Studies on the Respiratory Enzymes of Sea Urchin Eggs Citation Ycas, Martynas Freelandas (1950) Studies on the Respiratory Enzymes of Sea Urchin Eggs. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/ajnr-pa79. https://resolver.caltech.edu/CaltechTHESIS:09232025-213823820 Abstract 1. The literature pertaining to the respiratory metabolism of sea urchin eggs is reviewed. 2. Homogenates prepared from the unfertilized eggs of Lythechinus pictus and Strongylocentrotus purpuratus were found to possess enzymatic activity corresponding to the enzymes phosphoglucomutase, oxo isomerase, aldolase, triose phosphate dehydrogenase, lactic dehydrogenase and enolase. Lactic acid accumulates anaerobically in intact eggs. Respiration of homogenates in inhibited by fluoride. 3. Citric, alpha ketoglutaric, succinic and malic acids are metabolized. Respiration is inhibited by malonate and increased by cytochrome o. 4, Spectroscopic evidence is presented for the existence of cytochromes a and b. A method of preparing an insoluble fraction containing these cytochromes is described. 5. It was found that the increased respiration obtainable with dinitrophenol increases faster than the normal respiration during the first eight hours of development. 6. It is concluded that the respiratory pathways of the sea urchin egg are similar to those known to exist in adult organisms. 7. The chemical basis for the similarity or the glycolytic mechanism in most forms of life is discussed. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Embryology and Invertebrate Zoology) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biology Thesis Availability: Public (worldwide access) Research Advisor(s): Tyler, Albert Thesis Committee: Unknown, Unknown Defense Date: 1950 Record Number: CaltechTHESIS:09232025-213823820 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:09232025-213823820 DOI: 10.7907/ajnr-pa79 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 17698 Collection: CaltechTHESIS Deposited By: Ben Maggio Deposited On: 04 Oct 2025 13:17 Last Modified: 04 Oct 2025 13:22 Thesis Files PDF - Final Version See Usage Policy. 40MB Repository Staff Only: item control page

STtJDii:S O!i TJDt RESPIRATORY ENZYMES OF SEA UHCHIN EGGS Thesis by Mart;ynas F. Yeas In Pa:r·tial F'ulfillment of the H.equ.irennents For the Deg:rae ot Doctor of Philosophy California Institute of Techx1ology PasadQna, Ca11.t"ornia 195'0 lt.eknowledgments I wish to thank P:roi'esso:r Albert Tyler for sncou.r-agemcnt, sugges tions and criticism during the course of my work. I should also like to thank t:hG members of the staff of the Biology Division for their interest in my problems and for the assistance they have given me. Table of Contents Subject Page ln tt._~1-Jm ;m4 ~gr,,v:e~l.-2.L U t_sara tYJ:! 1 ~ ~.!?\..te..m9Jl t 0L~l1~ ~tQ.~l~.m 20 22 ~2.~ma:t: 'ew'_._t Glycolysis 26 Phosphoglucomutase 26 Oxoisomeraae 29 Aldolase 32 Triosephosphate dehydrogenase 35 Enolase 39 Accumulation of lactate 41 Laet1c dehydrogenase 43 Acid of fertilization 43 Acoumule.tion of phosphoglycerate 46 Metabolism of trioarboX'Jlic ac.i d cycle acids 49 Malonate inhibition Cytochrome system 49 61 At tempts to extract cytochrome e 62 Cytochrome oxidase 63 Cytochromes a and b 65 Concentration of cytoch:romes a and b 6? Some properties of cytoohroroos a and b , Table of contents (cont ) Bf.feet of arsenate 74 f;';ff et,t of dini trophenol 77 Discussion of effect of dinitrophenol 81 ~~~WL~J.JaU 83 Hespira·tory pathweys in eggs and adult tissues 83 Chemical. basis :for the uniformity of the glycolytic mechanism in various organ.isms 88 Embryol<.}[;ical significance of :respiration 93 A:BSTRACT 1. The literature pertaining to the respiratory meta• boliem of sea m·ohin eggs is reviewed. 2. Homog&nates prepared from the unrartilized eggs or .liYth@ehin!:1§ ;1gtug and g:ttqngyl;ocep.trotu, PYlM@W W!3re .found to possess enzymatic activity corresponding to the enzymes phosphoglucomut&se• oxoisomerase, aldolase, triose• phosphatedehydrogenase, lact1o dehydrogenase and enolase. Laetie acid accumulates anaerobiealy in intact eggs. Res• piration of homogenates 1$ inhibited by fluoride• 3. Citric, alphalretoglutaric, succinic and malic acids are metabolized. Respiration is inhibited by malonate and increased by eytochrome o. 4, Spectroscopic evidence is presented for the exist• ence of oytochromes a and b. A method of preparing an insoluble fraction containing these cytochromes is described. 5. It was fou..Yld that the increased respiration obtain• able with dinitrophenol increases faster t han the normal respiration during the first eight hours of development. 6. It is concluded that the respiratory pathways of the sea urchin egg are similar to those known to exist in adult organisms. 7. The chemical basis tor the similarity or the gly• eolytic mechanism in most forms of life is discussed. lnt:rgduct121 The amphibian embryo has proved the most f1•uit:rul mat.. erial tor tha analysis of morphogenetic processes, oo the other hand more in.formation has been obtained about the very early events of development from studies of the eggs of marine invertebrates. Of these the eggs of sea. urchins, tor tech- nical reasons have b&on the material ot choice. The study ot earliest development encompasses several rather distinct tields. The maturation divisions have pro- v1ded material for the cytologist and an opportunity of linking cytological and physiological phenomena. The earl• iest determinations often occur about the tirae of rert111 ... Eation and are a field ot study for the embryologist in the classical sense. Interacting substances in eggs and sperm have an interest 1n themselves and are possible convenient models for interactions of cells and tissues in general . Finally there are studies of a more biochemical and physio• logical nature seeking to link morphogenesis and difi"erentiation with chemical phenomena. It has been stated by Dal cq (l941) that a distinction must be made between true embryological research and physiological studies which use the egg and a convenient cell for the purpose of tho 1nv0stigation. There is no doubt that there is some truth in this point of view. It would, however, perhaps be better to stat® that the1·e is no real distinction between any of the biological sciences , and that therefore, no apologies ne0d be offered for research 1t it does not fit into the strict frame ~r some well accepted discipline. In embryology particularly advances in related fields are likely to throw unexpeeted light on phenomGna long not understood.. The biochemical phenomenon most 1ntens1vel.y studied on embryological mat"rial has b-een respiration. , The reason for such ooncentrat1on on respiration has probably been that respira t1on, in the sense of gas exchange has been the . easiest phenomenon to study. Its significance is only now becomina understood in detail and much of the older, as •~11 as more modern work has tailed to throw much light on pro• blems of interest to the embryologist .• .According to J .. Needham (1931) the first investigator to study embryonic respiration was Spallanzani. (1803), whO found 1n the course ot studies, duri?lg which he discovered tissue respiration, that eggs gave oft and took in gas-ea,. A long period followed 1n which the only advance made was the substantiation of Spallanzani•s results, The mod.em work on embryonic respiration oan be said to begin with the investigations or the sea urchin egg· by Warburg. Using the Winkler teehn1que 9 Warburg (l9o8) showed that there is a very great rise in the reapiratory rate ot eggs after fertilization, This result created great interest, as it supported the theory of J. Loeb (1906) that oxidations were intimately connected to the events ot tert111zat1on. Further papers of Warburg (1909 9 1910 a, 1910 b, 1910 c) followed, in which he showed that parthenogenesis had the sue offec·ts on respiration as fertilization with sper.-t1. .Another important result obtained was the demonstration that c®rtain narcotics such as urethane could :;uppress cell divisions without significantly affecting raspirstion. The offset on respiration of various conditions, such as pH and osmotic pressure was also studied a.nd observations of the influence or iron on the respiratory rote led Warburg to studies of the catalytic effects of heavy metals and to th0 ultimate discovery of the atmungsfermentG It was at first thought that the increase in respiration on fertilization was a general phenomenon., but the investigations of Loeb and Waoteneys {1912) showed that the eggs of the starfish A,.s~~:rJ§J! .tgn_~ showed no such pheno- menon. A great deal of work has since been done on this question. The general result has been that eggs on ferti- lization may show any one of three types of respiratory behavior. The respiration may rise, remain unchanged or falle This is partly a species characteristic, but Tyler and Humason (193?) have shown that even within the same species, in the case of Dre..~bi§. _qau~t eggs of different females tri..ay show any one of the three types of respons~s. The sa~e phenomenon has been reported by Shapiro ( 1941) for A.~~s:.r5:iS: :f'o£b§§1¼• It is thus eloar that no general significance can be a ttachod to the direction of char1.ge of the respira- tory rate upon fertilization. Whitaker {1931) has attempted to f'1t the phenomena into a general scheme. 1te noticed that the respiratory rate or -4the unfertilized eggs of marine invertebrates of different species was very different, but that after fertiliF..ation the rates per unit volume became more nearly the same. The unfertilized egg is therefore in some sense abnormal as fnr as its respiratory rate is concerned and fertilization 11 regularizes" the gas exchange. scheme rather well. Most of the data tit this However, 1t should be treated with caution if only because o:f the technical difficulties often encountered 1n measuring the respiration. The question of the temperature at which comparisons should be made and the influence ot size and surface or eggs are factors to be taken into account. The question of respiratory rise has been clarified to some extent by the recent work of Borei (1948). It has often been remarked that the behavior ot unfertiliaed eggs of the sea urchin is apparently rather capricious. Borei, using the Cartesian diver technique has found that immediately after shedding the respiration of sea urchin eggs is almost equal to those fertilized, but that on standing 1n sea water the rate falls and as-ym.ptotically approaches a low figure. The rate after fertilization is the same regardless of when the eggs are fertilized, so that the actual rise on fertilization depends on the time after shedding that fortilization occurs. It had previously been sholVll by Lindahl and Holter (1941) that ooeytes have a respirattry rate slightly higher than t hat of the fertilized egg. It therefore seems clear that the variation in the amount ot rise of respiration ot eggs is a function of the t'all in resp.iratol'Y rate in tha prefertiliza tion period.- The eggs of animals L"'l which the respiratory rate falls on fertilization have not been invest• igated from this point or view. Neither has the problem of the cauoes and conditions of the fall in respiratory rate on shedding. For a discussion of the relation o! respiration to development the discussions or Needham (1942.) and Tyier (1942) should be referred to. Sinoe development frequently stops under anaerobic conditions it has some significance tor development, and therefore the changes it."l respiratory rate must have Some embryological significance. In any ease the mechanism of the change of respiratory rate is an interesting problem in itself., The problem of the control of biochemical reactions is more complex than the establishment of biochemical pathways and the sea urchin egg has appeared to many to be a suitable object for such studies. The result is that a large though rather inconclusive litaratur9 has accumuluted on the subject which can be mentioned here only rather briefly. Efforts have been mainly concentrated tm.•uu•d determining what changes occur 1n the respiratory mechanism upon fertil• ization. The most datailed analysis , has been made by J • Runnstrom and his school. (For review see ;"-tunnstrom { 1949)) The early studies established the !'net that like most tissues the respiration of fertilized sea urchin eggs is inhibited by cyanide and carbon monoxide. 1t "r'fas found that both fertilized and unfertilized eggs are 1nhib1ted but 1n d1f• terent degrees, the resp1rat1on of the rert111zed being much more ausceptible. In addition the eggs respond to the addition ot d1methylpa.raphenylened1amine by a great increase in respiration. The maximum rate reached is identical tor both fertilized and unfertilized eggs. From these results Runnstrom concluded that the cytochrome and cytochrome oxidase is equally active before and after fertilization, I but that there is laeld.ng a link to the dehydregenaae ayste•• It would appear that the conclusion about the cytochromea is gene:tally sound, in spite ot more recent work which baa uncovered some complications 1n the experiments with cyanide and carbon monoxide ir'Jl1bit1on. It was tound by Runnstrom (1935') hitnsalf, and then studied by Orstrom (1935) 1n more detail, that cyanide and o~rbon monoxide have considerable stimulatory effects on respiration. 