Studies on the Source of Urea Carbon Citation Paigen, Kenneth (1951) Studies on the Source of Urea Carbon. Dissertation (Ph.D.), California Institute of Technology. doi:10.7907/WV7D-FW20. https://resolver.caltech.edu/CaltechTHESIS:09072017-142857992 Abstract A method has been devised for the testing of any substance as an intermediate in a reaction sequence. Using this technique, thirty-three substances have been tested as precursors of the urea carbon atom. Of the compounds tested, only citrulline and arginine were precursors. Garbamyl L-glutamic acid was shown not to be a donor of the urea carbon, although it does have some function in urea synthesis. Item Type: Thesis (Dissertation (Ph.D.)) Subject Keywords: (Biochemistry and Chemistry) Degree Grantor: California Institute of Technology Division: Biology Major Option: Biochemistry Minor Option: Chemistry Thesis Availability: Public (worldwide access) Research Advisor(s): Borsook, Henry Thesis Committee: Unknown, Unknown Defense Date: 1 January 1951 Record Number: CaltechTHESIS:09072017-142857992 Persistent URL: https://resolver.caltech.edu/CaltechTHESIS:09072017-142857992 DOI: 10.7907/WV7D-FW20 Default Usage Policy: No commercial reproduction, distribution, display or performance rights in this work are provided. ID Code: 10419 Collection: CaltechTHESIS Deposited By: Benjamin Perez Deposited On: 07 Sep 2017 22:42 Last Modified: 03 May 2023 23:56 Thesis Files Preview PDF - Final Version See Usage Policy. 22MB Repository Staff Only: item control page

STUDIES ON THE SOURCE OF UREA CARBON Thesis by Kenneth Paigen In Partial Fulfillment of the Requirements For the Degree of Doctor of Philosophy California Institute of Technology Pasadena, California 19Sl TO LU no words are adequate ACKNOWLEDGMENT I should like to express my appreciation to Professor H. Borsook for his suggestions and criticisms during the course of this work, and to Professor A.J. Haagen-Smit and Dr. J. Dubnoff for their help and suggestions. I should also like to thank the man;y staff members whose assistance made the completion of this work possible. ABSTRACT A method has been devised for the testing of any substance as an intermediate in a reaction sequencee Using this technique, thirty-three substances have been tested as precursors of the urea carbon atom. Of the compounds tested, only citrulline and arginine were precursors. Garbanzy-1 1-glutamic acid was shown not to be a donor of the urea carbon, although it does have some function in urea synthesis. TABLE OF CONTENTS Topic I. II. III. HISTORICAL INTRODUCTION A. The Ornithine Cycle 1 B. Alternative Pathways 6 c. Amides 6 D. Source of Urea Carbon 8 E. Ornithine~ Citrulline 9 F. Citrulline ~ Arginine 12 G. Localization of Steps Within the Cell 13 H. Miscellaneous 13 PROBLEM AND PROCEDURE A. Problem and Method of Approach 16 B. Validity of t he Method of Approach 19 c. Experimental Methods 20 RESULTS A. Enzyme System 31 B. Krebs Cycle Compounds, Pyruvate and Acetate 34 c. Alilino Acids and Related Compounds 36 D. Carbamyl Glutamic Acid and Carbamyl Aspartic Acid 45 51 E. Vitamins F. Formic Acid, Para-amino-benzoic acid, and 4 amino- 5 Imadazole Carboxamide 55 IV. DISCUSSION 59 v. REFERENCES 61 l I. HISTORICAL INTRODUCTION A. The Ornithine Cycle Modern work on the problem of biological urea synthesis dates from the discovery by Krebs and Hensele.i t (1, 2) that ornithine acts catalytically in producing urea from annnonium lactate added to liver slices. It had previously been established that arginase is present only in the livers of animals in which urea was the major end product of nitrogen metabolism (3). Putting these two facts together , Krebs and Henseleit proposed the scheme given in Figure 1, and as support for this hypothesis, showed that citrulline as well as ornithine acted catalytically in producing urea. The scheme may be stated simply as the synthesis of arginine from ornithine, 2 NH , and C02, via citrulline. The arginine so 3 formed breaks dovm to ornithine and urea under the action of the enzyme arginase, which had been discovered by Kossel and Dakin in 1904 (4). The ornithine obtained from the hydrolysis of arginine is now available to go around the cycle once more. While maizy' workers in the past have denied the existence of part or all of the ornithine cycle, most workers today hold the view that most, if not all, urea synthesis proceeds by way of the ornithine cycle. Among early attempts to test the hypothesis was the work of Neber (5), London and Alexandry (6), Ikeda ( 7), and Isawa, Togo, and Kawabu (8). Neber found, using cat and guinea pig liver sections, 2 that the addition of keto acids and ammonia resulted in the f ormation of amino acids and urea in approximately equal amounts, and that ornithine acted as a regulator between the two. London and Alexandry, on the other hand, were unable to confirm the existence -of the ornithine cycle and concluded that 'vhile it operated in liver slices, it did not in the intact animal. Using dogs, they in- jected various compounds into the portal vein and collected blood which was analyzed for urea, at one minute intervals from the hepatic vein. They found that the addition of small quantities of NH4Cl resulted in increases of from 20-77 per cent in urea after 6 minutes; and further, that ornithine and citrulline under the same conditions had no effect. The addition of arginine stimulated less than. did NH , and a longer time was required for the maximum effect 3 to be obtained. Dceda and Isawa tl al. were, on the other hand, able to confirm the catalytic effects of ornithine and citrulline. Ikeda, in perfusion experiments on dog livers, found that the addition of citrulline increased the urea obtained from NHJ++ salts added to blood. Isawa ~ ~ showed that the addition of ornithine doubled and citrulline tripled the Q urea of liver slices. Trowell (9) again using perfused livers, was able to confirm the catalytic effect of ornithine, but not that of citrulline. At high concentrations, citrulline did stimulate somewhat, but this stimulation was transitory, while that from ornithine persisted for long periods of time. The results obtained with arginine were quite variable, while glutamic acid, glutamine, and alanine had no effect. 3 Trowell concluded that the results did not support the ornithine cycle theory and that the normal mechanism of urea formation remained to be discovered. Krebs (10) has criticized these results on the basis of the concentrations used and the general conditions prevailing in perfusion experiments. More direct evidence for the existence of the ornithine cycle was obtained when the heavy isotopes of hydrogen and nitrogen became available. Glutton et ~ (11) were able to show the conversion of ornithine to arginine using deuterium labelled ornithine. containing 13.6 atom per cent excess was fed to mice. DL-ornithine After nine days, arginine isolated from the total body protein was found to contain 1.02 atom per cent excess in the ornithine moiety, indicating that in this time period, 7.5 per cent of the total body arginine had been derived from ornithine. Vfuen N1 5H3 was fed to rats and the total body amino acids isolated, glutamic acid and aspartic acid had the highest N15 concentrations, while arginine vras next. the amidine group (12). All of the 115 in arginine was in If glycine (13) or tyrosine (14) labelled with Nl5 were fed, the leV;el of isotope present in the liver arginine (all of the N15 being in the amidine group) was very nearly the same as that in the excreted urea, the arginine having the highest N1 5 content of a:rry of the amino acids studied. The first real criticisms of the ornithine cycle came from the work of Bach (15). Bach found that when citrulline was added to rat liver slices in high concentrations, the ratio of ~-N disappeared/urea N formed was unity, whereas tha ornithine cycle as 4 postulated would predict a ratio of O.S. This effect was later con- firmed by Borsook and Dubnoff (16) and Gornall and Hunter (17). Bach also detennined the rate of urea formation from ornithine, citrulline, and arginine, and as the theory would predict, found the rates to increase in that order. A satisfactory explanation of the N ratio obtained was offered by Gornall and 'Hunter when they proposed that, if the step citrulline to arginine were the slowest one of the cycle, the citrulline which went to form arginine would be resynthesized from the ornithine produced, and hence for each mole of urea-N formed, one mole of NH3-N would disappear. In support of this interpretation, they found that when orni- thine was added in high concentrations, the ratio NH3-N used/urea-N formed was 1.4 and that the extra N used (over the theoretical 1.0) was incorporated into citrulline which accumulated in the reaction mixture. Later work by Bach and Yfilliamson (18) again questioned the validity of the ornithine cycle when these authors found that high concentrations of ornithine (1.6 per cent) would strongly inhibit urea formation from arginine, but not from annnonium lactate, arguing that urea production from ammoniu.~ lactate could therefore not go through arginine. Krebs (10,19) argued that these experiments were not valid due to the thickness of the tissue slices used. Since the arginase ac- tivity observed in the experiments of Bach and W illiamson was only approximately l per cent of that actually present, it would appear that the arginase inhibited was only that of the outer layer of cells s which had disintegrated, and that therefore the bulk of the arginase concerned in urea synthesis was not affected at all. In marshalling evidence for this view, Krebs was able to show that i f the reaction medium was rendered more alkaline, where ornithine (because of its decreased ionization) would diffuse into the slices more easily, urea formation from ammonium lactate was actually inhibited by high concentrations of ornithine. Further evidence in support, of the existence of the ornithine cycle has come principally from two lines of work: the demonstration and isolation of the individual reactions concerned in cell free preparations (evidence in this connection will be discussed in detail under each reaction) and the discovery of reaction sequences, showing the characteristics of the ornithine cycle in non-mannnalian organisms. Srb and Horowitz (20) in a study of "arginineless" mutants of Neurospora, concluded from the observed growth requirements, that one mutant was blocked in the step citrulline to arginine; two mutants were blocked in the step ornithine to citrulline; and four mutants were unable to synthesize ornithine. Neurospora also possesses ' the enzyme arginase, so that it would appear that a complete ornithine cycle exists in this mold. A similar case has been shown by Bonner (21) to exist for the mold P,enicillium. 