284C51 ( a "tru e " AChE inhibitor ) inhibited the worm cholinesterases more strongly than the horse s e rum cholinesterase but less than the eel enzymes. These inhibitor results show that the worm enzymes although classifiable by internal comparison, do no·t fit the dichotomous "true" vs "ps eudo " cholinesterase classification scheme that characterizes the vertebrates. Thermal inactivation At 45°c, form I was rapidly inactivated (Figure 9A) whereas form II was more stable. 0 0 At 45 C and at 56 C, forms III and IV were inactivated in parallel (Figures 9B and 9C). The differences between forms I and II, in spite of the similarity of their kinetic properties, demonstrates that size and stability differences need not lead to a measurable effect on the active site. Detergent s e nsitivity Incub a tion. wi th 0.5% DOC caused an inactivation of forms I and II whereas forms III and IV were more stable (Tabl e 5). This effect occurs in homogenates and could be used for separat e ly assaying forms III and IV activity without requiring fractionation. Tween 80 suppresses, but does not inactivate forms III and IV. Fig. 9. Thermal Inactivation. A small aliquot (l ess than 20 µl) of each separated form was diluted to 1.5 ml of 0.025 M ( K)Po ~, pH 7.0; 4 1 mg/ml BSA (pr eincubated for 10 min in a wa ter bath at the inactivation temperature). One hundred µl aliquots were removed at various times and transferred to assay vials on ice. After all samples had been taken, ChE 3 activity was assayed by addition of 10 µl of [ H] -ACh (5.0 x 10 -6 Min assay) for 90 min. as a percent of a control assay. Activity is recorded The control was an equivalent dilution into Po = at room temperature. 4 97 ro 0 u r t:! <.D ~ T 0 LO tel I[ 0 tj" 0 0 LO <..O LO tj- 0 r0 ..--.. C E ...___. Cl) E 0 }- C\J :-.: 0 ~. I ·L ' \ (" ·-;sf: . : : _ _ ] \ , .s,r 0 0 t.0 O 0 tj" O co 0 tO '\ 0 0 Q) H ::::, ~1 <( °' tj" bl) 0 ·rl <.D r-"--1 0 LO 0 0 tj" LO ---C tj- 0 r0 E ---QJ E 0 IC\J 0 ;;;--_ 0 co _ ......___ ___,__ _ ___.,__ _ _ _......___ _ ___.___.,._,._. Q 0 0 (.{) tj" 0 (\J 0 ID 98 Table 5 . Detergent Effects on Se parated Forms Perc e nt Activity Surviving Immed Assay 1% Tween 80 I 90 II 90 III 42 IV 42 0.5% DOC 67 84 91 93 76 90 31 33 8 12 51 56 Incub 25 hrs 1% Tween 80 40 0.5% DOC Aliquots of each separated form were diluted into 0 .05 M borate , pH8.8, 1 mg/ml BSA divided three ways and • • d at 4° Co ma1nta1ne Tween 80 was added to 1% in one sample, DOC to Oa5% was added to another. received no detergent. The control Ten µl of each sample was assayed 3 for 90 min with [ H]- ACH at 5.0 x 10 - 6 M. The assay was repeated after 25 hours incubation at 4°c. Activity is expressed as a percent of t h e no detergent control. 99 Discussion The solubilization of cholinesterase activity from other organisms has received considerable attention without yielding a generally acceptable procedure. Although some activities ( e.g. mammalian serum cholinesterase) are seemingly soluble in vivo, -. extraction of homogenates with distill ed water or with neutral pH buffers usually releases only small amounts of soluble activity. High ionic strength (Silman & Karlin, 1967; Massoulie & Reiger, 1969; Dudai & Silman, 1974), high pH (Smallman & Wolfe, 1956), divalent ion chelators ( Chan, et aL, 1972; Wenthold, Mahler Moore, 1974), nonionic d etergents (Ho Dudai, in press; Lim, Davis & & & Ellman, 1969; Agranoff, 1971; Hall, 1973; Yamamura, et al., 1973; Jack:son & Aprison, 1966), and treatments with proteases (Massoulie , Rieger Ho & & Tsuji, 1970; Ellman, 1969; Dudai, et al., 1972; Betz & Sakrnann,1971), collagenases (Hall & Kelly, 1971), and lipases (L awler , 1964), solubilize por tions of the activity in some systems. The extraction of electric eel electric organ AChE with 1 M NaCl is one case where all, or almost all, of the activity is solubilized; in most other cases the extraction is incomplete and additional soluble activity can often be gotten by repetitive extractions or by prolonged incubations at 4°c, presumably to allow time for "autolytic" dig estion. The fractionation of solubilized cholinesterase 100 activity into multiple molecular forms has often been achieved by velocity sedimentation in sucrose gradients (Massoulie & Rieger , 1969; Hall, 1973; Dudai, in press). Separable forms may arise from the expression of separate genes ( e.g . "pseudo"-cholinesterase vs "true"acetylcholinesterase) or by the formation of stable associations between a common active-site-containing subunit and other components. These associations may represent sequential stages in the formation of functional complexes, and they may result in the forms having separate localizations and functions. The degradation of these complexes provides another opportunity for production of separable forms of di fferent size. The solubilization and fractionation of cholinesterase activity by velocity sedimentation in the nematode Caenorhabditis elegans has revealed four molecular forms: form I (13 S). (5 S), form II (7 S), form III (11 S) and form IV Most of form I was released by repeated extraction with 0.05 M borate, pH 8.8. A large portion of the other three forms was retained in the pellet after many washes and was released only after treatment with the anionic deterg ent deoxycholate (DOC). High ionic strength, EDTA and non-ionic detergents were without effect. All of the forms have been obtained by velocity sedimentation of fresh homogenates after only a few minutes extraction 101 