'fti. effect ot co has recently been studied by Roths0h1ld (1949) with interesting results. He found that oarbon monoxide increases respb• ation, probably by being itself oxidised. !his increase is inhibited by carbon monoxide and the inhibition is lifted by light. respiration. In addition, light inhibits the normal The effects are therefore very oomplioat~ and conclusions drawn from earlier work on carbon monoxide will probably have to be revised• With respect to the eya.n1de inhibition, Robbie (1946) recently demonstrated that the earlier work: had frequently been vitiated by los.s of cyanide from the reaction vessel into the center well, - 7and that if proper p:recav.tions are taken the 1°espi.rati o.n of unfertilized sea urchin e ggs cnn be shown to b,i, strongly in..t-1ibi tecl by cyanide. The earlier conclusions or Hunn.strom, however, still appear to be valid. At one time Korr (1937} had. suggested that the a.ctiva• tion of cytochrome c resulted i n the respiratory increase. Until recently the search for cytochromes in the sea urchin egg had not been successful. '1.'he work of Ho t hsehild and the work reported in this thesis demonstrate that some at leas t of the cytoohromes are present. A great deal of discussion has centered a:round the question whether the dehydrogenases are more active in. t he fertilized egg. Runnstrom (1930) and Orstrom {1932) found that the reduetion of me t hylene blue was no faster in t he fertilized egg s t h.an in t he 1.mfertili.zod. However, in who.t appears to bee. more careful study Ballentine (1938) demonstrated that the reduction of both methylene l>lue and · rarri• cyanide is much more rapid i n t he fertilized eggs, and • furthermore that i t parallels the crumges in respiratory rate 1n other eggs, rising and falling as t he respiratory rate rises or falls. Recently Lindahl {quoted in Runnstrom ( 1949}) has shown that the difference bet,-aeen fertilized and unfertili.zed eggs in rate of reduction of methylone blu~ is a function of the temperature, being greater at higher temperatu1•as e.nd slight at about 12° c. As can be seen from the above brief su:a.mary , the qt'test:ion of the mechanism of the rise in respiratory r e.te of the e gg -8on fertilization remains obscure in spite of many efforts, The problem or fertilization has not been studied in this thesis, but a suggestion may perhaps be ottered. It is now known, since the work of Meyerhoff (1937) and D. Mo Needham and Pillai (1937), that certain dehydrogenations are obligatorally coupled to phosphate transfer. now believed to be a general phenomenon. This is Certain of tho dy~s used to study respiration act in an as yet unknown manner to uncouple the two phenomena. It rr.ay be that there is no activation of any respiratory enzyme on fertilization at all, but that respiration increases because of aetivation of soma enzyme system that uses A'lP and thus increases the availability of -phosphate acceptor. :Jeyerhorr and Wilson (1947) have clearly demonstrated the eftect of phosphate acceptor on the respiration or various tissues, especially brain. Tho main advantages of such an explanation, '.L'llltil decisive experimental evidence 1s produced is that it utilizes a known meohar11sm and further 0xplains the diffi• cult7 hitherto experienced 1n loco.ting the point of activation among the respiratory enzymes. Compatible also is the work demonstrating that phosphatase is more active in fertilized eggs, that labeled phosphate begL"ls to exchange and 'that free SE groups ~ppear on fertilization. ·All phosphokinases are SH enzymes. Even if the above is the mechanism of the activation of respiration, the problem of the ultimate site of activa• tion remains. The problem would seem to be the eeneral one of the nature of cell stimulation, as was suggested long ago by Lillie (1910). For a discussion the review of Hunnstrom (1949) should be consulted. As can be seen from the rev1e~ 9 little real lmowledge exists and s peculation is rife. Besides the 1r1ork: on the physiological changes that occur in the respiration, a considerable volume or work exists on the hioohemioal pathways of this respiration. A summary ot the pertinent data from the literature 1s given below. • There is apparently a considerable amount of carbol'l..y• drate material in the egg, but the nature or it is not completely clear. Ephrusoi and Rapkin& (1928) round a large amount of carbohydrate in the eggs or bracegtr.g__tu.s lividus, almost all of it in the form of glycogen. Essentially the same results were obtained by Zielinski {1935) in the same species and also by Chaigne (1934). confirmed by Stott (1931). Thes<!l results were On the other hand Perlzwcig and Barron (1928) cottld find no glycogen in trichloraeetic aeid extracts of the eggs of Ar~~. large amount of glucose was found. On hydrolysis~ howeveT, a Oe Meyerhof (1911) like• wise found no glycogen in tho eggs o:f _f,e.ra~~n.~r.gtus JcividY! at Naples. The most thorough investigation of glycogen was that made by Orstrom and Lindahl (1940). They were primar• ily interested in the substrate utilized immediately after fertilization. About 33 mg of glycogen per 100 mg egg nitrogen was found by them in the eggs of Ear.A.e~ntrgtu~ l,iv~µ..i• The exact nature of the substance in the egg called glyco3en is not eleuro It is well known that glycogen itself exists in two forms, the so called lyo and desmo glycogen. The latter is botmd to some other material, probably protein and 1s not easily extractable • .Braehet and Meedha.m ( 193 5) found the two forms of glycogen. 1n the frog's egg and discovered that the desmoglycogen disappeared first, after which the lyoglycogan was metabolized. that the glycogen of the sea urchin egg is not identical with that from other sources is indicated by the work or Orstrom and Lindberg (1940). They added glycogen of unstated origin to egg brei and found that it was not e.ttacl-red. They also quota Holter as saying that no appreciable amylase activity is to be .found 1n the eggs of farsg~nt£oIDl,i 11v1qµs and Arbac3,& RUA9IDllit.A• Runnstrom (1949) states that Vasseur has pointed out that the usual tests for glycogen may give misleading results. An apparent glycogen content can be demonstrated in the jelly coat substance or sea urchin eggs, although no glycogen exists there. The function of glycogen is now considered to be entirely that of carbohydrate storage. It has been pointed out by Needham (1931) that in the latter halt of the nine• teenth century there was a widespread opinion that glycogen was of some peculiar embryological and morphogenetie sig- nifieanco and a vary great number of papers appeared on the distribution or glycogen during development. This belief gredually died out, but other substances h.~v~ taken over the -11position of glycogen as oorp1"10genetic subs·cances. With the discoYery oi' new substraneaa, vitamins, hormones, steroids, mueleic acids and what not havo bean successively hailed as the key to development and of course to the problGm or cancer. Some have already been relegated to the status they deserve, but many are still eoing strong. !io dcubt the future ~till holds much in store. ln sun1raary it can be stated that some polli"Saoche.ride resembling glycogen is present a11d available in the sea urchin egg. The work of O:rstrom and Lindberg, quoted above, was designed 't.O solve the problem or whether the earboh.y- dra te is the substrate or respiration in the egg. They were able to show that the glycogen diminished very atlddenly on fertilization and in the expected amount. For a more pro~ longed period Ephrussi and Rapkine (1928) found t hBt the glycogen content diminished between fe1.. t11ization and the pluteus stage. Some measurements of the ros_piratory quotient exist. ,iccorcling to Orstrom (1941) the quotient for unf'ort1lized aggs isl and falls on rertilizQttion to o,89, or after correction .t'or certain errors of measuren1ent 0.73. This unfortunately does no more than possibly indicate that carbohydrate is not the sole substrate after fart11izat1on. As to the pathway of glycogen metabolism no very clear evidence exists. On the bosis of v1hat 1s known from other organisms, it would. be expected that glycogen would bo broken. down phosphorolytically as found or.iein.ally by Cori, Co:ri and Schmidt (1939). Amylitic breakdown apparently -12... does not oeeur according to the evidence of Holter, quoted above, that a.vlase activity is undetectable 1n sea urchin eggs. Orstrom and Lindberg examined the question and found that the breakdown or glycogen was inhibited by phloridsin and accelerated by addition of spers extracts. Due pre• sumably to active phosphatase activity ~o accumulation of esters was detectable and addition ot cofactors ot glycolys1a did not 1ntluence the rate of glycogen breakdown. The evidence does point to a phosphorolytic cleavage or gly• cogen in the egg. '?he further pathway ot substrate in respiration has not been clarified. There is evidence that 1t takes the same course as is well known in other tissues and also evidence that the course is quite different. Certain cofactors of glyeolysis and respiration are known to be present in the egg. In 1933 Runnstrom demonstnted the presence of what was probably Coenzyme I 1n the eggs of PAJ:acenttgty. Lindberg and Ernster (1948) found that addition of coenzyme I enhanced the respiration or bre1 in the presence or pyocyanine. Jandorf and Krahl (1942) esti• mated the amowit of eoenzyme I to be between 250 and 500 micrograms per gram wet weight of l}.£}?ao1a eggs. They also found diphosphothiamine to be present. The presence or adenos1netr1phosphate has been estab• lished by the work ot Needham and Meedham (1930) and con• firmed. For a recent review on phosphate esters in e~gs see Whiteley (1949). There is al,Jo present an adenos1ne- tr1phosphatase which has been studied by Connors and Scheer ( 1947) • They found that the enzyme re,1uired calcium as a cofactor; a point that maybe or int0rest in view of '' t..~e importance of calcium in the f0rt111zat1on process, The activity of the enzyme is also reported to increase in breis made !rom fertilized eggs over those .o f the unfer- tilized, but the authors express themselves with a certain amount of reserve on this point. Attempts have been made to detect the presence of phosphorylated intermediates in the egg. · The acid soluble phosphorus of the egg has been divided by Runnstrom (1949) into three fractions, one of 111.hich is orthophosphate ( 501;) , easily hydrolyzable phosphate (25%) and the remainder an ester very stable to hydrolysis. The same results hav~ bean obtained by other investigators, Lindberg (1943), Zielinski (1935), and Orstrom and i,.indberg (1940). The presenee of the known intermediates of glycolysis was not detected. The methods of detecting such compounds are however rather poor and liable to interference from other materials in tissues and thus failure to detect them cannot be taken too ser- iously. Lindberg (1946) made au extensive investigation of' the phosphate ester stable to hydrolysis. Be fot.l?ld such an ester 1n the eggs of sea urchins, in sperm and j_n mrun.mnlia.'1 tissues excepting muscle. The ester was isolated from cow brain and characterized as a propanediol phosphate. position of the phosphate was not established. The It should be noted th.at the identification of propanediolphosphate • ~ -141n eggs is based only on rate of' hydrolysis. It appears that propanediolphosphate is the major constituent of the so called barium soluble alcohol soluble fraction from most tissues. That it may have some bearing on embryonic meta• • bolism is indicated by its abundance in efgs. A large amount of a s1m111ar material 1s found in the chick embryo and in tumors (Needham, 1942, p614). Orstrom and Lindberg concluded from their studies that the pathway or substrata from glycogen on is different from that established in mus.cle. They state that such a conelu• sion is probable on comparative biochemical grounds, s1neo the echinoderm is an animal relatively low phylogeneticaly and its egg could be expected to have a "s1mplern type of raetaboli5m;. The factual arguments that they bring forward in support of such a conclusion are that they could detect no changes in phosphate esters during the disappearance of glycogen and no increase of lactic and pyruvic acids. They quote experiments by Runnstrom showing that the production of acid on cytolysis is not inhibited by iodoacetate and the work of Stott (1931) showing increases of reducing sugars. The metabolic scheme they favor 1s as follows. Glycogen is broken down phosphorolyticaly and the resulting esters are then hydrolyzed, the sugars are then metabolized in an unlmown manner• Lindberg and Ernster (1948) found that on the addition of py oeyanine and hexose, hexosemonophospha te and hexose• diphosphate an esterif1cat1on of ADP could be obser-~ed. This is entirely to be expected on the basis of our knowledge of what happens in .other organisms. Much of the argument for the existence of a pathway different from ordinary glyoolysis in the sea urchin egg stems from the inability of 1odoacetate to inhibit the reduction of methylene blue by hexoses and their phosphoric esters. Thus Lindberg and Ernster (1948) found that iodo- aeetate had no influence on the oxygen uptake of their preparations. Tyler and :;chultz (19J2) found that iodo- acetate does not inhibit the te:rtilintion of the eggs of Ufeghis 9JUR9• This was found to be the case for the sea urchin by Runnstrom (1933) and Ellis (1933) and also by Lillie (1931) and Pasteels (1935). This however does not necessarily have much beari."1g on the nature of' carbohy- drate breakdown. Hutchens, Kelteh, Kra.hl and Clowes (1941) w~re unable to detect an triosephosphatedehydrogenase in Arbaou eggs. Lindberg and Ernster (1948) round that in their homo• genates, using glucose and phosphorylated hexosas as m.1b- strates methylene blue was decolorized rapidly, but that there was no accumulation of tr1osephosphates. 