11 Arginineless 11 mutants have been found there which are deficient in the ability to synthesize each of the members of the cycle. The mold also possesses the enzyme arginase. Cohen and Lewis (22) have developed a technique for the force feeding of earthworms (Lumbricus terrestis). Using this technique 6 to study the nitrogen excretion of starved earthworms, they establishmi that administration of arginine or citrulline, but not orrdthine, glutamic acid, glutamine, glycine, alanine, histidine, hydantoin, or NH4Cl. resulted in increased urea excretion. The quantity of urea obtained from citrulline is increased by the s:imu.ltaneous administration of amino acids, but this effect is not noted with ornithine. In a later paper (23), they demonstrate that a homogenate of earthworm intestine possesses arginase activity and that, interestingly enough, the arginase activity increases ten fold if the worms are starved 24-30 days. B. Alternative Pathways Alternative pathways of two types have been proposed by Bach (15): a cycle involving ornithine and citrulline, but not going through arginine, and a cycle involving glutamine. Bach (15) pro- posed that citrulline underwent a dismutation with keto acids to form glutamic acid, urea, and a hydroxy acid. However, in the opinion of this reviewer, the quantitative data upon which this concept is based do not warrant the formation of a new theory. The same author has also suggested (15) that ·urea is formed from glutamine directly. c. PJllides Leuthardt and Glasson (24) in 1942, proposed that the whole amide group of a postulated half amide of oxalacetic acid was transferred intact to ornithine to form citrulline. This hypothesis was based on the observation that in the absence of ornithine, succinamide is more effective than succinic acid plus ammonia, or than 7 ammonia alone. F...owever, these authors neglected to report what hap- pens in the presence of ornithine. No further evidence has been ad- vanced in support of this view. Leuthardt (25,26) has also advanced the view that glutamine may play a specific role in urea formation. This was based on the follovr- ing points: (1) Liver slices form urea .faster from glutamine than from ammonia. (2) When glutamine is used, no oxidizable substrate is required. (3) Muscle protein contains much glutamic acid; usually the free -COOH is present as the amide; and no glutaminase is present. A teleological validity therefore exists for forming urea from glutamine directly. (4) F.ree energy relationships favor glutamine to urea in place of ammonia to urea. Krebs (10) has compared the formation of urea in liver slices from glutamine, ammonia, and glutamic acid plus ammonia. He con- firmed the greater effectiveness of glutamine as compared with ammonia, but also found that glutamic acid plus annnonia is as eff ective as glutamine. Other authors (16,27,28,29) have not been able to show urea formation from glutamine. -- More recently, Leuthardt et al. (30) using homogenate, have found that glutamine is not utilized at low concentrations (less than 0.01 M.) and these authors have concluded that gluta.mine is therefore probably not an intermediate in urea formation. 8 D. Source of Urea Carbon There is extensive evidence to show that the ultimate source of the urea carbon atom is co 2 • Krebs and Weil (31,32) have found that the rate of urea synthesis increases rapid~ as the concentration of the bicarbonate buff er is increased, and that there is almost no synthesis i f a phosphate buffer alone is used. These results have been confirmed in full by Leuthardt and Glasson (33). Gale (34) working with the Lancefield group D streptococci, has shown that they require arginine for growth, and that i f arginine is replacedby ornithine and C02, the rate of growth then becomes a function of the co 2 concentration. Tracer studies have been conducted using c11 and c1 3 in liver Evans and Sloti~ (35) found that when liver slices were incubated in the presence of ornithine, NH4Cl, pyruvate, and c11o2, slices. after 45 minutes the urea had a specific activity equal to 40 per cent of that present in the bicarbonate. Rittenberg and Waelsbh(36) used liver slices with NH c1, ornithine, and glucose as added sub- 4 strates and found that after 2 hours, 50 per cent of the urea had been formed from the c1 302 added. MacKenzie and du Vigneaud ·(37) fed rats 1-methionine with cl4 in the methyl group. 'l'he excreted urea and the expired co2 had identical specific activities on both the first and second days, al though on day two, urea excretion was up 35 per cent and co 2 excretion down 13 per cent. Grisolia and Cohen (38) have followed citrulline formation from c1 4o 2 in a semi-isolated enzyme system. The results obtained showed that the citrulline formed had a 9 specific activity identical with that of the bicarbonate, and furthermore, that urea produced from this citrulline by the action of wh©le homogenate, also had the same specific activity. Biotin has been shovm. to play an important role in the co 2 fixation reaction involved in urea synthesis, as well as in other reactions involving co2 fixation (39). MacLeod and Lardy (40) have studied the uptake of injected NaHc1 4o3 into various body components in normal and biotin deficient rats. Their results show that normal rats incorporate c14 in liver protein arginine at six times the rate biotin deficient animals do. MacLeod ~ al. (41) compared the activity of the washed residue enzyme of Cohen (which converts ornithine to citrulline) in normal and biotin deficient rats. Biotin deficient animals had about 50 per cent of the activity possessed by normal rats. That this result is specific for biotin is shown by the fact that riboflavin, or pyridoxine deficient rats have the same activity as the control animals. Whether biotin functions as a coenzyme in this system, or is necessary for · th~ production of the enzyme is not clear. Biotin or a heated eXtract of normal or deficient liver will not stinru.late normal or deficient preparations. However, the addition of heated residue will stimulate the activity of whole homogenate from deficient rats, but not from controls. E. Ornithine---'>,. Citrulline Borsook and Dubnoff (42) first demonstrated that urea synthesis could be carried out in a cell free medium. Using guinea pig liver 10 homogenate, they found that the optimum reaction mixture included ornithine, ammonia, glutamic acid, oxalacetic acid, and ATP. The reaction was inhibited by arsenate and required the presence of oxygen. Cohen and Hayano (28,43) discovered that by omitting Mg++from the reaction mixture, the further conversion of citrulline could be inhibited, and thus the first step of the cycle isolated for study. In addition to ornithine, the reaction required glutamic acid, am- monia, co 2, inorganic phosphate, and either adenylic acid or ATP, with ATP only 50 per cent as effective as adenylic acid. These authors were further able to isolate the first step in the cycle by their discovery that the enzymes concerned may be separated by centrifugation. If homogenate is centrifuged at 2000 g. for 10 minutes, the ornithine ~ citrulline activity is in the precipitate (referred to as 11washed residue 11 ) while the enzymes concerned with citrulline ----r urea appear to have been separated into two fractions. The fraction present in residue is non-specific and can be supplied by a variety of tissues; it appears to be an insoluble H transport system including cytochrome. The fraction in the super- natent can only be obtained from liver and is apparently the specific enzyme system concerned. Studying the washed residue preparation, they found that Mg+r is required for optimal activity in this step as well as in the further conversion of citrulline. Cohen and Grisolia (29,h4) in further studies on the washed residue preparation have succeeded in establishing the following facts: 11 (1) Carbamyl L-glutamate is 2-3 - times more effective than glutamate in the conversion of ornithine to citrulline. Its effect.iveness relative to glutamic acid is increased i f co 2 is excluded from the reaction mixture. (2) Ammonia is required for the reaction, ti.~o moles being used i f glutamic acid is the substrate, one mole if carbamyl glutamic acid is. Carbamyl glutamine and ar-guanidoglutaric acid are less effective than carbamyl gluta.mic acid itself. (3) In a footnote to their most recent paper, these workers report that they have been able to prepare 1 by fractionation of washed residue , a soluble enzyme which carried out anaerobic synthesis of citrulline from carbamyl glutamic acid and ornithine, but not from ornithine + glutamic acid + C02 + ammonia. Leuthardt et ~ (J0,45) have confirmed that carbamyl glutamate reacts with ornithine to form citrulline. These workers also found that the reaction will not go unless ammonia and an oxidixable substrate are present. Feldott et ~ (46) report that biotin deficient rats which do not carry out the synthesis of citrulline from ornithine and glutamic acid as well as normal rats, show the same enzyme activity if carbamyl glutamic acid replaces glutamate in the test system. Srb (47) has tested the ability of 11 arginineless" mutants of Neurospora to grow on carbamyl glutamate. 11 In tests on all types of argininesless" mutants, he found that carbamyl glutamic acid had no effect. 12 F. Citrulline ~ Arginine This reaction wa.s first demonstrated by Borsook and Dubnoff (48) using rat and guinea pig kidney slices. The reaction required the pre- senceof a dicarboxylic amino acid (glutamic acid and aspartic acid we:re equally effective) and was inhibited by arsenite, arsenate, and cyanide. The cyanide inhibition was partially relieved by the addition of hydrogen acceptors. Cohen and Hayano (49,50) have also studied this reaction. They report that rat liver homogenates require ATP, citrulline, glutamic acid, cytochrome c, Mg*, and 02 for optimwn activity. Aspartic acid was much less effective than glutamic acid and the reaction vras inhibited by a-ketoglutaric acid and annnonia. Fahrlfulder ~al. (51-53) have confirmed the inhibition by a-ketoglutaric acid, but find that contrary to the above workers, annnonia not only tends to reverse the a-ketoglutarate inhibition, but stimulates yields from glutamic acid as well. They also found that glutamic acid can be replaced by aspar- tic acid. They further report that the reaction is inhibited by malon- ate, methionine, and cysteine. The methionine inhibition is partially reversed by increasing the Mg++ concentration. Ratner and Pappas (54-56) have succeeded in isolating a soluble enzyme free of pyridine nucleotides which catalyzes the anaerobic reaction: Citrulline + ATP + Aspartate in the presence of Mg* ions. )Arginine + ADP -+Ma.lie Further purification has shown that two enzymes are actually concerned in the reaction. The first con- denses citrulline and aspartic acid, while using up one high energy 13 phosphate bond, and the second cleaves the condensation