with DOC suggesting that they are all present in vivo and do not represent artifacts of post-homogenization degradation. Once separated, the forms have not been observed to interconvert although specific attempts to interconvert them have not yet been made. The kinetic properties of the four separated forms (Km, substrate and inhibitor specificity) were compared with one another and with the electric eel ("true") AChE and horse serum ("pseudo") ChE. The four forms of nematode cholinesterase were divided into two classes based upon the kinetic analysis; forms I and IIj as compared with forms III and IV, 1) have larger Kms 2) are more specific for acetylthiocholine than for other acylthiocholine substrates and 3) are generally less sensitive to inhibitors. (None of the kinetic differences was very large, and confidence in the differences between them rests largely upon the substantial agre e ment between the measurements of the two forms within each class.) All of the nematode enzymes differ significantly from both the eel and the horse serum enzymes. Although the worm enzymes do not show very strong substrate specificity (a characteristic of "pseudo"-cholinesterases) they were all resistant to the specific-ChE inhibitor iso-OMPA at a concentration where even the eel enzyme was substantially inhibited. This failure to adhere to the vertebrate 102 classification scheme has generally b een the case for invertebrat e cholinesterases (Chadwick, 19 63 ). Whether the nematode enzymes should be given the designation EC 3ol.l. 7 ( AChE) or EC 3.1.1.8 (ChE) is unclear. In any case, the kinetic differ ences between the two classes of forms suggests that their active-site-containing subunit may be differento The selective suppression of forms III and IV with Tween 80 and the selective inactivation of forms I and II with DOC support this suggestion. Forms III and IV were much more thermos-tab le were the smaller forms. than Forms I and II were significantly inactivated at 45°c whereas 56°c was required to significantly inactivate forms III and IV. It is interesting that although forms I and II have similar kinetic properties, form II was considerably less heat sensitive. Assuming that I and II have a common active-si te-containing subunit, this shows that some properties of a complex can be altered without measurably affecting the active site. The kinetic differences between classes of forms are not large enough to al low selective determinations of the activity of a single class without fractionation. However, selective inactivation of forms I and II with heat or with DOC could be used to allow a selective determination of the more stable forms III and IV o If forms I and II have a common active-site subunit, it is natural to ask whether form II could be a dimer of lO J form I. Assuming that both forms are globular (gel filtration suggests that none of the forms are elongated) their molecular weights c a lculated from sedimentation coefficient would b e about 82,000 daltons (form I) and 130,000 daltons (ratio 1.6 ). This value is considerably less than 2 and suggests that a different component is added to form I to yield form II. Alternatively, the cleavage and release of a portion of form I followed by dimer formation could produce a form II of the appropriate sizeo The relationship between forms III and IV ( estimated molecular weights 275,000 daltons and 343,000 daltons) is proportionally smaller and may involve secondary modifications ( e.go perhaps a polysaccharide component)o It would be most interesting to know the size of the active-sitecontaining polypeptides in the different forms and an approach to this is being made by SDS-polyacrylamide gel electrophoresis af t er covalent labelling of the active-site • • Lh-3H~- P. serine WlL L J - LJt In a ddition, experiments to deter- mine the order and relative rates of reappearance of active enzyme in v ivo, DFP after irreversible inhibition with may demonstrate or rule out the possibility that the differ e nt forms represent sequential stages in the formation o f functional comp lexes. Refe rences Augustinsson , K.B. (19 63 ) In: Handbu ch d er Experimen- tell en Pharmakologie, XV, Koel l e , G ,B. ( Ed .) Spri nger -V erlag , Berlin, pp. 87-128. Baudhuin, P. (1964) Bioch em . J. 92, 17 9-1 84, Bayliss, B,J. & Todrick, A. (1956) Betz, 1tJ. & Sa:V,Inann, B. ( 1971) Bio ch em . J. 62, 62-67. Nature New Biol. 232, 94-95. Birks, R.I. & Mac Into sh, F.C. (1961) Can. J. Biochem. and Physiol. 39, 787-827. Brenne r, S. (1974) Gen e tics 77, 71-94. Chadwick, L.E. (1963) In: Cholinest erases and Anti- cholinesterase Agents, Ko e lle, G.B. (Ed.) Springe r- Verlag , Berlin, pp. 741-798, Chan, S.L., Shirachi, D.Y., Bha r gava, H.N., Gardn e r, E. & Trevor, A.J, (1 972 ) J. Neuroch em . 19, 2 747-275 8 , Dough erty , E.C. & Calhoun, H.G. (1948) Dudai, Y. (197 6) Nature 1 61, 29. Dros. Inf. Ser. 52, in press. Dudai, Y, & Silman, I. (1974) J . Neurochem . 23 , 1177-11 8 7, Dudai, Y., Silman, I., Kalderon, N. & Blumb erg , S. (197 2 ) Bio chim . Biophys. Acta 268, 138-157, Ellman, G.L., Courtney , K .D., Anders, Jr. V. & Featherstone, R. M. (19 61 ) Biochem. Pharmacol . 7, 88-95, Hait es , N., Don, M. & Maste rs, C.J. (1 972 ) Bio chem . Physiol. 4 2B, 303 -33 2. Hall, Z. (19 73 ) J. Neurobiol. 4, 343-361. Comp. 105 Ha ll, Z.W. & Kelly , R. B. (1 971 ) Hild ebrand , J. G., To1.<mse l, J.G. Nature New Biol . 2J2 , 62 . & Kravit z , E.A. (1974) J. Neurochem . 