'l'riosephos- phate was te~ted for as phosphoric esters hydrolyged in alkali. The question whether lactic acid is formed by sea urchin eggs has been debated. In 1929 Ashbel showed that the eggs of i,gr9:9entrot;qp have a considerable anaerobic glyoolysis and ineidentally t hat this increases almost -16threefold on fertilization. For some reason her results are dismissed as technically faulty by Orstrom and Lindberg (1940). Perlzweig a11d Barron (1928) found that lactic acid accumulates in eggs in the presence or cyanide. It now seems clear that there is no s1enif1cant rise of lactic acid on fertilization. Orstrom and Lindberg (1940) state that no accumulation of lactic acid o-ccures 1n egg bre1• Their experiments however do not. seem to have been especially designed to promote such accumulation. Runnstrom (1933,1949) believes that the dehydrogenase system ot the egg is that described originally by Warburg and Christian (1932). This system has been described in more detail by Dickens (1938). Gluco-se-6-phosphate is oxidized to 6-phosphogluconic acid, oxidized again to 6-phos- . pho•2•ketoglueon1o acid and then deearboxylated to a pentose. Dickens supposed that this process eould continue in the same manner to yield art erythr_ose and even further, but as yet there is no evidence that such is indeed the case. The belief that the respiratory system of the egg is based on this series of reactions rests on several grounds. Egg homogenates readily oxidize phosphoglucon1c acid (Lindberg, • 1946). Iodoacetate does not influence the oxygen uptake ot homogenates (Lindberg and Ernster, 1948). No triose- phosphate can be detected, as stated above and triosephosphate dehydrogenase is absent. negligible. Formation of lactic acid is Lindberg (1943) has further shown that the propanediolphosphate isolated by him when added to tissues produeas an increase in pentoses and phosphorylated he:xo ses produce the s:a:ne eff ;_;1 c t i n egg brei. The ittcreases however are s6arely significant. He.r1:ry (1943) clairr.ts that there ar e two di s tinct res• piratory systems in t he eggs of &rb~1~g 1 A basal respiration wh!eh maintains the egg •s basal me tabolism is not sensitive to sulfonamides. On fertilization anot her T$sp1ration fu...'l.etions, which is sensitive to ·sulfonamides and is con• cerned with providing enerey .for developmental proeess~s. Perhaps d1f!'erences in permea.biJ.ity could explnin at least part of the phenomenon. The metabolism of t he acids of the trioarboxylic acid cycle has been little studied until recently. Ji. ccordinr, to Barron and Goldinger (1941) and Krah.1, Jandor:.r e.nd Clowes ( 1941) f ertillzed eggs r.iotabolize pyruvu to at a gre.a te1• rate than the unfertilized. The claim is mado that the eggs a.re not able to oxidize alpha k:atoglutaric and succinie acid. Runnstrom (1933) found that pyruvic and lactic acids wer© oxidized much more slowly by egg bre1 than hexose phosphates and that succinic and maleie acids were scarcely oxidized at all. Lind.berg (1946) found that r1.:unaric and me.lie acid rapidly reduce methylene blue in homogenates of J;i<;hin,oq!.tdium eggs. Ball and ~,ieyerhof {1940) during studies .or the cytochrorne s yste::c: of eggs of various species found that suceinic dehydrogenase was absent in sea urchin eggs. Recently Crane and Keltch (1949) reported that suecinate, alphaketoglutarate, glutamate and citrate are oxidized• The metabolism of phosphate has been studied in some detail. Brooks and Chambers (1948) found that the uptake of labeled phosphate is very slow 1n the unfertilized f:3ggs but rapid 1n the fertilized, and this has been confirmed by Lindberg (1948). What is probably more significant was the finding that only part of the phosphate in the ur>.fert1• lized egg showed a turnover. The fertilized eggs, on the other hand, showed a continuously increasing activity throughout the period of the experiment. Studies of the terminal oxidase or sea urchin eggs have continued since work on their respiration was initiated by Warburg. As mentioned previously, Warburg discovered that cyanide inh1b1tod the respiration. The work ot nuru1strom referred to above made it probab. that the terminal oxidase is cytochrome oxidase as in so many other tissues. That this is indeed the case was established by Krahl, Keltoh, Neubeek and Clowes (1941) who showed that the sea urchin egg contains an enzyme able to oxidize reduced cyto• chrome Ct Bore1 believes this enzyme to be somewhat dit• ferent from a typical cytochrome oxidase. For a long time the function or cytochrome oxidase 1n the sea urchin egg was rather obseure since the presence ot , the other cytochromos could not be demonstrated.· Brachet (1934), Lindahl (1936), Ball and Meyerhot (1940) and Krahl, Keltch and Clowes (1941) reported th.at no c.rytochromes were present in the egg. This was considered to be of some •19• importance in view of the hypot."'lesis of Korr that the res- piratory rise was due to thG activation of oytoohrorr~ e. Rothschild (1949) has now reported that oytochromes a and bare indeed present in the sea urchin egg. Many other aspects or the metabolism of sea urchin eggs have been studied. Since, howavor, they ao not bear directly on the work reported in this thesis they will not be summarised here. All pertinent wo.rk except the most recent has been compiled in the two boola of ?le_edham ( 193,lt l.942) and by Brachet (1947). A more recent review discus• sing metabolism to some extent is given by Ru.nnstrom (1949). -20- Statement of the Problem Prom the Qbove review of the literature it is cle,ar that the respiratory metabolism of the sea urchin egg has not been satisfactorily analy~ed from the $tandpoint or present day knowledge or respiration in other animal or plant tissues. :t'his was even more true at the time the thesis work was started1 during the progress of this work • the pape:r or Rothschild (1949) identifying the cytoohromes and that of Crane and Keltch (1949) reporting the oxidation of the acids of the tricarboxyl1c acid cycle appeared. There are several reasons for desiring more accurate information on the respiratory metabolism or the sea urchin egg. From the embryological point of view it would be of interest to know whether or not the egg has a different metabolism from that known in the adult organism. If such were the case then the usual respiratory system would be arising anew each time in ontogeny as t for example, ·t he actomyosin system. It would, then 9 be a part or the pro~ oesses of differentiation and being relatively well under• stood would be interesting to study from this point ot view. Secondly, it is clear (Tyler, 1942) that the processes ot growth and differentiation require some energy which is provided by the respiratory system. Observing changes that occur 1n respiration we can get a clue to events of perhaps deeper signi.:f'icance. At ~"1Y rate we can suspect other changes if the respiration has changed., For example in the frog•s egg on fertilization, Hrachet (1934) observed that on fertilization or the trog's egr, the oAygen upta ke remains the same, but that the respiratory quotient changes greatly & Thirdly, such studios are of biochemica l lnterest. I·t is desirable to know how uniform is the respiratory mechanism of various organisms. If differences are established, than the detailed study of such systems is i.n order . Investigations were theref'oria made of the prt-:sonee or absence in the sea urchL."'l egg or .various r asp:tra tory enzymes 1 whose properties have been reasonably well established 1n. other animal tissues. ~oter111s and methogs, The studies we.r e made using the eggs of §.~t!?D&Ylosen• trotUJ! m!rPNi~ and laix\heghttms nictYI• Sinoe these two species are ripe at different times a supply ot eggs 1s available throughout the year. The animals were collected in the vicinity of the William a.Kerekhoft Marine Laboratory or the California Institute or Technology at Corona dal Mar. Animals were kept in aquaria with r·unning sea water until needed. From time to time the req11ired number of animals waria brought to Pasadena. These we\.e kept in jars ot sea water in a refrigerator and air was bubbled th.rough. Under these conditions the an111lals survived several days. Eggs were removed from tha animals 1n one of two ways. Either the animals were opened with scissors and the ovaries h placed in sea water were they shed, or the animals were "' injected with isotonic (0.55 M) KCl (Tyler, 1949) which caused them to shed at once . The eggs were then allowed to set~le in a beaker; the supernatant was removed, and fresh filtered sea water added. The washing process was always repented three or four times, It is clear that the study or intact ecgs was not likely to give the informat ion desired. The ml.fertilized sea urchin's egg is extremely impermeable to many substances that would be used in st~die~ ot respiratory metabolism. The fertilized is more permeable, but not to a degree suf• . fioient to permit the introduction of cofactors and sub• strates i nto the intact eggs. Therafore homogenates of eggs were used. Homogenates were prepared in the following manners the washed egg suspension was poured into a graduated centrifuge tube and then centrifuged !or about ten minutes at 3000 rpm. The supernataJ1t soa water was ral'."lovod and tho volume or the packed eggs noted. Bu.f'f'er was uddod to the amount desired. The suspension of eggs ,vas then homogenized :ln a test tube with a lucite pestle attached to an electric motor. After this treatment there were no intact eggs in the homogenate. The concentration of the homogenate was e~-pressed as the per cent of packed eggtS in the final volume of homogenate. Some of the experiments were done on eggs that had been lyoph111zed and then stored in the deep freeze. The findings of Lindberg andErnster (1948) indicated that the respiration or homogenates, unless kept very cold would soon cease. It was found, however, with the species of sea urchins used that the andogenousrespiration, that 1s respiration of a homogenate without added substrate was considerable. • Using the Warburg technique i t was fou"ld .. that the respiration at 22°c showed som~ decrease (Fig. 1) after two hours. Storage of e 6&s in the deep freeze for two days still yields preparations with a considerable endogenous respiration. '.Chus no special' precautions are needed to study • the respiration. The ~\ arburg m&n.ometer bath tempe.rl'I ture was set; at 22°C wtich is a comranient temperature and within the range 1..'l which the eggs will davelopo On warmer days a • -24- 200 C\J 0 1 100 60 Time in minutes 120 Fig. 1 Endogenous respiration of homogenates of eggs of s. purpuratus in phosphate buffer, 1/15 M, pH 7.1. Dark circles - stored frozen two days. Light circles - fresh. Concentration of homogenates - 25%. cooling system was needed for t ho bath. The best approach seemed to be to talte yea.st and muscle as a model and attempt to demonstrate a similiar respiratory , system in the eggs. The question of substrate has to some extent been clarified as can be seen from the literature survey and was therefore not studied. fhos Rhot:lJ.120t1N~lil As shown by Cori SJ,d Cori (1936) 9 the first product of the breakdown of glycogen is glucose-1-phosphate. Gluoose-1-phosphate is then transformed into gluoose•6phosphate through the action of phosphoglueomutase. The mechanism of this transformation has recently been clari• tied by the work of Lelo1r et. alo (1948). A coenzyme 1s involved and has been identified as 1,6,glucosediphos• phate. The reaction prooQeds 1n such a manner that tree phosphate is not formed. The property that most easily distinguishes glucose•l~ phosphate :from glucose-6-phosphate 1.s the lability of the phosphate of glucose-l-phosphate to aoid hydrolysis. This ester is completely bydrol1zed in l N HCl at 100° C in fifteen minutes, while under the same conditions glucose--6phospha te is scu,e ely a.ff ected • This property was used to test for the presence of phosphoglucomutase 1n soa urchin eggs. Unfertilized eggs of L. pictus ware lyoph1lized, o.4 g of the powder was added to 24 ca of distilled water and the suspension homogenized in a glass homogenizer. The homo• genate was kept ice cold through the time or preparation. It was then dialyzed L"l the cold tor five hours w1 th three changes or dialyzing solution against a solution of o.;% KCl and 0.26% N62co3, pH 8.o. paration. This was used as t he enzyme pre- Like many other enzymes concerned with phosphate meta• . bolism phosphoglucomutase is rapidly inactivated by heavy metals (Najjar, 1948). It is also known to require rig ions. Cysteine and Mg ions were therefore added to the incubation mixture. As substrate the potassium salt of glucose•l•phos• phate was used. Incubation was at room temperature 23° C for 90 minutes and aliquots were pipetted into 4% trichlor• acetic acid at the beginning and end 0£ the experiment. This was then filtered and the inorganic phosphate deter• mined on aliquots after 15 minutes hydrolysis in a boiling water bath by the method of Fisko and Subbarow (1929). In addition, fructose was determined by the method or Roe (1943)• Two separate experiments were done, both of which indicated activity corresponding to phosphoglucomutase. The results of one are presented in Table I with details of the composition of the incubation mixtures. The amount of easily hydrolyzable phosphate decreases considerably 1n 90 minutes, a result to be expected if phosphogluoomutase 1s presont. That the decrease is not great~r is probably due to phosphatase activity. There is a sienificant increase in the rructose value. This is to be expected if the next en~;me of glycolys1s 1 oxoisomerase is present. for this onzyme. A direct test v1as therefore made Inorganic phosphate i.l'l. mg P ai'te:r: 15 minutes i-wgz,olysis j_n 1 J:J HCl at 100 C t-1. 0 min. 90 min. o.64 0.82 Fructosei, as mg fr-uctose-6-phosphate 0 min. 90 r.!dn. #1. 6 co enzyme prepara tion, l ce 0.1 ll eysteine hyd::."O• chloride, 1 cc 0.1 M MeCl? t l oc glucose-1-phosphate , 8 mg or the potassium sal-c a..t1d 1 ee water #2. Control. As above, substrate and water replaced by l ec of ~lucose, 4.4 r:ig and 1 ec of K:H2Po 4 , eontail:1. ing 0.78 mg P. .Q.;"!CQi§OVJ!i'l'§S~ Lohman (1933) fou.nd that the equilibrium between glucose-6-phosphata a.Yid rruetose-6-phosphate 1s established in a mat·tor of seconds. fore used. A aho:rt incubation period was there• The homogenate oonslsted of 0.5 g oi' lyophilized aggs ot lr• pietYs, cround and suspended in 30 ee of dis• tilled water . The suspension was then dialyzed against a solution of o. 5~~ KCl and 0.26% Ifa.2 co , pH 8.6 for six hours 3 and stored in the deep freeze until used. suspension was takeu·i. tion was added. substrate. Four cc of this One cc of fructose•6•phosphate solu- This contained approx1mately 5 mg of the At O minutes 5 cc ot 1~ triehloracetie acid was pipetted into one reaotion mixture and substrate added. At t he same time substrate was added to the second reaction mixture an.d the reaction stopped seven minutes later with the same volume of triehloracetic acid. Activity was measured by determination of fructose by tha method of Hoe (1943) and .by phosphate determination without hydrolysis and after h!,r"drolysis ir1 1 N HCl for 180 minutes. Under these conditions fructose-6•phosphate is hydrolyzed com- pletely while glueose-6-phosphate is little affected. Attempts to measure the glucose formed using the iodine titration method of. Macleod and Robison (1929) gave excel• lent results using pure solutions of glucose, but unsatis• factory ones with t1'1ahlo:raeet1c acid extracts. of an experiment are presented below. The results Pructose-6-phosphate Found 0 min. 5.35 mg 7 min. _2,12 mg Difference 2. 