product to arginine and malic acid. They explain the efficacy of glutarnic acid in place of aspartic acid in whole homogenate as being due to the du~ role glutamic acid can play by supplying both high energy phosphate and aspartic acid via oxalacetate and transamination. G. Localization of Steps Within the Cell Arginase has been shown by Dounce (57) to have the same concentration in the nucleus, and in the cytoplasm, w'nile the preceding step (citrulline ----;;;.arginine) has been found to be in the soluble cytoplasmic proteins (54,SS). The first step (ornithine ~ citrulline ) is appare:rt,ly in the "washed residue 11 fraction (28). Since this fraction includes nuclei, cell fragments, unbroken cells, and cytoplasmic particles, it is not clear exactly in which part of the cell the first step is localized, though it is not in the soluble cytoplasmic fraction. It is interesti..ng tha t, although urea synthesis is a closely linked sequence of reactions, the individual steps do not appear to be spatially integrated within the cell. H. Wiiscellaneous Leuthardt and Fahrlful.der (58 ) report that methionine or methionine sulfoxide inhibits urea formation in liver slices~ The methio- nine inhibition is relieved by the addition of pyruvate$ Bernheim and Bernheim (59) have found that certain amino acids inhibit urea production by liver slices from NH4+ salts, but not from alanine or glutamine& The inhibition can be overcome by adding orni- thine, but not by Mg+t. 2,4-dinitrophenol has a similar effect. It inhibits urea production from ammonia strongly, has less effect when alanine is used, and has no eff ect when glutamine is used as the nitrogen source. The inhibitory action of 2,4-dinitrophenol is not relieved by ornithine, but can be overcome by the addition of pyruvate. These same authors have ~lso noted similar inhibitions by caffeine, theophylline, and theobromine (60). overcome the inhibition. is not arginase. Ornithine and glutamine Tests have shown that the point of inhibition The inhibitory effect of caffeine and theophylline has been confirmed (43). Borsook and Jeffreys (61) have estimated the free energy change in the formation of urea from ammonia, acid and co2 , under conditions existing in plasma to be +14,300 calories. Leuthardt and Glasson (62,63) have found that in thiamin deficient rats and guinea pigs, the addition of thiamin to liver slices causes an increase in the rate of urea synthesis if pyruvate is the substrate, but not if oxalacetate is. They suggest that fixation of co 2 into urEB might be by w~,y of the Wood...;Werkman reaction. In this connection, An- fins en ~ al. (64) have reported on the uptake of NaHc14o3 into prote:in by rat liver slices. The guanidine carbon of arginine had the high- est specific activity of all substances tested. Oxalacetic acid was found to inhibit up to 50 per cent, whereas glutamic acid, aspartic acid, glutamine, asparagine, malate, pyruvate, and alanine had no effect. Leuthardt ~ al. (45) have made the interesting observation that the addition of cis-aconitic acid inhibits the formation of citrulline from glutamic acid and ammonia, but has little effect when glutamine 15 is used instead. They also report that washing the homogenate re- moves a factor required for the reaction from glutamate and ammonia, but which is not required by gluta.mine. 16 II. PROBLEM AND PROCEDURE A. Problem and :Method of ~pproach It has been shown that the primary source of the urea carbon atom is carbon dioxide (31-38). We are faced then with the problem of a reaction sequence in which we know the first substrate, carbon dioxide; several of the final steps, citrulline to urea; and the last product, urea. However, we do not know the following crucial points: (a) What is the initial carbon dioxide acceptor? (b) What metabolic transformations does the fixation product undergo?- leading to (c) What is the substance, or substances, reacting with ornithine to form citrulline? The point may be raised that if carbon dioxide reacts directly with the delta amino group of ornithine to form a carbamido compound as postulated by Krebs (65), the problem as stated above becomes meaningless. There are, however, several reasons for assuming that C02 does not react directly with ornithine. First, as has been point- ed out by Leuthardt and Brunner (66), the pK of the delta amino group of ornithine is too high to permit of direct reaction at physiological pH's. Second, the energy required for the synthesis of urea from ammonia, co 2 , and acid, under conditions existing in plasma (which may be taken as a reasonable approximation of those existing inside a liver cell) is +14,300 calories (61). As a qualitative assumption, we can look at citrulline as a mono-substituted urea in which one hydrogen is replaced by the group -CH2 - C~ - C~ - CH(NH2 ) - COOH, 17 a.nd the synthesis of citrulline as analogous to that of urea, but in which one of the ammonia molecules is replaced by the primary amine, ornithine. If these asswnptions are correct, the formation of a car- bamido ornithine should require in the neighborhood of 7,000 calories, an energy requirement 1vhich would be too high for a simple reaction between ornithine and co 2 • Third, there is the experimental evidence that under conditions which result in citrulline synthesis, there is 002 fixation if ornithine is omitted from the reaction mixture, but not if glutamic acid is omitted. And further, if enzyme and substrates vrl.th ornithine omitted, are pre-incubated, before the addition of ornithine, not only is there C02 uptake, but the yield of citrulline is higher than if no pre-incubation is carried out (29). The problem may then be stated: Through what sequence of compounds does the carbon atom of C02 pass before reacting with ornithine to form citrulline? At the time this work was begun, it was felt that what was needed most for a:rry- successful ,attempt to solve this problem was a reasonably rapid technique for testing compounds as possible carbon donors in urea synthesis. The technique finally worked out was in response to this requirement• . It depends upon the principle that i f there is a reaction sequence S ~ X---7 Pj and we start with radioactive s, then the specific activity of the product P will be depressed in the case where X is added to the reaction mixture. In practice, the experiment is performed by incubating a mixture of enzyme, buffer, and the necessary substrates, and then determining the yield and specific activity 18 of the product. These values are now compared with data from a simi- lar set of vessels to which a substance, suspected of being X, has been added. If the substance is X, the specific activity of the pro- duct would be depressed; the yield may or may not be increased. If it is not X, the yield may or may not be affected, and the specific activity would not be lowered. The starting substrate used (S in the sequence S--? x~ P) has been c14 labelled NaHC03, and urea has been the product (P) which has been analyzed. Urea was chosen as the product to follow in preference to citrulline for the reason that, as was mentioned in the introduction, there has been work on the urea synthesis problem which suggesta:l that the ornithine cycle might not be the only way to make urea. If' this is the case, :ilnportant facts might now show up if citrulline were chosen as the end product. The enzyme system used was rat liver homogenate. This was used in preference to the semi-isolated enzyme system used by Cohen's group (28) since, as was mentioned above, the aim was to follow urea format:lon not citrulline. It was also felt that the removal of enzyme systems might reduce the chances of identifying X. If a suspected compound is not X, but is metabolically converted to X by an enzyme present in whole homogenate, but not in an isolated enzyme system, the tested compound would be expected to cause some depression of the specific activity in a test using whole homogenate as an eµzyme source, but not in a test using a partially isolated enzyme system. 19 B. Validity of the Method of Approach Ebcperimental proof of the above technique can be obtained by testing citrulline and arginine, the two known intermediates in the urea cycle. The results of testing these substances are shown in Table 1. TABLE 1. EFFECT OF ARGININE AND CITRULLINE ON THE INCORPORATION OF NaHC14o3 INTO UREA Additions to Control Urea Formed Total Urea Radio act. Urea Spec. Activity {µm.) (cts./min.) (cts./min./µm.) Per Cent Control None 0.89 64 72 100 1-citrulline (20 µm.) L-arginine (20 µm.) 3.43 7 2 3 17.2 138 8 11 Conditions given in text (Section II-C). 0.03 M. fumarate present in Glutamate was .05 M. _ NaHC0 3 added had 252 cts./min./µm. Enzyme concentration was 21 mg. (wet weight) liver/ml. reaction mixt~ all vessels. As would be expected, both citru.lline and arginine greatly stimulate the yield of urea and markedly depress the specific activity. The figures on total urea radioactivity are interesting because they confirm several facts about the ornithi he cycle. Arginine behaves al- most as though equimolar quantities of urea and ornithine had been 20 added; it actually stimulates the total munber of counts fixed. This is in agreement with the lmmm rapid hydrolysis of arginine to ornithine and urea, which is the fastest step of the cycle (17). Citrul- line, on the other hand, not only causes a marked depression in the specific activity of the urea, but a strong inhibition of the total counts fixed. The step citrulline to arginine is known to be the slow- est of the cycle (17). It is at such a step that one would expect the greatest decrease in specific activity of the product, since great quantities of synthesized citrulline would have to pile up for an appreciable number of counts to go through. Further evidence on this point was obtained from experiments in which the enzyme concentration was increased 2.5 fold. Since it has been foui1d that, 1vith increased enzyme concentration, less citrulline tends to pile up, and its conversion to arginine is more rapid (43), it would be expected that under these conditions, there would be an increase in the yield of urea from citrulline, in the total urea radioactivity, and in the urea specific activity vrith respect to the control. This is exactly what was found. With added citrulline, in the pre- sence of a higher enzyme concentration, the total urea radioactivity becomes greater than the control (though still less than when arginine is added), and the urea specific activity approaches the value obtained in the presence of arginine. c. §xperimental Methods Preliminary experiments were carried out in the experimental vessels shown in Figure l.. A mixture of substrates and enzyme were 21 placed in the reaction vessel and N~c14a of 5.0 mls. 3 added to a final volume The tops were placed on with both stopcocks closed and incubated at 38° C with shaking. Following the incubation period, 1.0 ml. of 3 M. acetate buffer (pH 4.8) was added through stopcock A under suction to bring the pH to 5.0. The vessels were then eva- cuated to 45 - 50 nnn. pressure for 5 minutes, after which air was admitted and sucked through for 5 minutes under slight vacuum. The ves- sels were now re-evacuated and co 2 permitted to enter. A total of five evacuations were carried out alternating air and co 2 • After tb~ ·: addi­ tion of 4.o mls. of water, the vessels were boiled 5 minutes on a boiling water bath, and filtered through Whatman No. 5 paper. A 3.0 ml. aliquot of the filtrate was taken for. manometric analysis of urea, the sidearm containing 0.3 mls. of a mixture of 3 vols. 3 M. acetate buff er and 1 vol. of a glycerol extract of jack bean meal. * Temperat,ure equilibration was 20 minutes. As a check on the analytical method, recovery experiments were performed by adding known quantities of urea to enzyme buffer mixtures and carrying them through the procedure. The results are shovm in Table. 