2J , 951 - 96J . (1 967 ) Hill e , B. Ho, I .K. & J. Gen . Phys iol. 50, 1287 -130 2 . Ellman, G,L, (1 969 ) J. Neurochem . 16 , 1505- 1513 , J ackson , R.L. & Apri son , M.H. (1966) J, Neuro chem . 13, 1367 . Johnson , C.D. & Russe ll, R.L. (1 975 ) Anal. Biochem . 64 , 229 -23 8 . Lawler, M. C. ( 1964 ) Biochim . Biophys . Acta 81 , 28 0- 2 8 8, Lim, R., Davis, G.A. & Agranoff, B.W. (19 71 ) Bra in Research 25, 121-131, Mar tin , R.G. & Ames, B, N, (1 961) J. Biol . Ch em . 236 , Ri eger , F. (19 69 ) Eur. J. Bio chem . 11, 1372 -1 379 , Massoulie, J. & L1,L~1-4L~5, Massoulie , J., Rieger , F. & Bon, S . (1 971 ) & Tsuji, S. (1970 ) Eur . J. Biochem. 21, 542-551 , Massouli e, J., Rieger, F. Eur . J, Bioch em . 14 , 4J0-4J9, Mill ar , D.B., Garfius, M. A., Palmer , D.A. (1 973 ) Eur. J o Biochem . 37 , 425 -4JJ . & Mi ll a r, G. 106 Nigon, V, (1949) Ann. Sci . nat. Zool. 11, 1-132, Silman, M.I. & Karlin, A. (1967 ) Proc. natn. Ac ad. Sci. U.S.A. 58, 1664-1668, Smallman , B.N. & Wol fe , L.S. (1 956) J. Cell . Comp . Physio l , 4B, 197-213, Sulston, J, & Brenner, S. (1 974) Genetics 77, 95 -104. Wenthold, R.J., Mahler, M.R. & Moore , W.J. (1974) J. Neurochem. 22, 945 - 949, Wilson, B.W., Mettler, M.A. & Asmundson, R.V. (1 969 ) J, Exp. Zool. 172, 49-5 8. Yamamura, I'll . I., Reichard, D.W., Gardner, T.L . , Morrisett, J.D. & Broomfield , C.A. (1973) 302, 305-315. Biochim. Biophys . Acta 1 07 IV Bioch emist ry and Behavior of a Mutant Nematode Missing Forms of Cholinesterase 108 Abstract A screening procedure based upon selective inactivation by sodium deoxycholate (DOC) has been used to screen eighty nine mutants of Caenorhabditis elegans for their relative contents of different forms of cholinesterase. One uncoordinat ed mutant (BC46) was found which lacked DOC stable activity and this indication that the mutant was missing cholinesterase forms III and IV was confirmed by velocity sedimentation. Biochemical characterization (Kms 1 substrate and inhibitor specificity and thermal inactivation) of forms I and II from BC46 suggests that they are unaltered. The mutant's rate of growth and fecundity are normal; the most apparent defect was the inability to coordinate the propagation of waves of contraction in the body region of the animal. of the head are les s affected. The motions Sensory :responses to mechanical, chemical and osmotic stimuli have been demonstrated. After several backcro sse s, the b ehavioral and biochemical defects have not separated and both map to the X-chromosome. 109 Introduction When mul tiple forms of cholinesterase are found in an organism, two related questions naturally arise: 1) do the forms share a common active subunit, and 2) do they have separate functions? A case for which clear answers exist is the case of human "true " and "pseudo " cholinesterases. Here the large differences between the two enzymes in such qualitative properties as Km, substrate specificity, substrate inhibition and inhibitor sensitivity suggest different active subunits and this suggestion is strongly borne out by th e existence of human variants with altered pseudocholinesterase (review , La Du & Dewa ld, 1973). Originally detected by their extreme response to preoperative doses of the mu scle relaxant succinylcholine , some of these variants have markedly reduced serum levels of pseudocholinesterase (Katt amis, Davies & Lehm~nn , 1967 ; Rubenstein, et al., 1970); in other cases ps eudocho linesterase activity in serum (Kalow & Staron, 1957) and in other tissues (Liddell, Newman and Brown, 1962) has altered substrate and inhibitor specificities. Nonetheless, their true acetylcholinester- ase is unaltered and, in the absence of succinylcholine, they are asymptomatic, indicating both that th e true and pseudocholinesterases have different subunits and that the pseudocholinesterase, in contrast to the true cholinesterase , is quite inconsequential. In other cases, the evidence is les s complete. In 110 rat diaphragm, for example, where three forms of cholinesterase are found (4 S, 10 Sand 16 S), the largest form is selectively localized to the endplate region of the muscle fibers, suggesting that it is the synaptical1y relevant form (Hall, 1973) 0 However , it is uncl e ar whe ther this form has the same active subunit as the others, sinc e there is no genetic variant affecting these enzymes; all that can be said at the moment is that it resembles the other forms in its Km ( 2 .. 0 x 10 1973)0 ing -4 M, Hall, The absence of firm subunit evidence is frustrat- in this case, since a clear demonstration of a common active subunit would make the important implication that incorporation of this subunit into the synaptically relevant form e ntails a specific structural change. A genetic variant could also facilitate attempts to determine whether the synaptically relevant enzyme is synthesized in the motoneuron or in the muscleo In Q. e leg ans, as shown in the previous part, the four separable forms of cholinesterase can be divided into two classes on the b as is of differenc es in qualitative properties (Km, substrate specificity, inhibitor specificity, thermal sensitivity) o However, t h e differences b etween the classes are not as stri}~ing as for the vertebrate tru e and pseudo cholinesterases, and th ey might concievably r esult from changes arising when a common 111 active subunit associated with .other components. In addition there has heretofore b een no method for det e rmining whether these forms h ave separate functions. Sinc