96 Phosphate as mg KH2P04 7 min. Incubation 0 min. Incubation No . hydrolysis 0 .24 0. 72 150 min. hydrolysis _s,12 . 1, 52 Di.fference o.84 In seven minutes 1.1 mg of phosphate hydl'olyzable in 180 minute~ disappaa.redi which corresponds to 2. a5 mg or fruotose-6-phosphate as agai..r.i.st 2.96 mg of r i,uctose-6.-phos• pha te as determined by the fructose method. is satisfactory• 'I'he agreement the presence 01 oxo;tsome:rasa is t;horefora indicated. il ttempts were l\'iade to demons t rate the presence of hexokinase and phosphohexo_ k inase, 1.:r1:i was added to dialyzed homogenates with and without ~lucose and easily hydrolyzabla phosphate determined after varyine periods of tinw . cases In some a small decrease or easily hydrolizable phosphate was noted 9 but 1t was srnall and not consistent. It has been pointed out by Hroh-Kahn and i.ar-s ky (1948) that the demon• s~~ration of hexokinase in homogenates, due to the comple:z nature of the phosphate metabollsm, is difficult 1:1nd pt!r1- fieation of tbe enzyme is often neocessary. T1:is we.s not done e..111d therefore the presence of hexckinase is indica. too by only experiments shotting that glucose is metabolized by sea urchin egg homogenates. The attempts to demonstrate activity or phosphohexo• kinase were l:f.kewise not successful. No increases of 1,6,rructosediphosphate could be d¢1monstrated after true• tionation of trichloraeetic acid extracts or homoganates incubated with rruetose-6-phosphate, glucose and ATP by the barium method (tePage and Umbreit, 1945). This enzyme is reported to be very labile and so may be unstable during the dialysis periods that are needed to remove the coenzyma of triosephosphate dehydrogenase. Th@ next enzyme in the glyeolytie scheme is aldolase which converts 1,6,rructosediphosphate into phosphoglyeeraldeh~e and phosphodihydroxyacatone. As mentioned in the literatv.re survey, previous attempts to demonstrate the formation or triosephosphate in egg homogenates on addition of hexoaediphosphate have not been successful. The method used was the determination o! alkali labile ph.osphate. It would appear, however , that a method. th.at would trap the products would be desirable for the deraonstratlon of aldolase activity, Sibley and Le!~1::tnger (1949) have described a convenient end specific colorimetric method for ald.olase. The p:r:u1ciple is to trap the triose phosphates for~ed with hyd!"azL,e, add 2,4,dinitrophcnnylhydrazine and develop eolor with alkali. This rnethod was used to detect aldolas0 aeti 0 vi.ty in sen urchin eggs. 100 mg of lyophilized eggs of 1'zyl\q~q~ Iii£~~ were suspern:1ed in 6 ec glycylglyeino buffer, 0.1 M, pH 8 . 6 m'.ld hornogen.1:.too. This was used as the enzyme preparation. Substrate - fructose - 1,6,phosphate 20 mg/ec. Hydrazlno o. ;6 M. The reactit)n rnixttu"es were .incubated in tost tubes for 30 minutes at t•oom temperatu.ro of 2(P c. The :reaction was tht?,,n stopped by adding 2 cc of 10% trichloraeetic acido Tho contents ware filtered a.."ld 1 ec aliquots taken for analysis. l cc of 0.75 NaOH was Etdded to the aliquots i.n colorimeter tub@s and kept n t room ten:1pera tu.re for 10 ra:Lrint@s • f l cc of 2fJ4,din1trophenylhydra.zine 9 concentration lg 1n l liter of 2N HC1 9 was added and tubes incubated 1n a water bat.~ at 38° C for ten minutes. The contents vuere the..'l made up to a volume of 10 cc with 0.75 rt NaOH, kept at room tem- perature for 10 minutes and the color read with a green filter. The contents ot tho reacti:m mixtures and colorimeter read• i11£s are given 1n Table II. As oan be seen, color devalop• ment is initially proportional to enzyme concentration. Several repetitions of the experiment gave similar results. Since this method is reported to be speoifie, it can be con• cJ.uded that aldolase is present 1n the sea u-rch3.n egg. Sibley and Lehninger report that if the reaetion of fructose-di-phosphate with aldolase is allowed to proceed in the absence ot hydrazine and if phosphotriose isomerase is present a much stronger color-development results, as the triose phosphate 1s then predominantly phosphodihydroyacetone w'.hich. gives a stronger color t han phosphoglyoer• aldehyde. Using undialyzed egg suspension, in one oxpari• ment enzyme incubated with hydrazine gave colorimeter reading of 72, with hydrazine added atter incubation 40. Instead of ·an increase a decrease was obaerved. This 1s presumably due to further reactions or the tr1ose phosphates formed. Tabla II Formation of Trios8phosphate Tube ~ " l 2 Buffer ee cc 1.0 1.0 Hydrazine cc Enzyme co Water cc Densi~, 0.2 0.1 0.1 0.9 0.9 25 27 0.2 .': o.8 52 o.4 o.4 o.6 o.6 96 91 Oo2 1.0 1.0 0. 3 0.3 1 •.0 0.3 0.3 1.0 9 HDP 0.2 0.2 1.0 1.0 o.e o,8 1.0 o.8 Optical . o.e. Concentration of reagents in t ext • • #9. HDP added after addition or triohloracetic acid. 50 ,tr1oe.!P!l£tspha te d§n,_tdfo&..OJ!.@U , Si.nee aldolase is present in the ·eggs of sea urchins, it is to be expected, in spite of the work mentioned in the introduct:i.on that triosephoap}'-1..ate dehydrogenase should be present. no triosephosphate as such ,1as av·a.ilable, but addition or fructose•l,6-diphosphate should be equivalent to addition of trioaephosphate to homogenates, Trj.osephos• phate dehydrogenase in all case~ knt>wn (Meyorhof and Kiessling, 1933) is strongly inhibited by 1odoacetate and fail• Ul"a to detect such inhibition has been one ot the main are'l.lments against the existence of the usual glyeolytic system in sea urchin eggs. However it is well known that the effecrt or iodoacetate is not instantaneous. llxperi• ments were therefore dorte to see it firstly the endogenous respiration of egg homogenates is inhibited and also if such an inhibition takes time to develop. blue technique was used. The methylene Thunberg tubes were prepared, al.l components including homogenate were added and iodoocetate was odcled to the tubes .60, 30, 1,, an.d O minutes hefo1•e evcicua tion and tipping in or mGthylane blue. Fig• 2 shows clearly that the endogeneous respiration is inhibited by iodoaoetate and t;hat; the e:f'feet is progressively stronger the longer the homogenate has been in contaet with the inhibitoro This is some evidence that at least part or the :respiration passes through the triosephosphate stage. course other enzymes are also inhibited by iodoacetate, or 23° C 0 ,-j 0 • 0 0 3 Fig. 2 Effect of iodoacetate on reduction of methylene blue by 20% homogenate of L. pictus eggs in 1/15 M phosphate buffer, pH 7.1 iodoacetate 0.03 M. Ordinate - reciprocal of time in minutes till reduction. Abscissa - length of time in minutes homogenate exposed to iodoacetate. Black circle - control, no iodoacetate. but not so strikingly. It can be shown quite ensily by the same technique : that the dehydrogenation of hexose diphosphate is likewise . s inhibited by iodoscetate. It is however nec0espry to use a more dilute homogenate, of the order o:f 5%, as oth@rwise the reduction by the control is inco.n veniently rapid. A typical resnl t is presented :L"l Table III. Al t hough the products of the dehydrogenation of l-,6,rructosediphosphate were not 1dentif1ed, it is to be expected that it would b0 phosphoglyceric acid. In th.at ca:.H! it would be expected that the phoaphoglyeeric. acid would lose wate:r under the action of enolase and be transformed to phoaphoenolpy:ruvic aeid. Table III Reduction of Methylene Blue by lt6Fruetosediphosphate and Efrect of lodoaoetate Tube If lo 2. .3 . 4. Tube contents Side arm Tims d0color. min. 2 ec homogenate l cc.MB 2 cc homogenate l cc ·1odoaeetate l cc MB l co water > 120 l cc water 2 co homogenate 1 oc MB 1 cc HOP ;2 2 cc homogenate l cc MB l oc HDP ) 120 l cc water l cc :todoacetate 1-6-t'ructosediphospha te 0.05 M. l 00 water 100 fhe calcium snl t {Schwartz) was usedt calcium being removed by addition or sodium oxalate . Iodoaeetate 0. 1 M. Ioooacetate was washed with petroleum ether, dried and the solution neutralized to pH 7 with Na.OH. Methylene Blue, e. solution of 12 mg. 1n 100 cc water. Homogeni\te . pH 7.-1. 5~ §, .J?'UtP!FA:tYi eggs in 1/15 M phosphate butfm:• 1 lodoacetate was ·added and the tubes evacuated and allowed to sta."ld one hour before tipping in the eon tents of the side arms. Temperature 23° c. · Jmolau The presence of encl.as@ can be suspected 1r respir• ation or glycolysis iS inr,.ibited by fluoride. Fig. 3 shows the cou.rse of respiration or a homogenate.\ of atrongylQcan: .trotu, mµ:purgtJYi eggs with no substrate added. t..s can bo seen, the respiration is quite strongly i1~1ib1ted by NIU' o.t concentrations of o.o, and 0.01 Min a phosphate bnffe:r . The inhibition of respiration by fluoride is net com• pletely specific, as a variety of other enzymes, especially phosphatase@ are also inhibited, although not at as low a concentration. The presonce or enola.se was checked direct• ly by the method of Lohman and Meye1"hof. (1934) • is dialyzed ATP is removed. If tissu® If phosphoglyeer.ic acid is •• added and enolase is present phosphoGnolpyruvio acid aoou. raulates, sinCG in the absence of phosphate acceptors it does not go to pyruvic acid. Iodine ean be added to form iodoform and the exoesa iodine titrated with thior:mlf~te. Lyophilizod lu...R'9£tYi eggs, weighi..'Plg o. 5' g wsre ans• pended in 30 ce of distilled water and homoeenized. The suspension was then dialyzed in the cold against o. 5;,f KCl and 0.26% Na 2co • 3 The barium salt of phosphoglyceric ac.id (Schwartz) was treated with excess of sodium 13nlfat0 to precipitate the barium ~nd made up to a concent:ratio11 of 0.02 H computed on.the basis of tho weight of the salt taken. Due to absorption of the barium sulfate t he actual coneentration was of eotll"sG somewhat less. -40- 20 10 30 60 Time in minutes 90 Fig. 3 Effect of NaF on the endogenous respiration of a 25% homogenate of s. purpuratus eggs in 1/15 M phosphate buffer, pH 7.1 volume of homogenate 2.0 cc. Light circles - control. Dark circles - with 0.051and 0.01 M NaF. Throe series of test tubes were U3ed• The reaction was run at room temperature 23° for 10 minutes, and stopped by addition of 8 ce or 4$ trichloraeetio acid, filtered and a 2 cc aliquot treated with iodine in the usual manner and titrated with 0.01 N thiosulfate. Enzyme was added to the blank after addition of triohloracetio acid. About .f'.ifteei., per cent of the theoret1oal amount of phosphoglycaric acid added was converted to phosphoenolpyruv1e• The equili• brium amount reported for this temperature is about 307;. Activity 1s completely abolished by flue.ride. The above results together with the inh..1.b1t1on ot the endogenous 1·espirations of homogenates indicates that enolase is present and functions in tho respiration. It is to be expoctoo that it the normal glycolyt1c mechanism is opGrative 1n the intact egg then under the proper conditions certain intermediates could be shown to aecuum.late. As discussed in the literature survey the accum- ulation of lactic acid has been questioned. A..'1 attempt was therefore made to sea if under anaerobic conditions lactic acid would aceu.'?lulate in intact egzs. 3 oc au.spans-ions or ,Jn.fertil:tzoo ecgs of §., t!\li'.Pllr-atua, e..0ntaining 0.1 ee of packed eggs were pipetted into ThunJ-;erg tnboa to one series 3 co of 1o;i triehlo1.. acet:tl acid was added et once, another series was evacuated and allowed to stand at room temperature for three hours, then opened and trlchloraeetio acid added. Lactic acid was determined on aliquots by the method of Barker ru1d Surr.r~rson {1941). Table IV Formation of Phosphoenol Pyruvic :from Phosphoglyceric Acid Vessel :Ji substrate H2 0 IiaF --- Blank l 1 ce lee Blank 2 l cc loo 3 l ee 4 l cc ----- 5 lee lee 6 lee loc 7 lee lea Enzyme Thiosu.lphate Pyruvata Formed lee 8.07 lee 7.93 a.verage 8.00 lee lee 7. 97 lee lee '/.94 av@rage 7.95 lee 7.60 leo 7.61 lee 7.68 average 7.63 --- ---- --- --- ---- --- 0.03 mg 0 • .30 mg The tubes 1n which the reaction was stopped at the sta:rt showed only a trace of lactic acid while those incubated anaerobically showed a total !,eumulation of 0.086 mg ot lactic acict, or o.86 mg per oo of eggs. This can be com• pared with the value given by Perl2Weig and Barron (1928) who found about 0.1 mg of lactic acid per cc or unfertil• ized eggs o~ Afb.&Siii incubated with KCl~ for varying periods of time. The other values in the literature vary a great deal, ZielinslQ. reporting about o.; and Hunnstrom 0.3 mg per oc or eggs. The point made he1~e is that there is an accumulation of lactic acid in anaerobiosis, This would 1nd1oate that some anaerobic glyoolysis occurs with lactic acid as the end :product. This con.forms with the early findings of Ashbel (1929) that aaid is produced by sea ui~ohin eggs under anaerobic conditions. Run.nstrom (1949) reports that lactic acid is metabo• lized to a small extent by egg homogenates. This was con• firmed by Thunberg tube experirMmts, but as in the e:xperi• ments of Runnstrom the increases of rate are not large. One experiment, for example, showed time or methylene blue deoolorization of control 19 minutes, with 0.03 M lactate 14 ndnutes. Lactic acid can thus be metabolized by egg homogeruates. Runnstrom (1933} was the first to show th.at on rarti• lization of sea urchin eggs there is a production of acid which can be detected by evolution of carbon dioxide from the tJioarbor.i.a te of the solution. The a.mmmt of acid produced is rather small, about 20 microequiva.1.ents per 100 me of egg nitrogen. The production of aeid ""'as al5o observed by Rothschild (1939) if eggs •~ere injm"ed or eytolyzed by var• ious means. Runnstrom (:reported in Orstrom a.n.d l,indb~rg, 1940) attempted to analyze 0c;es before and after fe:rtil.iza• tion for lactic aeid and found no eha.Jige. The eggs hov."Over were washed ex.huustivoly before a.nal;;r.sis e.nd the lactie acid, if given off into the mediulll would not be rouxi.d,. TJ-i.at no ereat change occurs 1n the lactic .acid content on fertil1• zation was shown by Perlzweig and Barron ( l 928) • Dy di1•ect atte:npts to obtain a precipitate with ketonic reagents Orstrom <'~oncluded that the acid was not py·ruv1c. It is concentrations-. Eggs wore eytolyzed by distilled vm tor nnd aftan-- periods or fi,;te minutes trichloraoetic acid was added and the filtrate tos ·t oo foi.. lactic acid by tho method of Barker and Smnmerson (1941). The difference in lactic acid content between eggs c:,tolyzoo and the controls was ro-..u1d to b-0 noeligible. The a...