2. * The extract was prepared according to the method of Koch (67). It has the advantage over connnercial urease preparations in that it is extremely active and shows no loss in activity on standing several years in the refrigerator. ~o test the specificity of this enzyme preparation, a 50 mls. batch of reaction mixture was run ahd the product analyzed using this urease, a commercial urease (Nutritional Biochemicals Corp. #1607), and a twice-re-crystallized, 100 times purified connnercial urease obtained through the courtesy of Prof. He Borsook. The results obtained were 1.00, 0.96 and 0.97 µm./ml. of de-proteinized solution, respectively. 22 TABLE 2. RECOVERY OF UREA ADDED TO HOMOGENATES lilicperiment No. Urea Added Urea Found -Blank (µm.) (µm.) (µm.) o.o 6.o 2.20 o.o 8.18 5•98 12•0 14.4 12.2 o.o 6.o 2.94 o.o 9.22 6.28 12.0 15.l 12.2 A B All values are averages of duplicates. The total urea radioactivity was determined by incubating a 3.0 ml. aliquot of the filtrate with l.O ml. of the glycerol-acetate mixture four hours at 38° c. The vessels i.vere now aerated 10 minutes with co 2 free air into two glass bead columns as shown in Figure 3 . Each column contained 8 mls. of 0.7 N. C02 free NaOH. Following the aeration, the columns were washed down twice with 8.0 mls. of C02 free water. The NaOH and washings were pooled, and 1.0 ml. of a 20 per cent BaCl2 solution added. They were stored overnight in the refrigerator and the BaC03 filtered the next day onto tared papers and dried 20 minutes at 100° c. and fastened to aluminum plates for counting. They were weighed Since the self-absorp- 23 tion of samples having a total weight over 20 mg. becomes high, all samples vvere kept to below 20 rng. in weight. In cases where the BaCO precipitate weighed more than 20 mg., only part of the sample 3 was used. A new plate was made by suspending part of the original sample in alcohol and refiltering. In these experiments, the standard reaction mixture was 5.0 mls. ci.nd was composed of: Concentration Co!S'.!ound KCl .145 MgS04 .0033 ~P04 .0125 L-glutama.te .05 DL-ornithine .0067 NH4Cl .0067 Adeeylic acid .001 The reaction mixture contained 1.0 ml. of a 25 per cent homogenate of rat liver in isotonic KCl. Starting pH was 7.h and the reaction was carried out for 90 minutes at 38° C with shaking. After preliminary experiments were carried out as described above, the procedure was abandoned as being unsuited for routine work. The procedure and conditions described below applies to all experiments reported except those otherwise noted as "Prelim". Reactions were carried out in 15 ml. serum bottles (Kimble No. 15105) which were incubated thirty at a time in a Dubnoff apparatus (69). The reaction time was 60 minutes at 38° C with shaking and air as a gas phase. The buffer was of the following composition: 24 Final Cone. (M_J eyi./liter cone. (M.) KCl 23.2 .312 .145 K2HP04 7.3 .042 .0125 MgS04 24.4 .099 .0033 Solution A. Solution B. It was stored in the refrigerator and just before use, 9 vols. of Solution A. were mixed with l vol. Solution B and the pH adjusted to 7.2 with HCl; 1.25 mls. of buffer were used per reaction vessel, and the figures given in the last column represent the final concentration of substances in the reaction mixture. The standard substrate mixture was of the following composition: Final Cone. (M.) L-glutamic acid ~· 868 DL-ornithine • HCl 122.4 .0067 NH4Cl 42.4 .0067 Adenylic acid 43.2 .0010 Compound .050 It was made up to 30 mls., the pH adjusted to 1.2, and 1.0 ml. used per vessel. In arry experiments in which the concentration of gluta.mic acid was varied, the rest of the substrate mixture was made up to half volume and 0.5 ml./vessel used. 24 µm. of bicarbonate were added per vessel, the bicarbonate solution being prepared fresh for each experiment by adding water plus a standard solution of Na2c14o to dry NaHC°J: 3 * The standard Na2Clbo3 _t5plution was prepared in the following manner. A 2.5 mg. sample ~f Ba C.J.403 was placed in the reaction vessel shown :in Figure 1. The cl was supplied by Carbide and Carbon Chemicals Corp., Oak Ridge, Tenn., and obtained on allocation from the U.S. Atomic Energy Co:rmnission. The gas inlet was connected to a source of tank N2, the gas outlet to a round bottomed Peligot flask, with 10 bulbs containing 20 mls. or 0.0984 N. NaOH. The outlet from thePeligot flask (continued on next page) 25 14 For practical purposes, the concentration of carbonate in the c standard was zero. Compounds tested were made up fresh and adjusted to pH 7.2; in a few cases, a test solution was stored in the deep freeze for use in more than one experiment. In all cases, water was added to a final volume of 3.8 mls. In an actual experiment, all additions e.~cept the enzyme and bicarbonate solutions were added the night before and frozen. The enzyme was prepared by killing a male albino rat by stunning and rapid.ly excising 4 to 8 grams of liver. This was placed in a 20 ml. beaker which had been packed in ice and frozen. The liver was weighed and the weight adjusted to give a final homogenate concentration of 20 per cent. The weighed liver was now minced and added to a cold solution of isotonic KCl; for each gram of liver, one drop of N. KOH was added and the liver homogenized in a glass tube using a lucite pestle with a stainless steel rod. The pH at the end of homogeniza- tion Wa.s from 8.4 to 8.8 and the temperature from 11-13° C. The pH was adjusted to 7.3 and o.4 ml. of this used per vessel, to give a final concentration of 5.5 mg. (wet weight) liver/ml. of reaction mixture. The enzyme was added to the reaction vessels which had been thawed out, and following this, the bicarbonate solution. The vessels * (Footnote continued from page 24). was connected to a NaOH safety trap. 5 mls. of 1 per cent perchloric acid was added_to the reaction vessel and N2 passed through for 2 hours. At the end of the aeration, the contents of the Peligot flask were washed out with a total of 30 mls. of vlater. The pooled washings and N/10 NaOH are the standard solution. It contained 176,500 counts/ min./ml. with a final base concentration of 0.04 N. 26 were stoppered and incubated. * At the end of the incubation period, o.4 mls. of S M. KOH were added through the stopper using a 1.0 ml. ~~ hypodermic syringe with a 23 gauge needle. · The vessels were shaken 20 minutes closed to absorb all the co 2 into the solution; and then the caps removed and the vessels shaken 90 minutes with a fan blovdng across the top. This procedure removes all ammonia present. Stoppers fitted with center wells were put on, each center well containing 0.5 mls. 0.7 N. NaOH. 0.25 mls. of 41 per cent ace- tic acid were then injected past the center well and into the main body of the solution to bring to pH to 5.2, at which pH all the co 2 is liberated. The vessels were shaken at 38° C for 30 minutes to effect ~ complete transfer of co 2 to the center well. The center well contents were discarded and the vessels placed in a boiling water bath for 10 minutes to coagulate the protein. Following this treatment, fresh center wells containing 0.5 mls. 0.7 N. C02 free NaOH are prepared and placed in the vessels :iJmnediately following the addition of 5 drops of urease and shaken 6o minutes at 45° c. The center wells now conta~he C0 2 obtained from urea hydrolysis and their contents were washed into 50 ml. * The pH of the reaction mixture after the addition of the bicarbonate solution is 7.4 - 7.5. The pH at the end of the reaction is 7.0 - 7.1.. ** A 23 gauge needle upon being withdrawn leaves a gas-tight seal. -iHH~ Center wells are constructed from 13 x 100 nnn. test tubes by cutting off a 15 nnn. length from thebottom and attaching this to a 5 cm. length of 4 nnn. glass rod. The rod is passed through a hole in the stopper to suspend the center well inside the vessel. ~HHH~ The urease is previously. the glycerol extract of ~ack bean meal mentioned 27 Erlenmeyers and o.5 mls. of a solution of BaC1 2 and NH4Cl added. * The flasks were allowed to stand overnight stoppered and then filtered ~h~ onto filter paper discs, washed with water, alcohol, and ether . The funnels were washed between each filtration to insure no contamination from sample to sample. Samples prepared had an area of 2.85 cm~ and were taped to aluminum plates for counting. Samples were counted using an end window (mica) counter wi. th a counting efficiency of 5 per cent. All counts reported are corrected for background and have been corrected to a standard sample. The serum bottles 1vhich now contain ammonia derived from the hydrolysis of urea have one drop of lauryl alcohol and one drop of Turkey red oil added. .~'* 2.0 mls. of a saturated solution of potas- siu.m metaborate were added and the vessels aerated in a train into 10 mls. of N/10 sulfuric acid, contained in a 16 nnn. i.d. test tube. The aeration is carried out 90 minutes and following this nesslerized usirg ~HHH'· 2.0 mls. of Nessler 1 s solution:h "Colors were read in a Coleman Jr. Spectrophotomet er. A composite picture of the experimental details is shown on the flmv sheet in Figure 4. * The solution is prepared by adding 15 gms. BaC12 and 3 gms. NH4Cl to 100 mls . water. The NH4Cl neutralizes the NaOH present and gives better precipitates. *"~ The discs were circles 2.1 cm. in diameter cut from W hatman No. 1. paper. ~.P~ The addition of lauryl alcohol and Turkey red oil is necessary t o prevent foaming during the aeration. It is also necessary to use metaborate as base; KOH will not work due to excessive foaming. Nessler ' s solution was a modification of that published by Koch and McMeekin (70). In the preparation used, the solution was allovred to become as hot as possible after the addition of mer cury. *i~X~~e 28 All procedures in the analytical scheme have been checked by control experiments, and the results are given in the summary below. fu!dition of 5 M. KOH - As shown below, co 2 transfer from the main solution to the center well is quantitative in 20 minutes. Un- der these conditions, co 2 will be completely absorbed into the main volume of the solution which has a surf ace area ten times that of the center well. Efficiency of ammonia removal (after the stoppers are removed) was tested by determining the quantity of ammonia left in a mixture of homogenate and buffer after various shaking times. After 20 minutes shaking, 13 per cent was left; and after 60 minutes shaking, no residual ammonia was found. Ninety minutes was the time used throughout to provide a margin of safety. Addition of 41 per cent Acetic Acid - In the first eight experiments, it was found that the addition of 0.25 rnls. of acetic acid solution always resulted in a pH between 5.1 - 5.3. 