e a gene tic variant would help greatly to resolve th ese questions in Q, elegans, this section describes a screening method for such variants, together with a de s cription of the first vari a nt it has detected, an uncoordinated mutant lacking cholinesterase forms III and IV. Materials & Methods Methods for the isolation and study of r ecessive mutants of caenorhabditis e1egans have been described (Brenner, 1974). The behavio ra l and morphological mutants analyzed here were isolat e d as p ar t of a large mut ant hunt (Coleman, et al,, 1972) using a selection procedure involving failure to execute rapid chemotaxis to bacteria. Muto.nts u s ed fo r mappi ng are from Dr. S o Br enner's l aboratory through Dro R ,So Edgar and Dr. D. Hirsh. Other materials were as d e scribed in p a rt III. Homogenization, extraction, velocity sedimentation, assays for choli nes terase and kinetic characterization experiments were performed as d es cribed in part III, Methods. For screening of cholines terase activity levels in mutants, 1-10 plates of animals were eluted from their growth plates, washed with distilled wat e r and homogeniz e d 112 by freeze-powdering in liquid nitrogen (0.5-1.0 ml final volume)o One hundred µl aliquots of the homogenate were diluted with an equal volume of 0.1 M borate, pH 8.8 and separate aliquots received 20 µl of 10% Tween 80 or 10 µl of 10% DOC. 0 After incubation at 4 C for 12-36 hours, the extracts were assayed for cholinesterase activity. Parameters of growth and reproduction of mutants were determined with the "3-egg" plate technique described by Byerly, Scherer & Russell (1976). In this procedure, three juvenile nematodes, from synchronously laid eggs, are transferred to a fresh plate and the number and size distribution of the F 1 and a portion of the F 2 progeny are determined using an electronic nematode count er (Byer l y, Cassada & Russe~l, 1975) just as the F 2 genera- tion begins to hatch ( ~ 1 week) o Chemotaxis to NaCl gradients was in 9 cm petri plates containing 4 ml of tracking me dia, the center of . which received 1 µ1 of 5 M NaCl 4-5 hours before the addition of a single nematode (Dusenbery, Sheridan & Russell, 1975; Ward, 1973). The repel~nt effect of high osmotic strength was tested by placing several animals inside an annulus formed from 30 µl of 5 H NaCl (u npublished procedure of Dr o Jo Culotti) . Tracks were photographed by placing the petri plate upside down on photographic paper and exposing it to the light from an enlargerc 113 Th e ms.pping of an uncoordinated ( unc) mutant (BC46 se e Result s ) located on the X- chromosome was by segregation of the double mutant cis - heterozygote with the markers lon - 2 and dpy-7. In general the production of double mutants for X-linked genes i s prevented by the inability of th e hemizygous mutant males to mate with h ermaphrodit es. However 1 males of the X-l inked lon-2 allele E678 mate well, as do the (non -dumpy ) males of the temperature sensitive X-linked dpy-7 allele E1J24 produced by growth at the permissive temperature (20°c). The double mutant was obtained by mating homozygous unc hermaphrodites with hemizyg ous l on or dpy males. From this cross, c lones of unc +/+lon (o r +dpy) hermaphrodites were estab li shed and their long (or dumpy ) progeny were further cloned . At twice the recombination frequency these c lon es were heterozygous a t the uncoordinated locus and segregated the homo zy g ous do,.-cble mutant which was cloned to establish a pure double mutant culture. The double mutant was then mated with wild type males to produce the double mutant cis - heterozygote. Results Screening procedure In designing a screening procedure to detect genetic variants of C. elegans cholin es t eras e , an important consideration was to have a method which would monitor the two 114 major cholinesterase classes (forms I and II versus forms III and IV) selectively. Among the class differences reported in part III that might be exploited for this purpose, differences in thermal sensitivity and detergent sensitivity seemed well suited, since forms III and IV could be separately monitored (after inactivation of forms I and II) without prior fractionation. Detergent sensiti- vity was chosen and in order to see whether the class differences detected in the separated forms would hold in the whole, mixed homogenate, a homogenate was extracted with 0 , 5% sodium deoxycholate (DOC) and assayed at intervals thereafter. As shown in Figure lA, there was a rapid initial loss of activity until about 12 hours, when a stable level was reached at about 30-40% of the initial activity. Velocity sedimentation of homogenate extracted for JO min or for 8 hours in 0,5% DOC (Fi gure lB) indicated that the rapid lo ss had been, as expected, primarily of the slower sedimenting forms I and II. A standard screening procedure based on these observations was then devised. Using paired aliquots of homo- genate incubated in 1% Tween (suppresses forms III and IV) or 0,5% DOC (inactivates forms I and II), a total of 89 mutants were screened for possible defects. These included 36 mutants resistant to cholinesterase inhibitors, and behavioral or morphological mutants isolated in a large previous mutant hunt (Coleman, et al., 1972). Figure 2 53 115 Fi g. 1. Inactivation of cholinesterase activity in homo genate b y incubation with A. pH 8.8; 0,5% DOC. Nematode homogenate was diluted 0.5% DOC and stirred at 4°c. to 0 .05 M borate At specific times, an aliquot was assayed with 5.0 x 10- 5 M ACh.Cholinesterase activity was plott ed as a percent of that measured 5 min aft e r dilution. B. Homo genate v-1as diluted as in A and after JO min (top gradient) or after 8 hours (lower gradient) at 4°c, 100 µl aliquots were loaded unto 4.5 ml 5 - 20% sucrose gradients (0.05 M borate; pH 8.8; 0.1% Tween 80) with 0.5 ml Renog rafin cush ions . After centrifugation for 4 hours at 65,000 rpm, fractions were collected and 10 µl of each was assayed for 1 hour with 5 x 10 - 5 M ACh. Th e bl.ahk was 0.4 x 103 cpm; complete conversion 21.7 x 103 cpm . 