-wt::nt of acid produced is so small that tho pro- blem or its identification is a difficu.lt one. f The most ob11ious approach is to a:ttonpt extraction with ether. 'l'his method was tried. A oontinuous ether ext1"actor was used. When isolating such minute quantities of substance the danGer of artifacts from the ether or from impurities is considerable. The en.tire apparatus was carefully cleaned with dichromate and rinsed with glass distilled water. The etha:r was CP gra1de 9 :red1stilled from ferous sulfate a..11d. k~pt over anhydrous caleit1.1,-, chloride for twelve hours. A lat'ge qu.antity, about fifty ec or the eggs of St Ri!r.R.Y.t~t'!! was used. The eggs were washed four times t.u"1d suspend&d in filte1..ed sea water. They were then i'ertilizad, allowed to stand for ten minutes and then centrifuged. The supernatant was removed, filtered throueh filter paper a.nd then through a Seitz filter. similar batch of oggs was used as a control. A These eggs imre not .fertilizoo, and an equivalent quantity or sperm was add®d to the supernata.11t just boforo filtration through the Seitz fil tar. A t1lank or f:tl teroo sea water served as a second control. The f il te'.!."ed solutions \Vere aeidii'ioo i,;i th sul.furic acid and extracted with ether for three hours• . The ether extracts were then evaporated in a sti"aam of air. It was found that th0 residue or all three extracts was aeidie to indicators. An attempt was made to see 1f the acidic substance would move on paper chromatoeraphy. Using the mothod of Luge and Overall (1947), one dimensi01~l ehromatograms wer@ run with a solvent of water saturated butanol in an atmosphere saturated with formic aeid. After chromatography overnieht, the paper was dried and developed by spraying with pH indicator. One spo·t was fou..111d common to all three extracts which had moved slightly. In addition the extract from the f'ert111zed eggs showed another spot that had not moved at all. A similar spot, but extreme- ly faint was tound w1th the unfertilized extract and was absent 1n the blank. The amount of material atter evaporation of the ether was so small, about 0.2 ee, that regular procoedures of fractionation were di1'1'1cult. Tests with 2t4,din1trophen:,l• hydrazir1e ind1ca ted absence of aotive carbonyl groups. A direct test for lactic acid by the method ot Barker and Summerson (1941), which is sensitiv~ to quant~ties of the order or 0.5 gamma gave a negligiblG oolor. Further work on this extl"·set has not bean completed. The above work confirms earlier :reports that the acid of tertiliza~ion is neither lactic nor pyruvic. In addition it does not appear to be any of' the acids of the tricarboxy• lie aeid cycle, all of which move on paper with very appreciable Ii.F • Its nature is not Imown, but the determination of its identity would be of great interestt as it might give ,a clue to the events oc~g 1mvJed1ately on fertilization. Since enolase activity can be demonstrated in egg homogenates and the endogenous respiration, ·as shown i."'1 Figure.3 ia 1..t1.hibited by fluoride it would be expected that ii intact eggs are incubated with fluoride ph.osphoglyceric acid should accumulate. A negative result of course would not be sign1fic81lt 9 sinoe fluoride might not be able to penetrate into the intact ogg. Twenty oe of a susp~nsion a, PW:PWIW eggs were pipetted into small Erlenmeyer flasks. The nitrogen content ot the suspension was determined by t he K3aldah1 method and found to be 6.6 mg. To two flasks 2 eo of 0.55 MNaF in dlstilled \"later was added, to the controls 2 cc ot sea water. The :f'le.sks were incubated in a water bath at 23° C for 4 hours . Ten ce or 10% triehloracetic acid was then added to each :flaslr. The susp<ftnsions were filtered and the f'1lt:rattSJ trea t od 't'11 th bar1 um to preoipi ta ta phosphosphoglyoerio acid according to the method of LePage and t7mbre1t (1945). An attempt to estimate the phosphoglyaerie acid by the method J of Meyerhoff and Schultz (1938) based on t he onhaneement ot optical rotation in the presence of roolybdate d1d 11ot prove possible due to interfering substances present. The less speeif:i.c method of Rappoport (1937), bas~ on the dei·elop- rnent of color with alpha naphthol in concentrated sulf uric acid was therefore used. The results \rere ns follows: mg Barium Phosphoelycerate Control Wii;h NaF 0.984 1.279 A rather unexpected phenomenon was noted on adding fluo1·ide to egg suspensions. agglutinate. The eggs would immediately Because of th~ possibility ·t hat this phenomenon could be r<1lated 1n some way to the ag~lutinations produced by intera.oting substances of eggs and sperm some teats wera made to determine its nature. It was found that the sperm of sea urchins wore likewise agglutinated.in large sheets. On suspending eggs in isotonic sodium chloride solutions On addition ot calcium aeglut1nat1on did not occur. . 0 agglutination would aocur at once. The calcium and magne- sium salts of fluorine are very insoluble. The addition or .fluoride to sea water therefore results in the preeipi• ta.tion of these salts. Under the microscope the precipitate was found to be in the form of a gell. A suspension ot carbon black particles was observed to agglutinate in a manner similar to that or eggs, although not in so striking a manner. It was there.fore concluded that the agglutination or the eggs and sperm observed has no bearing on the specitio intaraetion or their surfaces. The stickiness or both eggs ai~d sperm to th~ calcium fluoride gell is nevertheless rather striking. Although the increase of color in the test for phosphoglycerio aoid could indicate an increase in phosphoglyoeric acid this 1s actua.l ly doubtful. The precipitation of fluo• ride by magnesium and calcium 1n the sea water lowers the concentration of fluoride. The method or Rappoport is not var y specific and is reported by tePage and Umbreit (1945') to give at times too high values due to reasons that are not understood. In the teat reported above interfering materials were present that gave a brown coloration and undoubtedly i nterfered with the accu~acy or the test. Therefore the aecumuls. tion of phosphoglyeeric acid should not be regarded a s demonstrated. M.4.t&J&•!§m o:l'.' certgin .~c;tds. ..,Q.f t4e Jiri't.~rbg;grlic ~g1d cyc:t,2 If tha metabolism of the sea urehin egg is similar to that known in other organisms it is to bo expected that it would be able to metabolize the various intermediates of the tricarboxylic aeid eyelo. The ~tilization of intermediates was determined by measuring th.e oxygen uptakrn of egg homogenates· prepared from unfertilized eggs of .§..t... t?urmY;atBI• • As will be shown la te:r the addi tio11 of cytochrome c was round to stirm..1la te the respiration ·very considerably. Phosphate buffer, l/15 M pH 7 .1 was used ir1 prepa.r1ne the homogenates. Malonate is la10'ffi.'1 to be a specific inhibitor or succinie dehydrogenase and if respiration is inhibited by it, it is probable that the respiration is proceedir...g through the triearboxylic acid cycle. It was found that the endogenous respiration of homogenates is quite strongly inhibited by malonate. The inhibition is not instantaneous but develops gradually. Fig. 4 shows the course of respiration in the presence a.11d absence of malonate. The inhibition ooeurs both in the presence and absence of cytochrome c. Fig . 5 shows the effect of '7'ary1ng concentrations of malonate on the respiration. The respiratory rmta is cal• eulated for a period starting about an hour after the addi• tion of the inhibitor when the inhibition had reached an approximately constant value. As can be seen from the figure the inhibition is almost maximal st a malonate con• centration of 0.002 M. Thia is a low figure and probably 30 20 -· 10 III, V 60 Time in minutes Fig. 4 120 Effect of malonate respiration of a 2 cc homogenate, 25%, of s. purpuratus eggs in 1/15 M phosphate buffer pH 7.1 Malonate 0.044 M. Cytochrome c 5.xio-5 M. r, II, III cytochrome c added. -51- 90 ~ E3 ~70 ' (\J 0 1 50 ' - - - - - - - - - - - - - - - - - - , - - - - - - - , , - - ~ - - ' 20 x 10-4 M 80 x 10-4 M Malonate concentration \ Fig. 5 Effect of malonate on respiration of 2 cc of 25% homogenate of s. purRuratu~ eggs in 1/15 M phosphate buffer, pH 7.1. indicates a low concentration of succinate in the homo• gen.ate. S:l.n.ee the endogenous respiration at least in homogen• ;;1 tos is malonate sensitive it is to be expected that t.'l-1e respiration passes through succinic dehjrclrogenase. Succi.n- a te r;hould t hen be metabolized by the homoge.nata. I?ig. 6 s hows that t he addition of both suooinate and cytochrome singly and together results in a greatly increased :respi:rat1on. The increase above that of t..11.e control on the addition of both substances together is greater than that due to each singly .• This indicates that the electron · transfer from succinie dehydrogenase passes thr01.Jgh the cytochrome system, which is of course the ease in. most other organisms studied. The typical succinic dehydrogenase does not require a dialyzable eoenzyme, That this is the case also for the sea urchin egg is s.hown in Fig. 7. A short period of dia• l~rsis greatly reduces the endogenous respirat'i on of the homogenate but the ability to oxidize suceinate is unim• paired. It was originally noticed by Keilin and Hartree (194-0) that the activity of their preparations of succinic dohydro• genase made from weshed heart muscle was 1.nh.ibi ted by coenzym~ I. 't his was shotm to be due to the f orn1a tion ot oxaloacetic acid from succinio. Coenzym.e I is required for the dehydrogenation of malic to oxaloacetic. To see if such an effect would occur in egg homogenates a homogenate -53- I 150 50 60 20 Time in minutes Fig. 6 Effect of cytochrome c and succinate on the respiration of a 25% homogenate of s. purpuratus eggs in 1/15 M phosphate buffer, pH 7.1. I - succinate, 0.02 M cytochrome ✓,i:: X 10 -5 M. II - succinate 0.02 M. . III - cytochrome c 5 x 10-5 M. IV - no additions. -54- I 200 1 100 20 Time in minutes Fig. 60 7 Effect of di'alysis on succinic dehydrogenase. Dialyzed against 1/15 M phosphate buffer, pH 7.1 2 hours in cold. Controls kept cold for same period. Homogenate of s. purpurlatus eg gs, 2~. Cytochrome c added to all vessels, 5 X 10-·· M. I, III succinate 0. 005 M. II, IV no additions. III, IV dialyzed, I, II controls . • -55was dialyzed in the col<i .for 20 hours. Snccinate and cyto- chrome e were then added and the effect of coenzyma I on the • :respiration observed. no inhibition. Ar; car1 be seen from li':tg. 8 there is Since, as shown below, ma.lie dehydrogenase exists in the homogenate the lr1ck of inhibition is presumably due to inactivation of some stops leading to oxaloacetic acid by dialysis or to a rapid destruction of any oxaloacetate formed. The addition of eitrate or of alpha ketoglutarate causes an increase in the oxygen uptake. The magnitude of the increase v~ri~s from one preparation to another. Besides inereasine the oxyeen uptake the respirat1.on proceeds w1 thout mueh fall in the Pate. In the controls there is a tendency tor the respiration to fall over the period of the experiments. This fall varies with diff'erent homogenates even if they are prepared in what seems to be an identical manner. Fig. 9 shows the :rosp:l.ration in the presence of added citrate a..vid alpha ketoglutarata. 1-wo different types of alpha ketoglutaric oxidases have been described. Ochoa (l.944) prepared an enzyme from cat heart muscle that required orthophosphate, ATP and Mg ions for act:1 vi ty. On the other hand Stumpf, Zarudnaya a.nd Green (1947) made e similar preparation from pigeon breast muscle r:md found that 1t did not roqu1re any of the above substances. \\'nether such differences a.re an intrinsic property of the ei1.zymes or depend on such things as the physical state or the prote:Ln is not clear. At any rate the enzyme in sea -56- 60 C\J 0 (V) ~ 20 15 Time in minutes 60 Fig. 8 Effect of Co I on succinic dehydrogenase 2.0 cc of 25% homogenate of St pur~uratus egg, dialyzed 20 hours agains 1/1 M phosphate bµffer, pH 7.1. Succinate - 0.01 M Cytochrome c - 2 x 10-5 I O. 5 mg Co I. II No addition. -57- 200 ('\J 0 1 Citrate I lphaketoglyta1ate II 100 20 Time in minutes 60 Fig. 9 Effect of citrate and alphaketoglutarate on the respiration of 2.0 cc of a 20% homogenate of s. ~urpurat1:!§. eggs in 1/15 M phosphate buffer, pH .1. Cytochrome c 5 x 10-5 M. Citrate - 0.015 M. • Alphaketoglutarate - 0.023 M. •58• urchin eggs reselubles pigeon breast muscle more than eat heart. Fig. 10 shows that the addition ot rag and or a boiled musele extract prepared from .rats does not stimu• late the oxidation ot alphaketoglutarate but rather inhibits it. These effects have not been studied further. Using the same homogenates and the Thunberg technique it was found that malic dehydrogenase is present. An ex• ample of such an experiment is presented below. Cyanide was added to trap the oxaloaeetate formed. The eomposition of the tubes was as followst l cc of 25~ homogenate, 1 cc phosphate but.fer, 0.5 cc of l/4000 methylene blue solution and 0.5 cc of 0.1 M KON. arms l co of 0.08 M ma.late. In the side Incubation at 240 o. The ti.me tor deeolorization was Control With malate > 120 min. 40 min. As will be shown later 1n the discussion of .the cyto• chrome system it is possible to precipitate the particles carey1n~ cytochromes a and b by the simple prooeedure ot bringing the pH or. a homogenized suspension of egg material down to 6.o. Since the tricarboxylic acid cycle enzymes occur in the insoluble material such precipitates were resuspended 1n buffer and tested for citric and suceinic dehydrogenase activity. Even with addition of cytochrome c and boiled muscle extract the preparations were completely inactive. blue. Likewise there was no activity toward methylene -59- I II 300 0 III N IV 200 V 1 10 45 90 Time in minutes Fig. 10 20% homogenate, 2.8 cc of s. purpuratus eggs in 1/15 M phosphate buffer, pH 7.1. I 2 x 10-5 cytochrome c, 0.007 M alphaketoglutarate II as I, and boiled muscle extract. III as II and 0.02 M MgCl IV 2 x 10-, M cytochrome 2 c. V 2 x 10-5 M cytochrome c, 0.02 M MgCl2 and boiled muscle extract ..6o- Wh1le this work was in progress t he report of Crane and Keltch (1949) on the metabolism of intermediates of t he tricarboxylie acid cyole appeared. Lindberg (1943) had previously noted t hat fumaric arid malic acids are meta• boli.zed b }T e gg homogenates• Together with the inhibition of respiration by malonate reported here this constitutes an indioation that the tricarboxylic ac1d cycle functions 1n sea urchin eggs. 1.h~.,_(~;zt&<mt.2.m.e ~YS,tem Spectroscopic examination of thief{ suspensions of eggs of both 1!,_ J2W.P.1?.Atu,.