0.25 rnls. were added routinely. After this, The time for co 2 diffusion was deter- mined by putting a standard carbonate solution in serum bottles and liberating the co 2 • Trapped C02 was analyzed gravimetrically as BaC03. It was found that diffusion was complete in less than 20 minutes. Thirty minutes was therefore adopted as a routine time. Tests on the recovery and retention of c1402 showed 100 per cent recovery and no detectable retention. Urease - Activity of the enzyme was tested by the time required to effect 100 per cent hydrolysis of 5 µ.m. urea at 38° c. It was found that 3 drops of enzyme caused complete hydrolysis in less than 20 minutes. Routinely a 60 minute incubation at 45° C {the 29 temperature optimum) with 5 drops of enzyme has been used to assure complete hydrolysis and diffusion. Aeration - The time required for complete aeration was found to be 55 minutes. A 90 minute aeration has been used throughout. Absorption of ammonia by one trap containing 10 mls. of N/10 H2S04 is complete as shown by an average recovery of 100.2 per cent on standard nitrogen samples, and a complete lack of color in a second trap if it is placed in series after the first. In addition to the above controls, two tests of the analytical procedure were conducted periodically throughout the course of the investigation. The first of these were recovery experiments on standard samples of urea added to reaction mixtures at time zero and carried along with the experimental vessels. Recoveries as determined in this fashion ranged from 94 - 101 per cent. The second test was tre determination of the radioactivity of the urea formed during the enzyme incubation in blank vessels (vessels in which the substrate mixture was omitted). In no case did the urea formed under these conditions show arry trace of radioactivity, proving that none of the urea radioactivity measurements were affected by incomplete removal of co 2 • This point is interesting in that it also shows that none of the urea formed when enzyme and buff er are incubated in the absence of added substrates, is derived from co 2 • All of the carbon of this urea, then, comes from organic compounds already present in the homogenate. Probably the principal source of this urea is arginine, vr.aich would be continually made available during protein breakdown. Note: - All values reported are the averages of duplicate 30 vessels, and, unless specifically noted, all results have been confirmed using more than one batch of enzyme. 31 III. RESULTS A. Enzyme System Before entering into a discussion of the results obtained in testing various substance, some data on the nature of the system will be reported. The relationship between enzyme concentration and yield, as found in one experiment, is shoi.vn in Figure 5 . The curve goes through a ma,~..imum. , which occurs at a concentration of approximately o.S mls. homogenate in 3.8 mls. of reaction mixture. of interest concerning the shape of the curve. '1\n10 things are First, at low concen- trations of homogenate, the rate is an exponential function of the concentration ( a plot of log activity v. log concentration indicates a limiting slope of 1.8); and second, the curve goes through a maximum. These two facts suggest that the observed rate is the result- ant of two factors: (a) an increase in rate per unit enzyme with rising concentrations of homogenate, due to the increase in concentration of interacting enzymes and co-factors; and, (b) an inhibiting reaation whose rate is a higher exponential functions of the concentration of homogenate than the first set of reactions. No evidence is avail- able for determining the particular reaction concerned with the inhibition. It is possible, however, that it may be involved with the phosphate metabolism of the enzyme preparation. Urea formation is known to be dependent on a supply of phosphorylative energy (28,56), and Ball and Cooper (71) have shovm that the enzyme systems supplying high energy bonds are attacked by other enZJ7llles present in liver homogenate . These enzymes appear to be phos- 32 phatases. In a series of experiments designed to discover which of the fou.r substrates reacted with C02, it was observed that the omission of aderzylic acid had a unique effect on the stability of the system. Substrates were omitted singly and then added 10 minutes after the start of the experiment; i f aderzylic acid was omitted in this manner, the enzyme system was totally inactivated. Such a marked effect was not observed as a result of the omission of other substrates. It was observed in early experiments that the pH of the homogenate mu.st at all times be kept above 7 for max:imu.m activity. If the pH is allowed to drop below 7 during preparation of the enzyme, not only is there a decreased yield of urea, but the pH of the reaction mixture following incubation is 6.75 - 6.85 instead of 7.0 7.1. A possible explanation for this effect is that nucleoprotein breakdown is in some way triggered by a prior drop in pH to the acid side of neutrality, and the resulting pH of approximately 6.8 is the consequence of the buffering action of the liberated phosphoric acid. As seen from Figure 5., the maximum yield of urea obtained in 60 minutes was 4 µm. In general, rats fall into two classes with regard to their enzyme activity. One class gives uniform results of from 3.9 - 4.5 µm./hour, while the other group under identical conditions are somewhat more variable, but give yields in the range o.8 - 2.0 µm./hour. The effects of varying the magnesium concen- tration and the addition of cytochrome C were studied as possible explanations of this effect. Magnesium is known to be required for both the conversion of ornithine to citrulline and citrulline to arginine (28,49). Experiments in this connection showed, however, 33 that there is no decrease in yield i f the magnesium concentration were cut from .0033 M. to .001 M., so that magnesium did not appear to be l:iJn.iting. In several experiments, cytochrome C was added at concentrations up to 3 x io-5 M., but no effect on the yield vras observed. No correlations between age of rats (known only approximately) or weight and activity were observed. However, it is possible that the difference noted between groups may have a genetic background. One batch of male rats from an inbred line fell in almost all cases into the 3.8 - 4.5 group. Rats from this source became unavailable, and the new group tended almost exclusively to fall into the 0.8 2.0 range. The results of complete yield controls are shown in Table 3. The only value shovm which varies from experiment to experiment is the increase in urea production caused by the presence of substrates. This is the value which is referred to throughout as yield, and all specific activities have been calculated on this basis. In short, the table shows that about o.4 µm. of urea are present at t = 0 in 0.4 mls. of homogenate, and another 0.3 iim. are formed during the incubation in the absence of added substrates. The lowest yield (above the value in the absence of substrate) which has been observed was 0.8 µm. or an increase of 270 per cent. The highest yield ever observed was 8.2 µm.; however, yield of this order have only occurred twice. Figure ~. shows the results of one experiment in which the specific activity of the bicarbonate of the medium was studied 34 as a function of time. It is important to keep the shape of this curve in mind, as the specific activity of the urea formed from C02 will re.fleet the change. It appears that after 60 minutes, the bicarbonate specific activity has dropped to 80 per cent of its initial value. As is noted in the figure, 40 µm. af bicar- bonate were used in place of the customary 24, so that in routine experiments, the bicarbonate specific activity will have dropped still lower by the end of the first hour. TABLE 3. YIELD CONTROLS Conditions Substrates - no enzyme Enzyme + substrates, stopped at Urea Measured -Blank (µm.) (µm.) 0.26 =o o.64 Enzyme - no substrates (blank) 0.97 Enzyme - no substrates 4.45 µm. urea added t=O 5.46 4.49 Enzyme plus substrates 2.63 1.66 t B. Krebs cycle compounds, Pyruvate and Acetate The tricarboxylic acid cycle contains three decarboxylations, one of which is known to be reversible (a-ketoglutaric + co 2 ~ 35 oxalosuccinic), while oxalacetic and malic acids are known t.o be products of the addition of co 2 to pyruvate, which is also a reversible reaction. Data obtained in testing members of the cycle are shown in Table 4. TABLE 4. KREBS CYCLE AND RELATIID COMPOUNDS Urea Formed Urea Spec. Activity (µm.) (cts./min./µm.) Control Pyruvate 4.54 4.20 152 173 114 Control Pyruvate a-ketoglutarate Succinate 1.63 3.24 3.61 2.98 147 129 138 156 88 94 106 c Control Pyruvate Fumarate 1.66 2.69 2.33 72 63 87 88 121 D Control Citrate Acetate 3.96 2.54 3.52 138 178 Exp. No. A B Compound Tested Per Gent Control 142 97 125 In all experiments, bicarbonate specific activity was 252 cts./min./µm. All compounds were added at a level of 20 µm./vessel. The results indicate quite clearly that none of the substances tested behaves like an intermediate in the process of fixing carbon dioxide into urea. Since, under the conditions of the experiments, 36 (Mg*, phosphate, and adenylic acid) the Krebs tricarboxylic acid cycle would be expected to be operating, we may conclude that no member of the cycle, nor pyruvate or acetate is concerned with the particular co 2 fixation under study. As can be seen from the data, some members of the cycle tend to stimulate the reaction, especially with enzyme preparations in the 0.8 - 2.0 class. After this fact was established, fumarate vras added routinely to some experiments in an attempt to raise the activity of the enzyme. However, activities in the 0.8 - 2.0 class continued to occur despite fumarate addition at a level of 0.03 M. c. Amino Acids and Related Compounds 1. Histidine - Edlbacher and Kraus (72) have demonstrated an enzyme in the livers of rats, rabbits, and guinea pigs, histidase, which breaks histidine down to glutamic acid + f orrnic acid + 2 ammonia molecules. They postulate the formation of formylglutamine as an intermediate in this process. Fdlbacher and Neber (73) have also sho1vn that if histidine is added to liver slices, urea, rather than ammonia, is the end product. As an explanation, they hypothesize that ammonia liberated during histidine breakdown reacts with the fornzy-lglutamine formed to produce citrulline. This citrulline would now be available for urea synthesis. Tesar and Rittenberg (74), in experiments feeding rats histidine labelled with N1 5 in the Y-N, found that excreted urea had twice the N15 excess of ammonia. If ammonia labelled with N1 5 is fed, the opposite result is obtained, suggesting that histidine 37 plays a specific role in urea formation. Histidine was therefore tested as a possible intermediate, ivith seven enzyme preparations, using histidine from two different sources. In no case has a significant depression of the urea specific activity been obtained. A sample of the data is shown in Table 5. TABLE S. HISTIDINE &p. A (Prelim) B c Urea Formed Urea Spec. Activity (µm.) (cts./min./µm.) Control L-histidine (10) 1.08 2.12 161 154 96 Control L-histidine (6) 1. 