11 6 A 60 40 20 1----------.-----.--~\ 10 0 20 30 Ti me ( hours) ( 80 2 (\J I 0 ~ X E Q_ u ~~~ • o-~ ~~ - . - - _ : . . . _ - - . - - - - . - - - ~~ ~-r-- o 5 10 15 Fraction Number Fi g ure 1 20 25 117 Fig. 2 . DOC inac tivation of mutant extracts. Ten plates of animals for each strain to be assayed were eluted , washed with distilled water and freeze powdered in 1 ml . One hundred µl aliquots of homogenate were diluted with an equal volume of 0.1 M borate, pH 8.8. Separate aliquot s received 20 µl of 10% Tween 80 or 10 µl of 10% DOC. Ten pl aliquots of the extracts were assayed for 5 min with 5,0 x 10- 5 M ACh at about 12 hour vals. inter- Activity ( u/ml ). was plotted versus time of assay. The DOC extract of one mutant (BC46) was devoid of activity after about 24 hours. ::JI E X (\Jo . 2 0 4~ ¥'~ 2~ 2-1'\. /"\ ··· 2 0 5~ - - !"!>· 5 - D053 I 2k 2 BC46 Fi gure 2 5 I I 2 0 2.5 I ~I El38 Time (days) 2 0 - . m,~ 2-~ ~- I"- ~ _ I -~ , ,,;,, ~ ~ 2' N2 ~~~ ' @ I 2 I 0---~ RRI02 ~ CD I-' I-' 119 Fig. J, Stable DOC/Tween ratio in mutant extracts. Homogenates were extracted with 0.05 M borate, pH 8.8 and Tween (1%) or DOC hours. (0.5%) and assayed after 24-27 Activity measured in the DOC extract is expressed as a fraction of the activity measured in the Tween extract, The arrows represent N2 controls, 1 20 Cf) C -- t 0 )._ -- 20 ! ~~t Cf) Y- 0 L (l) ..0 E 10- :::J z '°--r. u ,:;a 0 1.0 0.8 c O.o DOC/Tvv Fi g ure J 0.4 ratio 0.2 0 121 shows the screening results for one series of mutants, including the one mutant (BC46) which was found to be significantly altered. For wild type and for most of the mutants the activity measured in Tween rose slightly and then gradually fell, whereas activity in the DOC extract showed the rapid loss and plateau behavior of Figure lA. For BC46, however, there was virtually no DOC-resistant activity, and as the histogram of Figure 3 shows, BC46 was unique in this respect. BC46 cholinesterase The implication of the initial screening result was that BC46 might lack cholinesterase forms III and IV or that these forms if present were as sensitive to DOC as were forms I and II. Direct ve locity sedimentation of a BC46 homogenate sh owed no sign of forms III or IV (Figure 4) indicating th at if forms III and IV were present, they were either abnormally sensitive to homogenization or abnormally tightl y bound to pelletable debris. Short-term extraction with DOC followed by rapid sedimentation into gradients containing Tween 80, a procedure designed to maximize the amount of solubilized cholinesterase (as explained in part III) also failed to reveal any BC46 activity sedimenting as forms III or IV (Figure 5). Larger amounts of BC46 were grown in liquid cultures and fractionated by repetitive extraction in 0.05 M borate, pH 8.8 (Figure 6A) releasing about 50% of the total 122 Fig. 4. Velocity sedimentation of N2 and BC46; extracted with 0,05 M borate, pH 8.8. Fre s h homo g enates of (top gradient) N2 and (lower gradient) BC46, were diluted three fold with 0.1 M borate, pH 8.8. After stirring 24 hours at 4° C, 1 ml aliquots of a 1500 g, 5 min supernatant was loaded unto 38 ml 5-20% sucrose gradients (0.05 M borate). The gradients were centrifuged at 27,000 rpm for 29 hours. Fractions were collected and 10 µl of each was assayed for 45 min with 5 x 10- 5 M ACh. The blank was 0.4 x 10 3 cpm; complete conversion 16 . 7 x 10 3 cpm. 123 6 4 2r0 I 0 X 0 ., E Q. u 6 4 2 O; 0 \ ' ~ 5 10 15 Fraction NurT1ber Fi g ure ~- Fig. 5. Velo c ity sedimentation of N2 and BC46; extracted with 0,05 M borate, pH 8,8, 0.2% DOC. One ml of (top gradient ) N2 and (lower gradient ) BC46 homogenat e was diluted with 0,8 ml of 0.1 M borate, pH 8 . 8. At 4°c, DOC was added to 0.2% and , after 2 min, a 1500 g, 1 min s up ernatant was prepared and 100 µl aliquots were loaded unto 5 ml 5-20% sucrose gradients in 0,05 M borate, pH 8 . 8 , 0.1% Tween 80 . Gradients were centrifuged for L~. 5 hours at 65,000 rpm; fractions were collected and 10 µl of each was assayed for 10 min with 5 x 10-5 M ACh. Th e blank was 1. 1 x 10 3 cpm ; complete conversion 32.0 x 10 3 cpm. 125 E Cl. u 8 4 o--1--··- - , ----..------,-----.------.