~ and bi• .n,;\qt;u§! using a hand spectro- scope reYea.1 rather weak absorption bands at approximately 565 and 695· millimiorons. cytochrome a and b. 'I'his corresponds to the bands or In view of the disagreement that has e::dsted as to the presence or ubsonea of cytoehromes in seo. urchin eces a more detailed study of the cytochrome system was attomptod. Cytochrome c is a well definod substance of known pro• pe:rtiris, but the other cytoehromes aro fol" practical purposes merely names for absorption bands in the spectra of tissues. An attempt was therefore made to extract cytochrome c from eggs; t his however was not successful. No spectroscopic En.r1donco for cytochrome e could be discovered• This t however t car~~ot be taken as evidenco against its existence for two re~sons. i3 In many tissues the concentration of cytoohrom~ c very low (Drabk:tn 9 1950), this being @specially striking 1n embeyooie tissue and tumors. Its e:ristence there has been demonstrated by extraction proceedures. It has been shown by Keilin and Ha:rtree (1940) that even ver:r low eoncentr-~tions of cytochrome c may be sufficient, as the tLtrnover m.llJlber of the enrj'me in ~.rivo is greater than that of the same substance added in vitro. This is presumably due to the organization of the particles 011 which tho other iron por-phyrin enzymes and preswnably cytochrome care located. The high turnover rate of cytoch:r•ome c is perhaps interpretable in the light of the vuJrk on its st1"l1cture by Theorall(l.941), an.d Horeoker an.d Kornberg (1946) who give reason to believe that the iron are stti'ficient to pi"trve:nt access of CO · and CN to the iron i.'tom,. ·rhus presumably cytochrome c doGs not act by aetu.al cont~.1ct of !Jubst:rate vd th the i1·on atom, but the iron a.tom u. nde:r.>goes change of valence state 11 gai~ing and losing eloc• trona throu.gh the peptide chairh Tbis probable fact has been mHdo the basis of a new hypothesis ot enzyme action r®ctmtly (Ge1s$man, 1949) • The rate of turnover of eyto- chrome o could thus be high and a eon.side:rabla :respiration . could itndst without mu.eh of the enzyme be:L"'.'lg present. The ar,1ou.nt of cytochrome c varies greatly in different tissues (Drabk:in-, 1950). '1:h~ a'bso:rption b&nd of cytochrome e is at about milllmiarons. 550 In sian urchin eggs strong general absorption thra t 1 t t1ould aJ!':is t in fair cono@n tra tion and still not be visible ~pectrosoopically. Att(p1npt.a!l to extract oytoch.rome e ware made in several diffe;ren't ways, s t~r•ting with the s tiand&.:rd proaeooure of Keilin and Ha:rtree (193'1) used in preparing cytochrome c on tations w1 t;h trichlo:raeetic acid and aL'1:taon11.:un sulfate. A large volume of eggs of h.JUY:~rt;~lm (460 ce) was used as -63-- with acid to remove t he jelly coat and stored in the deep freeze. Uo trace of cytochrome c could be detected L'l the fraetion in which 1t should have precipitated. An attempt WQs r.:mde to use the method or Rosenthal and Drabkin (1943) espeeially adapted for extraction of cyto• chrome e in small amounts of tissue. stored eggs wore used. 170 cc of frozen Again no yield was obtained. The author s of t his metl"lOd point out however that mucoproteins, in which the eggs are rich, interfere with the separation. Since it was found that commercial preparations of cytochrome care absorbed strongly by alumina, an egg extract wns prepared by heating e ggs with dilute acetic acid for one 1:our at so° C• This method of extraction has also been used by Keilin and Hartroe (1937). The extract waa filtered and dialyzed against phosphate buffer, pH 7 and then run through an alumina column. No cytochrome was obtained in this way. Various methods based on absorption on Baso4 likewise yielded no cytochrome e. Cytochrome c cannot, then, be pre- sent in large amo\k~ts in the eggs. Proparations frozen in liquid air failed to show the expected absorption band of cytochrome c. While no direct evidence for the presence of cytochrome c 1n eggs was obtained, it is nevertheless probably present. The presence or a cytochrome oxidase, perhaps identical with the cytochrome a (or a3) here domonstrated to be present in the egg , has been knmm for a considerable time. Fig. 11 confirms earlier results of Krahl ot.al. (1941) that an -64- 300 200 Cytochrome c added 100 0 ~ 0 -0- 0 60 Time in minutes v· ,_< 1g. -0 120 11 Oxidation of cytochrome c by 1 cc of 15% homogenate of s. purpuratus eggs in 1/15 M phosphate buffer, pH 7.1 Top curve - with 1.3 x 10- 4 M cytochrome c and 0.02 M paraphenylenediamine. Bottom curve - with 0.02 M paraphenylenediamine. enzyme capable of oxidizing cytoah.l'ome c is prese11t in eggs. That the respiration of fertilized eggs 1a inhibited by eya• n1c1e has been known since the early work of Warburg (1909). Robbio (1946) has recently show.11 that previous reports th.at unfertilized eggs are cyanide insensitive were based on technical errors• The only t-mown substrate for cytochrome oxidase is cytochrome c. Probably therefore the normal respiration proceeds in part at least through cytochrome e. Dimethylparaphenylenediam1ne is oxidized by living systems and its oxidation is believed to proceed through cytochrome e. Hunnstrom (1932) has shown, as mentioned i..'1 tho litcra• tu.re sur"vey that the r~spiration of intact oggs increases greatly on addition of dimothylparaphenyl0nooiamine. Beeides possessing n system for o:rldi~ing reduced cytochrome c, eggs are also able to reduce it. The endo€;enous respiration of an egg brei is considerably increased upon addi tio:n of cytochrome Ch• (Fig. 12). A systen1 that spocifi• eally requires cytochrome o is the succil1ie oxidase system. Fig . 6 shows that addi·tion of cytochrome -c gr,30.tly aoeeler• ates the rate of oxidation of succinate • ..\s mentioned above, eytochromes a and b are character• ized chieny by the positions of' their absorption bands . Ba.~ds corresponding to su.eb components al'8 observable in suspensions and bre1s of the eggs of both~, RW:R11£&9M! and 1u pigtJ.Ul after addition of hydrosulfi te • The b eornponent is clearly visible, but the a is vory fa!..11t. On freezing the preparntion :.i..n liquid ai1• both bands, but especially n -66- 80 0 30 Time in minutes 60 Fig. 12 Effect of cytochrome c (4 x 10-5 M) on endogenous respiration of a 25% homogenate of s. purpuratus egg. Bottom curve - no additions. Top curve - with cytochrome c. -67becomes mueh more distinct. A major dif'fitrnlty in their study is that the solutions are turbid and therefore cannot be studied quantitatively with instruments such as the Beckman spectrophotometer. Attempts to clarity suspensions by the use of detergents or desoxycholeate did materially decrease the turbidity, but not to a sufficient degreo. It was t herefor0 necessary to estimate the strength of the absorption bands visually, a proceedure that is particu.larly unfortunate hei:e, as tha faintness of the bands makes even estimates of the order of raagnitude doubtful. So far as it was possible to judge, fertilization, anaerobiosis, CO and KCN 1n intact eggs or addition of succinate to homogenates did not cause any increase in strength of the absorption bands. Attempts to concentrate the cytochrome fraction of breis was mads in the hope that it would be possible to obtain a solution with sufficiently low turbidity for instrumental study. In all cases thus far described in the literature cytochromes a and bare present on small particles contain• ing numerous other enzymes, whose separation without inacti• vation has proved impossible. A possible exception is the recent worlt of Eichel et. al. (1950) • A concentration or this partiC'ulate fraction was attemp• tod using the method of Dattelli and Stern (1912) for preparinG cytoeh.rome oxidase from heart rrn.iscle. Eggs of L. pictus were washed several times in sea vmtor ru1d thsir jolly coats removed by bringing the pH of the sea water to 3.5. Thay were than oentri.fuged and the vo1Ui'1le fmmd to be 3.2 cc. Throughout the rest or tl1e treatment t he preparation was kept ice cold. 20 cc of M/15 phosphate buffer, pII?.15 was added and the eggs broken U!) in a Waring blender. The preparation was then centri• fugt1d for ten minutes at 3000 rpm and l cc of sediment cm:ne down. This fraction on reduotion with hydrosttlfita did not show any absorption bands. The cloudy yellow super- natant was adjusted to pH 6 with dilute acetic acid. precipitate formed at once. A This was centrifuged again for ten minutes nt 3000 rpm. yielding 1 coot precipitate. On reduction this precipitate clearly showed bands correspond• ing to cytoehromes a and b. Further acid1f1cat1on of the superna tant to pH 5 gave another precipitate, which however showed only a very faint b band. A considerable eoneent.ra• tion of cytochromes a ond b thus appears feasible. That the cytoohromes are really on particles is shown by a centrifugation experiment. Seven cc of §1 purp,gatl!l eggs, washed and jelly coats removed at pH 3.5 were suspended in 13 cc of M/15 phosphate buffer pH 7.1; and homogenized. The very viscous preparation thus obtained was centrifuged in a refrigerated centrifuge w1th a high speed head at 10,000 g., the sediment resuspended and reeentrituged. sediment shows strong a and b bands. The The preparation can be considerably clarified by add1t10.i~ of taurocholeate. This same preparation, acidified to pli 6 precipitates at once. When tested \W:lth methelene blue, however, it shows no sue• cinio dehydrogenaee activity. The bands indicate that the cytoohromes gu•e in a reduced state, even after shaking 1n a.1r, and addition of succinate does not seern to intensify them. Although the physiolo~ical reduction and oxidation of the a and b bands could not be demonstrated, evidence was obtairled that they ar-e probably iron porphyrins linked to proteins. Cytochrome c is relatively heat stable. The a and b bands disappear when the homogenate is subjected to conditions that der..ature proteins. destroys the bands at once. One minute at 100° C i\ddi tion of orga..111c aolvonts such as methanol ca.uses the hands to fade out g1•adually. I.f after strnh treatr:1ent pyridine is added to the preparation, a very strong absorption band at 555 due to the pyridine homochromogen appears. This indicates that treatment has liberated the prosthetic groups from protein. Small rur10unts of hydrogen peroxide destroy the cytochromss irroversibly. The band a cytochromos saem to be eutooxidiza.ble at a low pH. If a lyophilized preparation of the eggs of • I.• pigtlJ§! is suspended in water, tha a 'and b bands can be observed at least partially reduced. If the pH is brought down to 2.6 the bands disappear and do not :reappear if the pH is readjusted to 6.o. That the cytoehromes are not destroyed, but only oxidized, is shown by adding hydrosulrite to the preparation which causes the bands to booome clearly vis:tbla again. -10Tlt(z,_]\,.is8 ,in tho 1les21ra~ory Re.te d~1,.r,t&J;srll':. Q.eVeM2nm...~l!!i t It is a well lmo\m fact that after fel\iliza tion the respiration of saa urchi...11 embryos rises during cleavage, t he rate following an s -shaped curve until hatching. At 22° C this ta ~:es about eight to nine hours in the case of tion is shovm in Fig. 13 o The riso has been froquantly commented on and various explanations advanced; it is usually s tatad t hat en .z:1,1rnes are bo:lnc synthesized during this period. On t he other hand, t here is a conce pt that cell division and d1fi'erentiation which rw.r include synthe• sis of new cell material, are mutually exclusive. This is obviously not tr·lle in the case of desoXj,Tibonueleic acid, which has boen clearly shown first by Brachet (1933) a.nd t hen by other workers to L"'lcrease linearly during early development in the case of the sen urchin. At least some t ype of synthesis of cell constituents is thus possible during the cleavage per:tod. or course such syntheses may be lim:lted · to the intercleavage periods, but as yet the problem has been scarcely touched. There arc several possible interpretations of the increase in the re.to of resp ira t:1.on during clcavaeo. Pirst, it :night be related ill some as yet undefined wny to the increase L"'l dosoxyr1bonuo. leic acid. It is clearly not directly proportional to the nur:iber of nuclei, as was first shown by Warburg {1910a) and discussed at length by Needham (1931). As Brachet {1938) showed , KCl treated embryos which un.dergo a certain amou.'