70 1.68 30 49 163 Control L-histidine (20) 1.63 1.56 180 164 91 Compound Tested No. Per Cent Control Bicarbonate specific activity was 252 cts./min./µm., in all cases. Figures in parenthesis represent µm. histidine added. One figure in Table S. warrants further connnent. It is the in- crease in the urea specific activ~ty resulting from the addition of histidine in Elcperiment B. This increase has been noted with other amino acids, but in nocase has an a.-amino acid been found which vrould give this result consistently. W ben an increase resulted from the addition of an amino acid, it was generally 30 - 40 per cent above 38 that of the control. This point will be discussed in more detail at the end of the section on amino acids. 2. Methionine, Lysine, and Phenylalanine - Bernheim and Bernheim (59) have compared urea formation from ammonia and from amino acids in rat liver slices. They find that urea production is strong- ly inhibited by a variety of amino acids; that this inhibition can be overcome by increasing the ornithine concentration; and that the inhibition does not occur when alanine or glutamine are used as nitrogen sources in place of ammonia • .Among the amino acids which they tested and found to inhibit, were methionine, lysine, and phenylalanine. In the cases of methio- nine and phenylalanine, they tested the D-isomer vthich was found to be as inhibitory as the L-compound. Results obtained in testing these substances have confirmed the existence of the inhibition in the homogenate system used. In this test system, both arrnnonia (.0067 M.) and L-glutamate (.OS M.) are present. These data, in addition to the radioactivity measurements are given in Table 6. It is apparent . from the results that none of these compounds is an intermediate. These results again demonstrate the role amino acids may have in raising urea specific activity. 39 TABLE 6. METHIONINE, LYSINE, PHENYLALANINE Eicp. No. A Compound Tested Urea Formed Control 1-methionine c Urea Spec. Acti'\l'ity (µm.) ( cts. /min. /µm. ) 2.30 150 (15) 1.92 17 61 (30) 0.39 83 1-lysine (15) 1.36 DL-phenylalanine B Per Cent Inhibition Control 3.96 DL-phenylalanine (40) 2.02 Control 1-methionine 4.24 (20) 3.32 172 194 Per Cent Control 114 129 142 49 193 136 134 22 188 140 Bicarbonate specific activity was 252 cts./min./µ.m. in all cases. Concentration of glutamate in Exp. c. was .03 M. Figures in parenthesis represent µ.m. test compound added. 3. Serine and Glycine - These two amino acids are metabolically related (75) and donate carbon atoms for uric acid and heme synthesis (76,77). They have been found, however, not to donate carbon atoms to urea synthesis. (See Table 7.) The Bernheims (59) have reported that glycine and serine are also inhibitory to urea synthesis from annnonia in rat liver slices, glycine being reported as the most inhibitory amino acid. In re- peated experiments with homogenates, glycine inhibition averaged about 40 50 per cent, while serine inhibits only very slightly. TABLE 7e SERINE AND GLYCINE Exp. No. A B c Compound Tested Control DL-serine (30) Glycine (15) Urea Formed Urea Spec. Activity (µ.m.) (cts./min./µm.) 2.30 150 200 201 1.98 1. 73 Per Gent Control 133 134 Control DL-serine (40) 4.54 4.40 184 121 Control G1¥cine (20) o.89 0.96 72 79 110 152 Bicarbonate specific activity was 252 cts./min./µ.m. in all experiments. Glutamate concentration in Exp. c. was .03 M. Figures in parenthesis represent µm. test substances added. 4. Praline, Glutamine, and Glutathione - An interesting feature of urea synthesis in homogenates is the high (.05 M.) glutamate concentration required for maximum activity. This fact suggests that there may be some metabolic product of glutamate which is concerned with urea formation, and not glutamate itself. Proline, glutamine, and glutathione are products of glutamic acid metabolism, and for that reason are grouped together. Bonner (21) has described a series of "arginineless 11 mutants of Penicillium. The growth requirements of one mutant are satisfied only by arginine; in another by arginine or citru.lline; in a third by arginine, citrulline, or ornithine; a fourth will grow on any member of the ornithine cycle or proline; and a fifth on any member of the cycle, proline or glutamic acid. This raises the possibility that proline might have some function in urea synthesis. Past work on glutainine has been sunnnarized in the introduction. Results obtained from testing these compounds are shmm in Table B. As can be seen from the data, none act as intermediates; moreover, glutamine does not stimulate the reaction. It vrould appear then that at least under the experimental conditions employed, glutamine plays no significant role in the process of urea synthesis. TABLE 8. PROLINE, GIBTAMINE, GLUTATHIONE Urea Formed Urea Spec. Activity (µm.) (cts./min./µm.) Control 1-Proline (20) 1-glutamine (20) 3.96 3.58 3.20 142 192 197 135 139 Control 1-glutamine (20) 4.54 4.06 152 122 80 c Control Glutathione (20) 1.63 2.75 147 140 95 D Control Glutathione (20) 1.66 1.46 72 75 104 EJcp. No. A B Compound Tested Per Cent Control Bicarbonate specific activity was 252 cts./min./µm. in all experiments. Figures in parenthesis represent µm. of test compound added. 42 5. Aspartic Acid and Alanine - P..esults of testing these compounds are given in Table 9. The combination of fumarate and as- partate was also tested in connection with some experiments on the role of glutamic acid, and a sample of these results are given. TABLE 9. ASPARTIC ACID AND ALANINE :EXp. No. A B c Compound Tested Urea Formed Urea Spec. Activity (µm.) (cts./min./µm.) Per Cent Control Control 1-aspartate (20) 1.66 1.82 Bo Control L-aspartate (40) + Fum.arate (120) 0.79 96 1.50 86 90 Control 1-alanine (20) 0.89 0.74 72 76 106 72 lll Bicarbonate specific activity was 252 cts./min./µm. in all experiments. Glutamate WE!:S .OJ M. in Exp. B. · Figures in parenthesis represent µm. of test compound added. 6. Other Amino Acids - Valine (15 µm.), leucine (15 µm.), hydro.xypraline (20 µm.), and threonine (20 µm.) were tested in single experiments and were not found to be active. 43 ?e Discussion of Amino Acids - In general, the amino acids had two effects: an inhibition of urea synthesis, and a tendency to raise the urea specific activty. These effects are apparently unrelated since they have occurred in all possible combinations: inhibition with no increase in specific activity, no inhibition accompanied by a rise in specific activity, both occurring together, and both absent. lu:ry explanation then of the role of amino acids in raising the speci- fie activity of urea, must be independent of the observed inhibition by some amino acids. There are two possible mechanisms by which amino acids could raise the specific activity of urea: (a) By preventing the formation of urea, or urea intennediates from non-radioactive sources. (b) By increasing the rate at which intermediates are removed by side reactions, hence lessening the dilution caused by intennediates present at the start of the reaction. Case (a) can be excluded since there is quantitative evidence to show that all of the urea carbon must come from C02. MacKenzie and du Vigneaud (37) in experiments on methionine metabolism, fed rats methionine labelled with c14 in the methyl group. The ure~a and co 2 excreted on the first day had identical specific activities. The specific activities were also identical on the second day although total urea excretion rose 35 per cent and C02 excretion dropped 13 per cent. It has been shown in this paper that methionine has no action as an intermediate in urea synthesis. It would seem improb- able, therefore, that any urea carbon were to come from sources other than co 2 • vV'e are left then ·wJ.th a mechanism in which amino acids remove intermediates by side reactions. Why the removal of intermediates should result in a higher specific activity for urea can be best understood by considering a pool of intermediate present at the start of the reaction. The rate at which this pool will increase its spe- cific activity will depend on three factors: the size of the pool, the rate at which the intermediate is removed from the pool, and the rate at which radioactive intermediate is introduced. If the rate of removal of intermediates is increased, the pool, at a:rry given time during the reaction period, will have a higher specific activity than if the rate of removal had not been increased. Further, if the increased removal is to form a product which is not urea, since the urea specific activity will reflect that of the pool, the urea specific activity will increase at a faster rate than if no increased removal of intermediate by side reactions had occurred. Amino acids could affect this removal either by reacting with an intermediate or by affecting an enzyme system functioning in its removal. The wide range of amino acids which give this effect (his- tidine, methionine, glycine, serine, lycine, phenylalanine, proline, etc.) would suggest the second mechanism rather than the first, in view of the well known tendency of enzymes to be highly specific. Amino acids as a class are known to influence enzyme systems (78-80). Among the enzymes affected by ~no acids are phosphatases. Bodansky et al. (81) have shown that amino acids as a group are phosphatase inhibitors. This fact seems suggestive in view of the 45 peculiar phosphate relationship in the conversion of ornithine to citrulline in whole homogenate. (43). The reaction requires both inorganic phosphate and either aderzy-lic acid or ATP, aderzy-lic acid being twice as effective as ATP. In Figure 7, a scheme is set up which would explain t he observed effect of amino acids in increasing the urea specific activity, based on their known inhibition of phosphatases. Biotin has been shown on the scheme at the point of initial co 2 fixation since biotin is known to be concerned with the process of co 2 fixation (39); has been shown to be involved in the ability of rats to convert ornithine to citrulline (41); and in turkeys to be concerned with the Vlood-Werkman reaction and the "malic" enzyme of Ochoa (8·2 ). One point warrants further discussion. This is the question of whether arzy- amino acids by raising the specific activity of urea might not mask their own action as a source of urea carbon. This effect is theoretically impossible since not enough of the amino acid could be manufactured using all the radioactive co 2 present to raise the specific activity to the levels observed. D. Carbamyl Glutarn.ic Acid and Carbamyl Aspartic Acid The literature on these compounds has been reviewed in detail in the introduction. Carba.myl L-glutamate, or a closely related com- pound, has been postulated as an intermediate (29) in the conversion of ornithine to citrillline, since it is more active in the semi-isolated "washed residue" preparation than is glutamic acid. In addi- tion, its activity relative to glutamic acid is increased if co 2 is 46 omitted from the system. Tests of this compound in the homogenate system used in this study, however, have led to the conclusion that, while carbamyl glutamic acid appears to play a role in urea formation, it does not function as a source of urea carbon. 