--- 0 5 15 10 Fraction Number Figure 5 20 25 126 activity to high speed supernatants. When these super- natants were examined by velocity sedimentation (Figure 6B), the activity sedimented primarily as a 5 Speak, but the later washes contained substantial amounts of activity at 7 Sas well. Treatment of the pellet with 0.2% DOC released additional activity most of which sedimented at 7 S (bottom gradient - Figure 6B). and the The 5 S (BC46 - form I) 7 S (BC46 - form II) activities were separately pooled and characterized with experiments parallel to those performed on the separated forms of the wild type enzyme (part III - Figures 8 and 9, Tables J and 4). Forms I and III from wild type, were included in the experiments for comparison. Km Forms I and II from BC46 have Km s of 9 .1 x 10- 5 M 1 and 8 . J x 10-5 M respectively, both quite close to the Km of wild type form I (Figure 7). As expected, the wild type form III has a significantly lower Km. In this determination, several higher substrate concentrations were used than were used for the Km determinations in part III and substrate inhibition was demonstrated for all forms. Substrate specifici~ Th e relative specificities of BC46 forms I and II for acylthiocholine substrates are quite comparable with those of wild type form I and different from those of 127 Fig. 6. Repe a t ed extraction of BC46. About 8 gm of fresh nematodes (BC46) were homogenized and thawe d in t o 5 volumes of distilled water and a 40,000 rpm, JO min supernatant was prepared (S40K-l). The pellet was rehomo genized (by freeze-powder) in 10 ml 0.05 Nl borate , pH 8.8 and another supernatant was pre- pared (S40K-2 ). This pellet was resuspended, rehomogeni- zed and recentrifug ed f i ve more times over several days (yielding S40K-J thru S40K-7), In panel A, the total units of cholinesterase activity extracted was p~otted a g ainst the time of supernatant preparation. The d a shed lines above S40K-5 and S40K-7 represent assays of activity remaining in the pellet. Panel B shows sucrose gradients of aliquots of all the S40Ks and (bottom gradient) the f inal pell et a f t e r extract:e.. on with O. 2% DOC for 2 min). The activity measured in each fraction was convert e d units/ml and multiplied by the total 1;0 volume of the S40K before plotting against fractiona l position (corrected to a sedimentation run of 8 hours at 6 5 , OOO rpm ) . - 3~ w ..c u I C ::J (/) -- 2 0 0 J ,_ 16 Time (days) r 45 I I I I I ! I I I I 2 Figure 6 :::i C (/) - X r7 - I 0 N I I I I I I I 0.6 1 r l-. ~ (DOC) pe II et 7 e,&:a 0.4 0.2 0 1~1 ~. ~ ~ o ..-..,.. Fractional Position ~ /\.~ 2 ~ ol, 1.0 0.8 4 8 12-L__ 10 201 24 28 32 36 40 CD I--' N 129 Fig. 7. Km for acetylcholine. Forms I and II, separated from extracts of BC46 and the wild type cholinesterase forms I and III were assayed with 10 concentrations of ACh ranging from 2 x 10- 6 M to 1.5 x 10-·J M. The Km was calculated as a linear regression of the measurement with ACh less than 4 x 10- 4 M. The standard deviations of the Km determina- tions were BC46-form I, -:::0,5 x 10-5 lVI; BC46-forrn II, -:::o.6 X 10- 5 M; N2-forrn I, -:::0.5 X 10-5 WI; N2-forrn III, -:::o.l X 10-5 M. 1 30 80 0 BC46-I Km= 9.1 x I o-5 M '- '' BC46-Il Km=8.3x10- 5 M 0 - , - - - . - - - . . - - - . - - - ~ - r - -· - - - - . - - - , - - - - - ~ x80 > N2-I Km= 7.2 x ,o- 5 M N2-1Il Km= l.9x 10- 5 M 40 + 0 -.;• - - - - - . . - - - - . - - - - . - - - r - - - - !- - , - - . l ---.-Sb-1 - 0 0.4 0.8 0 0.4 0.8 V/S x 10-2 F irrure 7 0 lJl wild type form III as shown in Table 1. Inhibitor specificity Th e same pattern was seen when the mutant enzymes ... were examined .Lor inhibitor specificity (Tab l e 2 ) . BC46 forms I and II and the wild type form I were inhibited to about the same extent by the concentrations of inhibitors chosen , whereas the wild type form III was general ly more strongly inhibited . Thermal inactivation At 45.1°c, forms I from BC46 and from wild type were inactivated in parallel (Fi gure 8). BC46 form II was more stable , its inactivation paralleling the wild type form II inactivation (part III, Fi gure lOA). The biochemical characteristics of the separated forms I and II from BC46 are nearly identical to the same form in the wild type and significantl y different from wild types for:r,s III and IV. (Whether the small differen- ces observed between the two forms I are a reliable indic ation of additi onal heterogeneity remains to b e seen). BC46 development To obtain quantitative measures of the size, the rate of development and the fecundity of BC46, the progeny descendant from three synchronously laid hermaphrodites were counted and sized with an electronic nematode counter about one week (two generations ) after the three animals were placed on a fresh growth plate. BC46 was, 132 Table 1 . Acyl-thiocholine Substrate Specificity of BC46 Forms % Activity of ChE form N2 BC46 Substrate I II I III acetylthiocholine 1 00 100 100 100 acetyl- ~ methyl thiocholine 39 4 L~ Ll.L!- 63 propionylthiocholine ~-5 48 60 99 propionyl -Q> methylthiocholine 26 28 35 99 b u tyrylthiocho l ine 19 23 24 64 Al l substrates were at 1 mM in the assay. Activity (~oD 412 /min) is recorded as percent of the activity measured with acetylth i ocholine. lJJ Table 2. Inhibitor Sp e cificity of· BC46 Forms % Inhibition of Inhibitor I BC~-6 II n e ostigmi17e ( 2 x 10- M) 16 18 Jl 8J temilc (2 x 10-5 M) 2J 2J JJ 71 DFP (lo- 6 M) 21 2J JO 78 parao:i;;:on (10- 6 M) 14 16 tensilon (2 x 10- 6 M) 26 29 BW284C51 6 (2 x 10- M) 51 68 TEA (5 X 10- 4 M) 65 65 61 62 hemicolin~um-J (5 X 10- M) 2J 26 2J 77 N2 I III 24 J6 99 Inhibition of activity in 15 min assay with 5 x 10- 6 M ACh (without pre incubation of enzyme and inhibitor). Percent inhibition was calculated by comparison with parallel assays without inhibitor. Fi g . 8 . Thermal inactivation. Aliquots (10 or 20 µl) were diluted into 1.5 ml 0.025 M (K)POLj_= , pH 7,0; 1 mg/ml BSA which had b een prewarmed to 45.1°c. One hundred µl aliquots were removed at specific times thereafter and transferred to assay v i als on i ce . After al l time points were taken, activity was assaye d and rec ord e d as a percent o f c ontrol (an equivalent aliquot diluted into buffer at room temp erature). 