.flt -71- 60 - - - - - - - - - - - - - - - - - - - - - , 40 a 0 ~C\J 0 1 • 2 6 4 8 Time in hours from fertilization Fig. 13 Respiratory rate during early development of L. pictus. Light circles - in sea water. Dark circles - in sea water with 0.03 M glyclyglycine buffer, pH 8.1. -72• o.f differentiation without cloavage (Lillie, 1902) show a slower rise in respiratory rate. Such embryos also show a slower rise in content of desoxy1?ibonucleic acid (Brachet, 1938) 9 eorrela ted no doubt with the abnorrr.ial and ra. ther obscure nuclear divisions that occur. It is also true that since respiratory rate rises as an S•shaped curve while deso.xyribonuclaic acid increases linearly, the respiratory rate is also not a linear function of desoxyribonucleic acid content. It is of course possible that the two are linked even if the respiratory rate is not linearly related to desoxyribonucleic acid content. A second possibility, preferoo by Heed.ham (1942) is that respiratory enzymes increase in amount during this period. Even if this is true, it by no means follows that an increase in enzyme eoncentration would or itself produce an increase 1n metabolism. Most tissues of the body have amounts of enzymes greatly in excess or that needed to maintain th.e metabolism which is normally observed. The classical example is of course muscle, but secretory cells, nerves and almos·t ar:i:;i;· other tissues could serve as examples. There exist mechanisms which control the metabolic rate independent of enzyme concentrations, providing of coursa that thase are adequate. This will be discussed shortly. It is possible th.at substrate could be 'becomir1g more available. This does not seem very plausible on general grounds but is difficult to disprove. An interesting possibility has been suggested by Szent - Gyorr;yi (1948). Ee points out that at lca3t some of the energy producing reactiori..s are obligatorally coupled to phosphoryle,tions and therefore their rato depends on the presence of phosphate acceptors. lt is very probable that t his is a general phenomenon, the dissociation of respiration and phosphorylation that can be observed in homogenates being an artifact. Since the so called hir;h energy phos• phates are now believed on rather plausible r,rounds to be ' the only methods of triu1s~erx,ng energy from the energy yield• ins to thE1 energy us:tne react tons, it is elea1• that actj ... vation in any manner of energy requiring reactions increnses t he concentration of phosphate acceptor and automatically nctivates the respira.tm.•y mechantsms. This hypothesis appears very attractive because it invok~s 0013:r known mech• anisms and because of its s:L'np11ci ty. In a sense, of course, it only pushes tho problem a ·stop .further back, the reason fo~ the act'.'1.vetion of phosphate acc<'.3ptor bein-e now the prohleo to be explaL11ed. Here, ho'!:ever, there is a model provided by actornyosin, which is an adenosintr.iphosphatasa in the contracted, but not in the ex-tended state. There are several possible consequences of this hypotl!esis that are 1n pr:tnciple subject to experimental test, rtll howev('3r i nvolving ccrttdn tu-rnumptions. a stud y of the Pastm.u" affect. Ono is th.rough It is now becomir1.g more 1videly believed tha t the Pasteur effect is due to t he fact respi:cation than in tha glycolysis (Johnson, M. J., 1949) • Oxygen th(,3refore suppresses glycolysis by decreasing the concentration of ADP in the glyoolytic cycle. The evidence for such a m~3e1:i..unism, while indirect, is rather conv.1.ne1ng. ! f th.ore is indeed an increase of phosphate acceptors during cleavaee, th01'"0 should be a decrease of the Pasteur ei'feot, that is, aerobic clyeolysis should increase. This idea 'I'"' occu.1.Aed too late to be tested, but there t.u•e several papers showing :in the case oi' the frog a significant .increase of aerobic clyoolysis during developraentct (For discussion see Drachet, 1947 •) Anothar possibility is to study the effects of arsenate, a competitive analogue or phosphate. If the respiration is inda~d ocmtrolled by tb.e concentration of phosphate acceptor, a sui:'f:teie:nt replaeemen.t of phosph.i,te by arsenate should br.i ng the respirat:lon at all stages of cleavage ·to a co1:art1on levt:1. An attempt was therefore made to study the effect or arsenate on respiration,. A ttmnpts WQre rnade to introduc<0 e,rsena te into the i..~- to.ct emz;s. Whether the arsenate penetrated into 'the eggs was not di:rectly determined, but at any rate its in.fluence on t he :respiration of th(B e ggs proved slic;ht. were done on the eggs of ~ k ~ • Experiments First an attempt was n1ade to introduee arsenate into tmfartilir~oo eggs by soak- ing t;hern in an mrsenete solution, removing them to sea 11ater and ob.serving the effecto of the treatment on the respira• terry rise on fertili!'.ation. Fertili2e.tion in arsenate -75solutions is not possible due to the precipitation of the calc:tumo The details of the procedure are given below. ·r o was hed unf ert111zed eggs one fourth of the volume of isotonic arsenate solution~ 0.37 I:1 pH 8.1 was added to [~ive a 0.09 M solution of arsenate in sea water. Actually, of cour~e, the molarity was less due to the low solubility of calcium and magnesium a:rsenates w1hich caused a preci• pi ta te to f'orm. The eggs ~-eere allowed to stand in test t ubem in 'this solution, the controls in o:rdinary sea water for 45 minutes, with occasional shaking. The eggs were then transfered to manometer vessels, the appropriate samples f ertilized by the addition of a drop o.f sperm into the vessels and the I"(?Sp:ira tion men sured.. course of the :respirationo were exarrd.~ 11.ad. 1~'.1.g. 14 shows the At the end of the run the eggs About 50% of ti-1.e egen were fertilized and had l"'eachecl the 2..-4 cell stage. Tho arsenate apparently had no significant 5.nf'luence on the un.fe:rtilized or ferti• 1..izod eggs. itepeti tion of the experiment gave essentially the same result. Experiments were also done suspending fertilized eggs for periods up to four hours in arsenate solutions (0.05 M) o.nd measuring their respiratione tion was found. No effect on the rcspira• More significant is the fa.ct that the expr1rimonts showad the. t the eggs went through 4 to 5 claav• age s both in the arsenatt::) and in the controls. Since it is known, as will be mentioned shortly, that agents uncoupling -76- 80 40 120 60 'l'ime in minutes Fig. 14 Effect of treating eggs with 0.09 M arsenate before fertilization. Details in text. Dark circles - arsenate. Light circles - no arsenate. I, II - Fertilized. III, IV - Unfertilized. bnbly due to pormeaL:tlity nonsiderations. Another method of' studying the effect of pi:msphory.... lations in controlline tho respiratory rate is to use agents such aeents are known, the rrt0st ep~?ct&enlar probably being the dinltrophenols nnd !"elated compo1,.1nds. .A ·very consider- Clowes and Krahl ( 1934) have st't.Jdied the effects of substit• U.(:rn.ts on physiologicsJ. activity, Tyler ilnc1 Horowit;a (19.37) have discussed the phirsioloeically active form, of these compcn.1nds • It is now Hospira tion in rnan.y easoz is greatly pLo~phatE1 ie; supp:rer,£1$de An tcixplanation oi' these two effects h.as bE:en advt:rnr?ed by Loomis and Li:pmarn1 ( 194 8) • According to t.h@ir hypothesis, th,J'J dini trophenols in so.me fashion If such 5. s indeed the case, dinitrophonol i:H·rnomas a vnluab1e tool fr..n~ the ~tudy of trm nature of the rise in of DNP on the respiratory :rate shortly after fartilization n.nd at about eight hours at which time the normal rate has approxLna tely double;~. The experiments were eond1).cted at the K.erckhof.f ttarine Laboratory, using the eggs of k...t... pJ~• Since i t has 'been shown that the effect of pH on the physiological activity of din.:i.t:rophenols is very great, it was neccessary to use a buffer in the sea vmter. The most suitable appeared to be) glycylglycine ('1:yler and Horowitz, 1937) • Fig. 13 shows trmt the regpiration of developing embryos is not adversly aff' {)cted by ttte presence of glyeyle;lycine, 'rl1e embryos in fact develop somewhat better in it th&n do the controls. ;fhis is probably not a r;peoific eff<act of glycylglyeine but :rathe-u· due to the rnaintru1a1:1ca of a constant pH in the mano- meters. 'l'his is especially important since the main buffer of sea water is bicarbonate and thtd; of com·se, is removed by the alkali in the center wall. Another important considera'!.;ion is the \'::oncentration of dinitrophenol. Since i t was desired to meas1a,.e 'the maximum capacity o:f the respiratory system of the mnbryos, it was neccessary to use the amount giving the max:imum stirirulation. On the othr::1• hrrna, too l;n-•c;e a concentration. produces inhi• bltion, and lesser amounts, while 1"aising the respiration to a high level, causes a rapid decline in the respiratory l"'rzte after a s}1ort tlme. Af•te:r some preliminary experiments, it was decided to use 'two different co:ncentratlons fand CQm• pare tho results. 'f he experimental proceedings woro t he follo wlng o Bge:s wore rernov,.:1d from the anlmals and washed several t.imes • 'lhey were tl'len .t"erti11zed and pl~ced in u1anometm"'s. . composition in the manometers was as foll.o·us: The 3.06 cc of 0.05 M glycylglycine buffer, pH 8.l 1n sea water, 1. 36 co egg suspension. NaOH 0.3 oc in center v;oll, 5%, Either 1.0 or 0.5 cc of 0.001 14 d1n1t:rophenol in soa water ·was added to -the side arm. bath t'ms 22° c. The terrrper&tur~ o.r the ma.nomet;er Slnoa this was slightly below room -temper• a ture, a cooling arrangement v;as needed" The mm1or1letisrs "ll<Voro shaken at the rate of 84 strok@s per minute, with an amplitude of 7. 5 cm. A separate experiment showed tht,t the 01.:ygen uptake was not limited by the rate of shaking. ings were begun about an hour after fartilitation. Read- Din1tro• phenol was ti.pped in at tho beginning of the readings and also into other- vessels seven hours later to dotert,i:J.nc the rate of rospirntion after eight hm.:cr-s of development._ The contents of the vessels were examined at the end of the rm1s. It was found that the development was verr good. :reached was t hat just before hatching. The stage The c,onc!ent:ra tions of dinitrophenol used was such as to stop t he develop01ent of mos t of tho eggs. A few di<'.i, however, go throu..gh one or t ·wo cleavages after the addition of the reagent. Tho results a:re sriown in Table V• In sp1 te of all p1•0- cautions th.e re is some variation, bu.t on t he whole the re- sults appear to be reasonably reproducible. They show quite Table V Experiment I 1 III IV 100 II 193 362 495 ½§8 ffi m ~ 3 100 200 434 618 4 100 216 477 5'91 ✓ e:~ 100 195' 45]. 578 6 100 174 394 622 ~ m * ~ 2 7 mean mean Effect of 2 1 4,dinitrophenol on the oxygen uptake of embryos of 2-, purpurat:q§ at one hour and eight hours after fertilization. Rate at one hour expressed as 100. Temper• atu.ra 22° c. Experiments 1,2 - dinitrophGnol concentration 1.1x10-4 M. F..xperiments 3• 7 • dinitrophonol concentration 2 x 10-4 ~. Columnsa I • rate at one hour. eight hours. III - rate at one hour with dinitrophenol. IV .. rate at e.i.ght hours with dinitrophenol. II• rate at -81• clearly that not only does the unstimulated respiration about double through the period of eight hours, but that the dinitrophenol stimula.t(id respiration also rises. The absolute rise of the latter is somewhat greater than the normal in• crease of respiration. 'rhe interpretation of this ph@nomenon is difficult. Taking the results at face value, one can say that the total capacity of the respiratory system of the embryo increases somewhat more rapidly than the rise in respiration, This shou.ld be the interpretation if the Lipmann and Loomis ( 1948) explanation of the dinitrophenol effect is correct. Unfortunately the roal mode of action of dinitrophenol is not entirely clear. Quite recently Lardy (1950) reports that in the presence of dinitrophanol a very labile phosphate ester accunmlates. yot known. The nature of this aster is not There are reasons to suppose that the action of dinitrophenol may be compl-ex. At an~r rate the data presented above demonstrate that in some sense there 1s a considerable increase in the capacity of the respiratory system during the first eight hours of development, and that this increase in capacity parallels the increase in respiration. This could be• as mentioned above, because of an increase in the availability of sub• strnte or it could be because of enzyme synthesis in some sense. To test the possibility of enzyme synthesis eggs and eight hour embryos of Lt pigty were lyophilized and stored in the de ep freeze. An assay for aldolasc was then done by the method previously de scribed. The act i vit~r of air;ht hour Embryos i n t l Jis enzyme 1nas found to bo so :nuch lower t han that cf t he e m~ s t ha t it wa s con cluded t l1r1 t this was either due to a lesser stability of t heir enzyma or else through 1naclvartanee t he 1yophi11z&d powder had been inactiva ted in some way. 'l'he attempt to measure a.cti vi ty of enzy:nes is i1 dif• ticult approach. It is neccessary to test a lar[;a number of enzyme systems, to determine their optinn.:un eondi tions for assay and to check ott all poss1blo s ources of error, which are many. It would appear better to start such wo:rk bv ., doter.mining the grosser chan.ges in the protein composition by some method such as electrophoresis or saltine; out, and after determining t his to attempt u correlation vii t h enzym• atic activity. The problem of the rise of respiration is of very real interest, since 1 t is a general phenor;1enon, unlike t he rise on fertilization, an.d because many embryologically i mportant processes are t hen taking place. An undarstandine of this phenomenon might t hrow light on several problems now obscure. Discussion The results reported above w~ke it probable that the respiration of the sea urchin egr; proceeds through the same pathway as that known L11 adult oreanisms. As the sur,.rey of • the 11 tera ture shows t this has been doubted 1.1y a large m.1m• ber of emb1lyologists. It may be worth while to examine some of the evidence which has been gathered in support of the latter view • . A large part of it is of a purely negative kind, based on the inability to demonstrate some enzymatic process. evidence should be treated with caution. Such For example the absence of the cytochromes has been frequently reported and some significance has been attached to this supposed fact. The work of Rothschild (1949) and t he work reported in this thesis demonstrate the existence of the band a oom• ponents. 