7(- First tests of carbamyl glutamic and carbaroyl aspartic acids as interrnediates in urea synthesis showed no tendency on the part of either compound to depress the specific activity of the urea formed. Carbamyl aspartic acid has been tested using five enzyme preparations, and carbamyl glutamic acid on a total of eleven. In no case~as there been a:rry depression of urea specific activity by carbamyl aspartic acid and in only one case 1'f.i..th carbamyl glutamic acid. A sample of the data is shown in Table 10. These results clearly rule out carbaroyl glutamic acid and carbamyl aspartic acid as sources of urea carbon. The results also show that carbamyl glutamic and carbaroyl aspartic acids, like the amino acids, have an erratic tendency to significantly raise the urea specific activity. It would seem reasonable to assume that the mechanism suggested to explain this action by amino acids would apply to carbamyl glutamic and carbamyl aspartic acids. * Three samples of carbamy L-glutamic acid and one of carbamyl 1-aspartic acid were used. The sample of carbaroyl 1-aspartic acid and one of the samples of carbamyl 1-glutamic acid weregenerously supplied by Dr. H. K. Mitchell. Another sample of carbamyl 1-glutamic w!l,s synthesized according to the procedure of Nye and .. Mitchell (83) :· The third sample was a gift of Dr. P.P. Coh~n. All three samples of carbamyl 1-glutamic acid had the same melting point, 158-9°C (corr.) and :mixed melting points showed no depression. The three samples of carba.myl L-glutamic acid have been used interchangeably, and no difference in behavior has been observed. 47 TABLE 10. CARBAMYL L-GLUTAMIC ACID AND CARBAMYL L-ASPARTIC ACID Compound Tested E:cp. No. Urea Formed Urea Spec. Activity (µm.) (cts./min./µm.) Per Cent Control A (Prelim) Control Garb. L-asp. (10) Garb. 1-glut. (10) 0.70 0.62 0.62 118 156 140 132 118 B Control Carb. L-asp. (6) Carb. L-glut. (6) 0.93 0.96 1.04 64 91 78 142 122 Control Carb. L-asp. (20) Carb. 1-glut. (20) 4.54 4.32 4.02 152 162 192 107 126 Control Carb. L-glut. (20) 1.63 2.54 147 154 105 Control 1.66 Carb. L-glut. (20) 1.88 Aspartic Acid (20) 1.82 Garb. L-glut. (20) + Aspartic acid (20) 1.88 72 76 80 ill 78 108 c D E 106 In Ex:periment A., the concentration of bicarbonate was .0075 M. with 224 cts./min./µm. In all other experiments, the bicarbonate concen- tration was .006 with 252 cts./min./µm. present µm. test compound added. Figures in parenthesis re- In view, however, of the importance of carbamyl glutamic acid, further tests on the nature of its action were carried out. Among the first experiments was an attempt to confirm the work on carbamyl glutamic acid using the semi-isolated "washed residue" preparation. 48 The incubation was performed in the usual manner, using washed residue* as the enzyme. The citrulline formed was converted to urea by adding aspartic acid (.01 M.) and a combination of washed residue supernatent and rat heart homogenate. The results, given in Table 11, confirm that carbamyl glutamic acid is more effectjve than glutamic acid in the washed residue preparation. TABLE ll. USE OF WASHED RESIDUE Conditions Urea Formed (µm.) .03 M. L-glutamate 0.14 .03 M. Garb. 1-glutamate 0.50 1-citrulline (2 µ.m.) 0.22 L-citrulline (4 µ.m.) o.44 Concentration of adenylic acid, ornithine, ammonia, and buffer as usual. Following this test, glutamic acid and carbamyl glutamic acid were compared in whole homogenate. In most cases, carbamyl glutamic acid alone caused a small, but distinct, quantity of urea to be formed, * ·washed residue was prepared by centrifuging a 20 per cent homogenate at 3000 g. 10 minutes in a refrigerated centrifuge. The precipitate was washed once with an equal volume of cold isotonic . KCl and resuspended in isotmnic KCl for use. 49 which was non-radioactive. This level of synthesis was never more than about half that obtained with an equimolar quantity of glutamic acid. ·When both compounds were tested together, a synergistic effect was noted. The data pertaining to these effects are shown in Table 12. TABLE 12. CARBAMYL L-GLUTAMIC ACID AND 1-GLUTAMIC ACID Substrate* Elq>. No. A B Urea Formed Urea Spec. Activity (µ.m.) (cts./min./µm.) .03 M. 1-glutamate 4.24 .01 M. 1-glutamate 2.67 •03 M. Garb. 1-glut • o.46 .Ol M• Garb. 1-glut. o.o4 •01 M. Garb. 1-glut. + .Ol M. 1-glut. 4.3S 134 1S4 .03 M. L-glutamate 0.89 .Ol M. L-glutamate o.S9 •03 M. Garb. L-glut • o.47 .01 M. Garb. L-glut. 0.12 .03 M. Garb. 1-glut. + .03 M. L-glut. i.as Per Cent Control 0 ns 0 0 0 217 162 72 66 92 0 0 0 0 102 142 *All experiments include ornithine, ammonia, and adenylic acid as additional substrates. In Experiment B., .03 M. fumarate was present in all vessels. Bicarbonate specific activity was 252 cts./min./µm. in both experiments. To clarify the situation regarding the role of carba.myl glutamic acid may play in the process of urea formation, some of the facts knovm about the action of this compound are swmn.arized below. 50 1. Carbamyl glutamic acid, when tested in homogenate does not donate its carbon atoms to urea synthesis.(This paper). 2. It exhibit s the typical behavior of an amino acid in raising the specific activity of the urea formed. (This paper). 3. When both carbamyl glutarnic acid and glutamic acid are added together in equimolar quantities, a synergistic effect on the level of µrea synthesis is noted. (This paper). 4. Carbamyl gl utamic acid is less effective than glutarnic acid in homogenates, but is more effective than glutamic acid when washed residue is used as an enzyme system, the relative effectiveness of carbamyl glutarnic acid being increased i f co 2 is omitted from the reaction mixture. (44, this paper). 5. When washed residue is used as the enzyme, a balance sheet of annnonia shcnvs onemole of ammonia used ·when citrulline is formai I from ornithine and carbamyl glutarnic acid, while two moles are used when citrulline is formed from ornithine and glutamic acid (29). Point 1. suggests that carbamyl glutamic acid is not concerned vvith the reactions involved in the forma:t.ion of the first substance containing radioactive C02• Point 3. suggests that carbamyl glutamic acid is concerned with the further reactions of this substance to form a product which will react with ornithine to form citrull ine. Point 5. r aises the possibility that this function of carbamyl glutarnic acid may be to introduce a nitrogen atom into the reaction sequence. This interpretation does not, however, explain why the effectiveness of carbamyl glutarnic acid relative to glutamic acid should be increased in the absence of C02 (Point 4.) E. Vitamins 1. Biotin and Thiamin - MacLeod ~ al. (41) have compared the rates of conversion of ornithine to citrulline in livers of normal and biotin deficient rats. Using a semi-isolated enzyme system, they found that the rate in deficient animals was much lower than that in normal rats. MacLeod and Lardy (40) have also found that 1403 into the arginine of liver normal intact rats fix injected NaHc protein six times as fast as biotin deficient animals. Thiamin pyrophosphate (cocarboxylase) is known to be the coenzyme for several reactions involving co 2 • .Among these are three reactions involving the decarboxylation of pyruvic acid, and another in which succinic semi-aldehyde is formed as a result of the decarboxylation of a-ketoglutaric acid. Leuthardt and Glasson (62,63) have observed that the addition of thiamin to liver slices obtained from B1 deficient rats, results in an increased yield, i f pyruvate is used as the substrate, but not i f oxalacetate is used instead. A sample of the results obtained in testing biotin and thiamin are given in Table 13. As can be seen, neither acts as an inter- mediate, nor increases the yield. press the yield slightly. In fact, biotin tends to de- 52 TABLE l3. BIOTIN AND THI.AMIN Eicp. No. A B c Compound Tested Urea Formed Urea Spec. Activity (µ.m.) (cts./min./µ.m.) Control Biotin (20) 1.66 1.40 72 75 104 Control Biotin (20) o.89 o.66 72 78 108 Control Thiamin (20) 4.57 161 162 101 4.36 Per Cent Control Bicarbonate specific activity was 252 cts./min./µm. in all experiments. In Elcperiment B., glutamate was 0.03 M. Figures in parenthesis are µm. of test compound added. 2. Riboflavin - The first experiments carried out on riboflavin indicated that, in the test system used, it behaved like an intermediate in the process of fixing co 2 into urea. The data obtained are shown in Table 14 (see next page). Further evidence, hovvever, suggests that the role of ribo!lavin in urea synthesis is not that of an intermediate. Elcperiments have show.n that the yield in the presence of riboflavin is not dependent on the operation of the ornithine cycle. Experiment A. in Table 15. shows that the presence of the standard substrate mixture (glutamic acid, ornithine, ammonia, and ade:nylic acid) is not required; and furthermore, as Experiment B. demonstrates, even the presence of 53 homogenate is not essential. Since the riboflavin reaction is comple-00- ly independent of the urea synthesis system, it raises the question of whether tne substance reacting with the urease preparation to give ammonia is actually urea. TABLE 14. RIBOFLAVIN Exp. No. A B c Compound Tested Urea Formed Total Urea Radio act. (µm.) (cts./min.) (cts./min.