100--:;:r,.---------------0 80 60 BC46-II 40 ~ +- 20 ·>· -0 0 0 4 5. 1°C 10 ~ orC 0 0 ~ 0 5 A 2 0 I I 10 20 I I 30 40 Time (min) F igure 8 I 50 I 60 136 on the average , 106% the size of wild type; they grew at 95% the rate and produced 97% of the expected number of progeny. BC46 behavior The behavioral defect in BC46 prevents the continuous wave propagation necessary for sustained movement. Al- though waves of contraction are often seen in the head propagating backwards, the se waves rarely pass the vulva and usually die out about one-fifth of the way down the body. The head is much more normal and active than the body. The low lli~plitude searching movements involved in feeding seem unaffected and the pharnyx pumps normally. Especially when excited by mechanical stimuli, the head undergoes a dorsal-ventral swinging with a frequency very similar to th e frequency of wave propagation in the wild typ e ( Table J). When tapped on the snout with a toothpick, wild type nematodes respond immediately, moving backward by passing forward travelling waves . During backward movement the low amplitude searching movements of the head are stopped. This head-tap-response (htr) lasts about 5 seconds (Tab l e J) after which the animal reverses and begins to move forward again. Tapping BC46 on the head produces an overall muscular contraction , a short backward movement and a curling of the tail which suggests the beginning of 1J7 a forward trave lling wave . Thi s "pose" lasts about 5 seconds durin g which the head searching movements cease ( Tab l e J) and is fol lowed by relaxation. If during a head-tap-response , the tail is tapped, the wild type interrupts its backward movement and immediately moves rapidl y forward. Similarly in BC46, if the tail is tapped during the "pose" response to head-tap, head swinging I movements resume immediately. BC46, when placed in radial gradients of chemical attractants (e. g . NaCl) showed strong chemotactic resp::mses (Figure 9A) , although moving slowly. The repulsive response caused by high osmotic strength was seen as well (Figure 9B). These obs ervations suggest that the nervous system of BC46 is qui te intact, being ab le to sense a varie ty of stimuli so as t o produce essentially normal (if slowly executed) behavi oral responses. Furthermore, in the case of the mechanica l responses, the duration of the behavioral response to he ad-tap, and it s reversal by a second stimuli (tail- tap) , were unalt e red even though the mutant's locomotory de f ect prevented any effective response to the stimuli. BC46 gene tics The initi al isolate of BC46, in addition to being uncoordinated, failed to grow at 25°c. When backcrossed 1J8 Table J. Behavior and Mechanical Responses of BC46 . BC46 N2 Respons e + (n:ccl0 ) (n=l 0 ) 0,59-0,07 (n=l 0) frequency of waves ( sec - 1 ) duration of 4 .4 ~1.6 he ad -tap-response (htr) ( sec ) l frequency of stopping htr wi th tail-tap 0.85 (n=J 0) (n=20) 4,7 +-;-2,1 5.8 "2:2.0 (n=2 7) (n=l0) (n=l0) 139 Fi g . 9. Chemot ax is and osmotaxis. Tracks made by N2 or BCL~6 hermaphrodites were photog raphed after A. 11 hours on a radial gradient of NaCl (1 µl of 5 M NaCl allowed to diffuse for 4 hours before add ition of single nematode) or Bo 2 hours inside an annulus of high ionic strength (JO µl of 5 IVI NaCl, nematodes placed in the center immediately). 4 Osrnotax i s s tudie s ·were developed and performed here by Dr. J. Culotti . N2 BC46 141 to N2 males and reisolat e d (as F 2 uncoordinated herma- phrodites), only three of twelve clones r etained the growth charact e ristic . ts In addition, all of the F of the backcro ss were uncoordinated. males 1 From this, it would appear that the unc mutation i s sex -linked, whereas the ts growth results from a separate, autosomal mutation. Twenty uncoordinated clones were tested for the presence of cholinesterase forms III and IV by the DOC inactivation proc edure . None were found with DOC stable activity. Th e absence of forms III and IV was confirmed in five additional backcross e s by v e loc i ty sedimentation. Linkage to the X-chrornosome was confirmed by segregation of the cis heterozygote of BC46 and t wo X-linked markers - lon2 and dpy 7 (Table 4). BC46 was clearly linked , but no t very tightly, to both loci. More precise mapping v..rill require more tightly linke d markers, but th ese pre limin2 Ty result s sugg est a map position on the right arm o_,_ .C> L,ne X-chromosome (Figure 10). -'- ' This region of the publishe d genetic map (Brenner, 1974) contains three un c lo ci . Th e un c? a ll ele ES, the u nc9 allele El Ol and the uncJ a ll e l e El51 have all be en examined for DOC resistant cholinesterase activity and all three were found to be normal in this re gard (Table 5), Di sc us sion Mu t ants altered in the distribution of the separab le 142 Tab l e 4. Segregation of BC46 h ete ro zygotes BC46/+ BCL~6-E678/++ BC46-E1 J24/++ + 110 unc ++ unc l on unc + + lon 509 69 1 J6 1 23 ++ 612 unc dI~Y 1 61 unc + + d:gy 91 96 Iw Fig. 10. Genetic mapping of BC46. The genetic map of the X chromosome is reproduced from Brenner, Q 974l BCL~6 and The recombination frequency between 1) the E678 allele of lon2 and 2) the E1J24 allele of dpy7 suggest that BC46 is located on the right arm of the chromosome. 60 v-? z O;6;- ov cPS . <~ Ov Q,. Ov ov -? ,. cP;-? -? ,. <--t,: ov-? . 0 I- SJo ~ <( z m o:Y< V,:, \J l % ..9--;:: "-- 2V, cP--t,:: ~ (i) $..~ :::> w •rl 0::: ~ 0 LO % .