'l'he c component has not as yet beon found, but obviously the conclusion cannot b8 dra.,m that therefore it does not exist. Lindborr; and Ernster {1948) reported that no triosephosphate is formed by homogenates on addition of hexoso• diphosphate. It was also found that there was no inhibition or respiration by iodoacetate. That aldoluse is present in e ggs has been clearl y s hown in this thesis by the use of a me t hod nuparior to t b.e one available to Lindberg and Ernster• '1:he qu.estion of the inhibition by iodoacetate has baeri clari.fiod to some extent by the demonstration that inh.i.,. bition devt1lops rather slowly. This must havo been even more true in the experim~nts of Lindberg and Ernster, since they worked at a temp@ratura of only 6° c. Di:f'ferenc(i)S in penetrat1m'J. and rate of reaction with proteir1s and glutathione oan probably explain most or the anom.olous results obtained by some investigators with iodo• Groat stress is laid by the Swedish school on the sim- ilarities between the decnrbor.rlative breakdown or glucose demonstrated by Dickens in 1938 and the respiratory m0ch• ru:i.isms fom1d in sea Ul"ehin eggs. The evidence bearing on this point has been referroo to in the literature st1rvey. O'or a recant expression or opinion see Runn~trom, 1949.) The following points however should be noted. Part of the evidence l"'ests on failure to demonstrate certain reactions • .Some of thesa reactions have now been sho·wn to take place. The proposed decarboxylative breakdown of sugar has not actually been demonstl"a ted in the sea urchin egg. r~ven if it were, 1 t is well ki.·io'Wn th.at many tissues whj.ch oan oxidize elucose-6-phosph.'a te to 6-phosphogluconic acid utlliae the Meyerhof glycolytic mechanism. Therefore it is :reasonable to conclude that there is no real evidence that oxid~tive decarboxyla tlon is the main l"Ospira tory pathway of the ,;;ea. The <iecarboxyla tion of 6•phoaphoglucon1c acid leads ultimately to the production of a pontose. Since pantoses -858,re components of nucleic acids and nucleic aoids have soma as yet not clearly defined role in grov1th i t is ve:ry conce1• vable that the ogg may havn a well developed system for the productlon of pentoses . Thi.s however is not a subject to be decided by speculntion but by expez iment. 4 The two points to determine are first how pentoses are produced and incorporated 1.nto nueleio acids and second hm1 well developed sueh e. system is :tn the egg. It must not be forgotten th~t tho gly.colyt1c and respirn tory ~ys tems produco not only one:•gy but also structural materlals and intermediates for other metabolic systems. It ha.s been demonstrated in the c~xperimental part of this thesis with some probability that glueose-1... phospha'te is transformed to glueose-6-ptiosphate. Glucoae-6•phosp~'lte and fructose-6-phosphate ar0 interconvertible. 1,6,rrueto• sediphosphate is broken down to triosephosphate. Rospir• ation is :i.nhibited by iodoacetate. Phosphoglyoeric acid is converted to pyruvate. ' I?luoricle inhibits this rcs.ction and also the respiration. anaerobic conditions. Lactic acid accuumla tes u.nder Citrate, alptm. l,;: ~to glutarate, suc- cinate and nm.late i~.re metabolized and re};pirution is in,hibited by malonate. C~rtochro'!"!1e c is both reduced and oxidized~ 'fho presence of cytochromes b ancl a :ls est- alll1.shed. Other work r..mntioned. 1.n the literature, survey has shown the presence of cytochrome oxidase and i.nhlbi t:ton oi' .respira •tion by cyanide and carbon monoxide. lt S(~Hams probable that the rezpiration of sea urc1'.1in eggs is ::iimiliur to that well knom1 in adu1 t organisms .. system then does not o.rJ.se anew in c. 0.ch o:£1togeny but is 1 transferred intact through tho egce it must be admitted, of course, that the demonstration or any enzymatic system is rwt tantamount to showing that 1.t is actually used. This is a. much more difficult problem and the solution or it in an unequivocal fr-r nhion ca.n probably· be donfl only by the use of methods involving labeled compounds 1n the intact organism. Should the point seem of suff icient importance fo:r the solution o:r some problem this could of' course be done. The ide.-1 that the respi:r~tory zystem :ls incomplete :Ln the er.J1bryo :received at one tir~1e a. great :tmpetus from thE'l work: of Needham. For a full disct1ssion of the problem his book (1942) should be consulted. His idea was that the e;lyeolytic sys tom invol vine pi·,cspha tf) arose anew in each ontogeny. Durinc early life itn plac~ was ta 1':sn bye. simpler system f'or the breakdovm of glucose not involvinp, pho~phateo Since it is now genernlly believed th.at energy transfer in. the organism involves phosphate the ex:tstcmce of a. r.) .ucolytic interest. Need.ham supported his idea with nmny expcrim.onts glucose and that of glycogen , wht ch he concluded did t:nvol.ve ptosphate. A favorite reat;ent to inhibit this n.cm.pbophoryG lative glucol;y-s:is was l•glycers.ld.ehydei11 1:he recent vmrk of -87Lardy, W:i.belhaus and Mann (1949) has to soma extant cla.ri• tied the mode of action of this compound. under the influ• ance o.:f aldolase it condansas with pho-sphodihydroxyacetone to form l•sorbose-J-phosphate, a powerful inhibitor of hexo• kinase. If this is lndeed the ease then l•glyceraldehyde, instead of' inhibiting nonphosphorylative glucolysis inhibits the first step involving phosphorylation. Meyerhof and Perdigon took up the problem in 1940 and succeeded in demon• striating the Meyerhof scheme in the chick: embryo. ~ore recently Noviko.ft, Potter and LePege (1948) have isolated th® intermediates of' this scheme from chick embr~1'0S in the expected quantities. It would thus appear that the:re is ri...o reason to believe that embryonic respiration in the chicken utilizes another mechanism than tho adult. The ideas of Needham !ia,re no doubt had a considerablo influence on embryologists wortdng w1 th the sea urchin i..-ri making them interpret any anomolous results in terrus of machanisms not present 1!1. adults. this is not nec¢essary. It would a ppear now that -88. o~ thii~" :fo~rn,.t~~...,gf -·An,~j_n·ter,m"e~3t.ion - - --- · ---xiqJ. =ril~r~~-___ av_G0c,yt~ --------~ 4 -- Pert1ner1t to the problem discn.1ae,M1 above irJ the Ot'.H3er .... vation tha't the meehan1sms for breaking down starch and glycogen ara extraordinarily uniform in the most distantly related orge.nisms. also of plan.ts. This is true not only of ani1nals but The possible e)teeptions are:~ organisms that ha:ve not hem, fully studied, such as tht~ fun.gi , or certain 'bacteria which can be easily int®:rp:retec1 ati hnviing lost part of' their enzyrnatic equipment.. The der.1cnstrt1tlon of such a uniformity can be regarded as one of the main contributions of biochemistry to general biolo~yo The cause of such a uniformity, which h.a.s been found now to extend to many other basic biochemical mecha.n.isms, could be due to the COffi!\lOn o:r!gin of all li v1nt0 things . On the other htmd, an explanation in terms of the limi'ta- tio.ns to which any gj.ven 'biochemical mechanism is sub;}eet should. 'b0 exam:tned • It will be pointt1d out here that tho reactions of gly• colysis aTfJ so in:te:rdependent that not much variation is possibJ.e. It is not possible to show with any complet~ness that no other mechanism than the one that exists is possiblG, but r.:mly that if others can be consi;ructec1 t hey i~m.st ·oe of a radically different nature. i'he glycolytic meeha.nism, let. it be mmnl\ned 'i> has to be ean:lly and generates ATP. The fj_rst point to notice is that a hexose h_.as to be split into a three earbon compound. The only chemical mech• anism that brealrn carbon to carbon bonds in a raversible manner would seem to be an aldol condensation. course what is done by the enzyme aldolase. This is ot The fact that an aldol condensation occurs limits the product (or substrate) to a keto sugar. Several points of interest now present themselves. Assuming ·tha/c we are dealing with hexoses, it might appear that any ketohexose of the d series could be an inter• mediate. Actually this does not seem to be the case. Fischer and Baer (1936) have studied the nonenzymatic con• densation of dihydroxyacetono and d-glyeeraldehyde and dis• covered the unexpectad fact that instead or four ketohexoses only two were obtained in detectable amounts. The eonven• tional structui•al formulae of the four possible ketohexoses of the d series are as follows: HCHOH ... - HCHOH HCHOH C a 0 • - HCHOH 0 HC◊H C 0 HOCH HCOII HCOH HCHOH HCOH HOCH HCOH HCHOH HGHOH HCHOH Psicose Fructose Sorbose Tagatose C HCOH HCOH I'hc two prod·uets were fructose and sorbose. C HOCH HOCH 0 HCOH The structural requ1roment is that the hydroxyls or1 carbon atoms 3 and 4 be trans to each other. r~ow i t is of interest that these ,- two sugars are naturally occu1Kng 11hile psicose and taga.tose are known only as synthetic products. In tho pr~sanoe of aldolase fructose and sorbose can be obtained, but not psicose and tagatose. Thus the enzymatic end nonenzymatic mechanisms are alike in this respect and an explanation is at hand for the exclusion of two otherwise possible inter• mediates from the glycolytic mechanism. Fructose rather than sorbose is the actual interI;1edlate. The reeson for this is not quite clear but seems to be connected with the optical, speeifioity of the enzymes of glycolysis. Aldolass in the presence of phosphodihydro;tyacetone and d•clyceraldehyde forms fructose. It l•glyeeraldehyde is present instead the product is sorbose {Inumea, and !\[~.,_, 1949) • 1937 and Lardy, Wibelhaut•'. 'i:hus the choice of fructose instead of so:rbo.s e would appear to depend on the optical specific1 ty of tr1osephosphate isomerase which f'orms d rather than l•gly• ceraldehyde from dihydroxyacetone. A point or interest, although it may be a minor one, is tLat if' a ttetohe;:;mse is split by the reverse aldol conden• sation the two products are different. They are however the kato and enol forms of the same compound and readily inter• convert even nonenzymatic.ally under mild conditions. Were the sugar a heptose or a pentose this would not be possible. In the case ot a hexose the two products are able to proceed rather directly a.lone ths same metabolic pathway. ?he above discussions show that a ketohexose must be an intermediate in the metabolic scheme. Yet the sugar residues of: s-tarch and glycor;Gn are glucose, an aldohexose. A conversion must oec-J.r and ·t.his explains the necessity of the other steps before the formation of 1,6,fruetosediphospha.te. eie have assumed that we start with slycogen or starch. The degradation of the molecule proceeds, as shown by Cori and Cori (1936), not hyd1'0lytionlly but phosphorolytically. The rationale of this has often been diaoussed 1n connaotion with the reversib:ili ty of the react.ton and net1d not be furthor discussed hers. ?-'rom the structure of glycogen the phosphorolytic split must give us glucose-•l•pho~:::phe,te (on paper at lenst slu.cose•4-pho8phate is also possibl00 The nonanzy!natic phot1p11orolysis of' c:lycogen do~s not appear to have been studl~d and therefore nothlng can be said on the chemist:r:; in-:1olvod here). Glucose•l•phosphate must now be transformed :ln'to fructose. There are threo enzymes involved in this opera tlon. soc. Tho X'fH:1son f'or this :ls not di.fficul t to Unde:r mild ultmline cond:1 tions cluco.'rn, mo.1m.o::50 and frt.H.::tose are tiut1ia11y into1"'ccm.ve:rtiblc. Th:ls if3 bol:Leved to proceed throueh an en-"Jic,l f orm cor'.1".1on to the t .h.x•ee sue;e.rsc 'I.'ht1 meehttnism of th@ enzyy;1atic conversion is not known, but is probably the same. If such is th:D ca:..,e, th1:m obviously r;luoose-l•phosphate cannot be directly trarrnformcd into fru.ctose, since the phosphri te on carbon atom 1 blocks enolizntion. The shift of the phosphate to position t1 11ber,1tes t he one poslt;ion and enoliza.t:ton ean occur, givine ris~ to fructose-6-phos• phate. The further phosphorylation of fructose-6-phosphate is of course connected With the ultimate transfer of the phosphate to ADP. This sort of discussion could be extended to ereater length if desired. The reason for bringing it up at this point is to stress that the organism does not have at its disposal an infinite number of po3sible mechanisms for na tu.rlil seloetion to operate on and therefore the wi.iformt ty of biochemionl mechanisms is not unexpected. riot only are the individual mechanisms subject to a variety ot rastriotions, but probably even more stringent are the requirements of interlocking mechanisms. I"or examr,le 9 the above discussion does not explain why the storaf:e po1fsa• charide 1s usually not a rx~ctosan. This would obviate the necessi f;y or several steps that actually occur in the ,slycolytic mechanism. this as vJell. However, there may be reasons for t3uppose i'or example that galactose is needed by some other metabolic system. Galaotose is synthesized by conversion of gluoose•l•phosphate to galactose•l•phos• phateo A route from fructose to glucose•l•phosphate may then have to exist in any ease due to a requirement for galactose. , Our knowledge of biochemistry is as yet alight, but it is enough to make us suspect that reasonable explan• at1ons based on sound chemical principles may eventually be found for the existence of certain systems and for the absence of others. With the IOXCt,ption of the viruses all livlni organi::;::is while a necessary condition of embryonic developraent , has no direct f,ffoct on t he course of growth e..n.d d:Lffcrent::L::. t ion. This oonclu.sion, howevar, i.s too sweeping. l'u-o aspect s of the study of respiration woult! soem to be of dirc·ct embryoThe problems of gr.owtJ~ nnd differentiation have proved phenomena arc the ones to study for tho solution of -these to the problem ,:nay yi;;3l<l re~n::1 ts or interest. It must also not bo forgotten t hat re$tJirntlo:'1~ 't;osides • providing energy for t rHs orcanism , cnn have other f unct:1.ons. As has been cler1r ly sho-,m by t h1 use of isotop:tc J lly labeled compounds, the inter:nodin te:,, of the retJpirntory process efm provide a variety of s tr·L1 ctural ma ter ia1s for other biochem• • ical systens., Changes 5.n the rospir0.tion alone co1,ld d.is t'i..lr'b the ste,1dy state of the organi sm, with resu:!.ts somot:hnes putholocical but perhaps also producinc some of the normal. -94phenomena of development. Whether this is indeed the case will be decided by investigations that have yet to be carried out. •95• Bibl io graphy Ashbel, R. (1929) Boll. Soc . 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