· 02 ~o~-?J C'--;:: % .....,_, l'o <---?,_, rl 0 u I 0 X b.D ~ 145 Table 5. DOC/'1 w Ratio f or X-linked I'l'Iarkers. 1 Activity. in (u/ml x 103) Lo cus Allele Tween 80 DOC Ratio DOC/T w N2 6.2 1.3 0.22 BC46b 2A 7.5 0.2 0,03 unc7 E5 19,8 4.8 0.24 unc9 ElOl 16.6 4,5 0.27 unc3 El51 2.3 o.6 0.26 uncl E94 14.2 5,1 0.36 tcfr2 30 .5 9.7 0,31 Cholinesterase activity measured after 2LY hrs incubation of homo genate in 0.5% DOC or 1% Tween 80 . BC 46b A is a second backcross of BC46 . 2 146 forms o f cholinesterase activity can provide important constraint s on the many possible interrelationship s of the differe nt forms. We have screened mutants of Caenor- habditi s elegans, for s uch defects and have found an uncoordinated mutant ( BCL~6), mapping to the right arm of the X- chromosome, which lacks cholines terase forms III and IV. The identification of this defect was facilitated by the use of a selective inactivation procedure which allowed the separate determination of cholinesterase forms III and IV without requiring fractionation. We have not b een able to separate the behavioral phenotype and the biochemical defect by backcros sing to wild type. A direct measure of their recombination frequency (<2%) suggests that they are either very tightly linked or effects of a singl e lesion. Revertants have not yet been obtained. Biochemical characterization (Kms, substrate and inhibitor specificities, thermal inactivation) of the forms retained in BCL~6 ( forms I and II) showed that they were unalt e red compared to comparable wild type forms I and II. These results support the suggestion, advanced initially after comparison of the kinetic properties of all four forms isolated from the wild type, that the active-sitecontaining subunit of forms I and II is a gene product different frorr.. that of form s III and IV. BC46 could 147 repre s ent a lesion in the structural ge ne for forms III and IV cholinesterase. For the moment, this must remain a sug gestion as it i s possible that the kinetic differences are secondary effects on a cornnwn active-site which occur as a result of converting forms I and II into the large r forms. In this interpretation, BCL~6 could repre- sent a lesion which prevents the conversion of forms I or II to the forms III and IV. The behavioral defect of BC46 is apparently limited to the inability to properly coordinate continuous passage of waves of muscular contraction in the body region. Sensory responses to mechani c al, chemical and osmotic stimuli have all been demonstrated and the capacity of the nervous system to integrate spatially distinct mechanical stimuli was shown by measuring the duration of the response to head-tap and its suppre ssion by tail-tap. Since the anim a l does occasionally pass an isolated, well coordinat e d wave of contraction down it s entire l e ngth, the muscle does n ot appear to b e defective. This suggests a functional localization of cholinesterase forms III and IV to the body r eg ion. If other areas of the nervous system contain cholinerg ic synapses, it seems likely that they utilize choline sterase form s I or II. The apparent normal coordination of muscular contraction by BC46 in the head suggests that the control of 148 contraction is different here than in the body. One possibility is that the head muscles contain an auto rhythmic pacemaker, ind ependent of direct neuronal control. When Q. elegans is treated with externally effective cholinesterase inhibitors (e.g. t emik, DFP, paraoxon), the animal becomes extremely hypercontracted. This phenotype was presumed to result from cholinesterase inhibition causing an excessive buildup of acetylcholine, leading to continuous contraction of the somatic musculature. Excessive hypercontraction did not result from the mutational elimination of cholinesterase forms III and IV in BC46. This may be because the remaining cholinesterase is sufficiently widespread to prevent too large of an increase of ACh. Alternatively, the hypercontraction may result from the effects of the inhibitors on other enzymes (e.g. DFP inhibition of ATPase - Hokin & Yoda, 1964). With BC46 in hand, it may be possible to design selective schemes for isolating mutants affecting the remaining cholinesterases, which would allow a precise determination of the behavioral effect of el iminating cholinesterase activity without the unintended side effects from the action of inhibitors on other components. References Brenner, S. ( 1974) Genet ics 77, 71-94. Byerly, L., Cassada , R.C. & Rus se ll, R.L. (1 975 ) Rev. Sci . Instrum . 46 , 517-522. Byerly , L., Sch e rer , S. & Russel l, R. (1976) Dev . Biol. i n press . _Coleman, R. , Du senb ery , D. , Gross, C., Ha ll, D. , Hedgec ock, E., Hollen, L., Johnson, D.C., Kubo ta, E., Russell, R.L. & Thompson, M.A. (197 2 ) Calif. Ins t. of Technology, Biology Annual Report, 2J - 24 . Du senbery , D.B., Sheridan, R.E . & Russell, R.L. (1975 ) Genetics 80 , 297 - 309 , Hall, Z. (197 3 ) J. Neurobiol. 4, J4J-J61. Ho k in, L.E. & Yoda , A. (1964 ) Proc. N::ttl. Acad. Sci. U.S. A. 52 , 454-461. Kalow, W. & Staron , N. (1 957 ) Can. J. Biochem. Physiol. 35 , 1305-1320 . Kattami s , C., Dav ies, D. & Lehmann, H. (19 67) Acta Genet . 1 7 , 229 - 30J . La Du , B. N. & Dewa ld, B. (1 973) Adv. Enz. Reg. 9,317-337. Liddell, J., Newman, G.E. & Br6wn;o.(1962 ) Nature 198, 1090- 1091. Rubenste i n, H .M., Dietz, A.A., Ho dges, L .K. , Lubrano, T. & Cz eb otar, V. (1970) Ward, S . Proc. J . Clin. Inves t. 49, 479-~·86. r-j?!J-• Acad . Sci. USA 70, 817-821.