µm.) Urea Spec. Activity Per Cent Control Control Riboflavin (20) 4.24 1.73 566 88 134 50 37 Control Riboflavin (20) 1.66 2•.56 ll9 8 72 3 4 Control 0.79 Riboflavin (20) 3.99 Riboflavin (1.0) 0.74 Riboflavin (0.05) o.14 76 3 96 1 2 2 0 0 1 2 0 Bicarbonate specific activity was 252 cts./min./µm. in all experiments. Glutamate concentration in Experiment c. was 0.03 Jjl. Figures in parenthesis represent µm. test compound added. Riboflavin itself does not react with the urease solution to give annnonia, indicating that one or more of the treatments prior to the addition of urease renders riboflavin susceptible to attack by the urease pr eparation.* Of interest in this connection is the apparent high concentration of flavoprotein in the urease used, as evidenced * For details of the analytical procedure, see Sec. ]J;...C or Figure 4. 54 by its intense yellow-green color. TABLE 15. RIBOFLAVIN BREAKDOWN Yield "Urea" (µm.) Conditions Exp. No. A B Homogenate + riboflavin Homogenate + riboflavin + substrate mixture 2.68 Homogenate alone Riboflavin alone Homogenate + riboflavin 0.94 2.90 2.24 3. 74 The substrate mixture was omitted in Experiment B. Otherwise, the experiments were carried out in the usual manner. Riboflavin was added at a level of 20 µm. The figur es on total urea radioactivity given in Table 14. show that the presence of riboflavin strongly inhibits the synthesis of 1 J, _ urea from C-+o 2• -!( Confirmation of this fact was obtained by testing riboflavin at low concentrations. The addition of 0.05 µm. of ribo- flavin ( a riboflavin concentration of 1.3 x 10-S)caused aL~ost complete inhibition. In an ~ttemp~ to elucidate the nature of the r iboflavin inhibi- tion, tests were carried out to see if radioactive urea precursors would accumulate in systems inhibited by riboflavin. In one set of Experiments have shovm that riboflavin does not interfere vdth urea recoveries, indicating that the inhibition is in the synthetic and not the analytical reactions. · -11- 55 vessels to which 20 µm. of riboflavin had been added, the solution and precipitate (denatured protein and riboflavin) remaining a£ter the removal of urea C02, were tested and found to be non-radioactive. This fact suggests that the riboflavin inhibition occurs at an early stage in the process of fixing co 2 into urea. F. Formic Acid, Para-amino-benzoic acid, and 4 amino-$ imadazole carboxamide Formic acid has been implicated in urea formation since the work of Bach (15), w'no showed that urea synthesis in rat liver slices, using a bicarbonate buff er with glutamic acid and NH4Cl as substrates, was greatly stimulated by the addition of sodiwu formate. Tests of formate as an intermediate have been consistently negative, however, and indicate no direct role of formate in urea synthesis. PAB when tested proved to be a very strong inhibitor of urea synthesis. As the data to be presented show, inhibition can be de- tected at concentrations aslow as 1.3 x lo-5 M. PAB and the other inhibitors w'nich have been reported in this paper (amino acids and riboflavin) have as a common characteristic either a primary or secondary amino group in their molecules. Subsequent evidence suggested that one of the points of inhibition may be the conversion of ornithine to citrulline. In some experiments carried out using the semi-isolated enzyme system of Cohen, which catalyzes the formation of citrulline from ornithine, ammonia, glutamic acid, adenylic acid, and C02; it was observed that increasing ornithine concentrations inhibited the formation of citrul- 56 line. Data are only available from two experiments, but in these the ornithine concentration required for 50 per cent inhibition was directly proportional to the maximum rate obtained. If the rate (which was varied by changing the concentration of bicarbonate) is a function of the concentration of some intermediate, this would suggest that ornithine(and possibly other inhibitors) compete with the intermediate for an enzyme surface. This interpretation of the inhibition results is in agreement with the evidence suggesting that riboflavin inhibition occurs at an early stage in urea synthesis. Studies of sulfonamide inhibition in E. Coli and its reversal by PAB and purines (84) have indicated that the substance 4 amino-5imadazole carboxa.mide is converted to purines by reactions requiring the presence of PAB and that sulfa antagonizes this reaction. For this reason, 4 amino - 5 imadazole carboxamide was tested in urea synthesis.~~ The results of these tests and those of formate and PAB are shown in Table 16. * A sample of the hydrochloride of this compound was kindly supplied by Dr. E. Shaw of the Rockefeller Institute for Medical Research. The sample had a melting point of 225-230° C (melting point in the literature 255-6°c) and was grey in color. It yielded a faintly purple solution. 57 TABLE 16. FORMATE, PAB, AND 4 AMINO-S IMADAAJ LE CARBOXAMIDE Eicp. No. Urea Formed Total Urea Radioact. (µm.) (cts./min.) (cts./min./µm.) 1.08 1.25 170 146 161 116 Control + PAB (20) 1.66 o.oo 118 72 Control + PAB (20) + PAB (l.O) + PAB (0.05) 0.79 0.28 0.16 o.66 76 0 0 48 96 73 76 Control + Formate (20) + Imad. (20) + Imad. (20) + Form.ate (20) o.89 o.84 1•.50 64 62 54 72 74 36 103 1.30 60 4.5 62 Control Imad. alone - substrate mixture absent. + Imad. (20) + Imad. (20) + Formate (SO) + Imad. (20) + PAB (0.20) + PAB (0.20) 4.57 736 161 1.34 .5.50 0 770 0 140 87 4•.54 646 142 88 4.26 3. 78 618 634 145 168 104 Compound Tested A Control Prelim + Formate (100) B c D E 0 Urea Spec. Per Cent Activity Control 72 0 0 Concentration of glutamate in Experiments D. and E. was 0.03 M. carbonate specific activity was 252 cts./min./µm. in all cases. gures in parenthesis represent µm. test compound added. so 90 BiFi- Experiments D. and E. of Table 16. show a depression of the urea specific activity resulting from the addition of 4 amino 58 5 :i.madazole carboxamide. That this depression is not due to the ac- tion of 4 amino-5 :i.madazole carboxamide as an intermediate is suggested by two results. First, the substituted :i.madazole does not depress the total urea radioactivity; it merely increases the yield (this behavior is reminiscent of that of arginine which was reported earlier). Second, this increase in yield is not dependent on the operation of the ornithine cycle. The urea analysis, then, gives a crude measure of 4 a.mino-5 imadazole carboxamide by measuring its abilj_ty to increase the urea assay over that of the control. Using this method, the data show that both PAB and formate facili tate the conversion of 4 amino - 5 imadazole carboxamide to some substance which will not increase the urea assay. PAB is concerned with the conversion of 4 amino-5 imadazole carboxamide (or a related compound (85) ) to purines. Isotope evi- dence (76) has shown that formate is the source of the carbon in the number 2 position in purines. This is the carbon which must be added to 4 amino - 5 imadazole carboxamide to form purines. It would, there- fore appear probably that a purine is the substance formed which fails to increase the urea assay. Attempts to secure hypoxanthine synthesis from 4 amino - 5 imadazole carboxamide in homogenates have been reported. Schulman, Buch- anan, and Miller (86) have found that incubation of c14 labelled h amino- 5 imadazole carboxamide with pigeon liver homogenates results in the formation of c14 labelled hypoxanthine. However, Greenberg (87) has reported that the substituted imadazole is not an intermediate in hypoxanthine synthesis in pigeon liver homogenates. 59 IV. DISCUSSION A total of thirty-three substances, including members of the Krebs tricarboxylic acid cycle, a variety of amino acids, vitamins, and some miscellaneous compounds, have been tested as possible intermediates in the process of fixing C02 into urea. In testing these substances, several effects have been noticed. Citrulline and arginine, as predicted by theory, behave as intermediates in ur ea synthesis, when tested for their ability to depress the specific activity of urea formed from c14o2• The sub- stances 4 amino - 5 imadazole carboxamide and riboflavin also showed the same property of lowering the urea specific activity. However, in both these cases, it was shown that this depression was the result of a mechanism independent of the ornithine cycle. In the case of riboflavin, it was found that some step in the analytical scheme rendered riboflavin susceptible to attack by the urease preparation used; and that, in addition, riboflavin is a potent inhfbitor of urea formation at concentrations as low as 1.3 x lo-5 M. The evidence suggested that the point of inhibition was at an early step in the sequence of reactions studied. In the case of 4 amino - 5 imadazole carboxamide, it ~ppeared that this substance broke down independently of the ornithine cycle to increase the urea assay value. By the use of this cr iterion as a measure of the 4 amino - 5 imadazole carboxamide remaining at the end of the reaction period, it appeared that this compound, under the stimulation of PAB and formic acid, was being transformed into 60 some product which did not affect the urea assay; and that this substance was probably a purine. The inhibition of urea synthesis by certain amino acids, which had been reported in liver slices, was confirmed for homogenate. In addition, the list of inhibitors was extended to include PAB, ornithine (a substrate for the reaction ), and riboflavin (mentioned above). Amino acids and the compounds carba.tey"l glutamic acid and carba.myl aspartic acid were further shown to raise the specific activity of urea formed under the experimental conditions employed. An explana- tion of this effect has been offered based on the known inhibition of phosphatases by amino acids. 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I COOH .t,rrd ~·- nine 20 a Figure2-Reaction Vessel solution inlet gas i ri I et stopcock B (one way) stopcock A gas {two way) outlet --E<'----- T 2 4 1 40 - - - 50 ml. Erlenme.yer 22a Figure 3 Aeration of C02 co 2 Free air 50 ml. Erlenmeyers I \ 27 a FIGURE 4. ANALYTICAL SCI-fr.1.IE Substrates, Buffer, Enzyme and NaHc1 4o l 3 Reaction - 6o min, Addition 5 M. KOH Shaking vrlth fan. I Discard t Solution+ co - I 3 Addition 41 Per Cent Acetic Acid pH to 5.2 I Solution (containing urea) Discard 1' Boil to coagulate protein. Cool and add urease,. r- - - - - - - - - - - - - - I : To center well ; for p ep. BaC03 I I I CQ 'A Solution + NJt+ from uI'ea 2 !_ _ ~ -- -- - - - --- - - -~J I .KB02 and ,-- - - -- -- - - ---- - -----, I aeration I I Into N/10 H2S04 f or Nesslerization : : I I B. L _ - - - - - I I - - - - - - - - - - - -• Residue Discarded Ao At this point, the measurement of the total number of counts fixed into urea is obtained. Bo At t..his point, the yield of urea is determined. 31a Figure 5 - Homogenate cone. vs. yield 4.0 3.0 ~ ::i. - 2.0 0 .....I w - >- 1.0 0.2 HOMOGENATE 0.4 CONC. 0.6 (mis homog . /3.8mls) 0.8 1.0 33 a Fig u r ,e 6 Bicarbonate specific activity vs. time 80 0 a:: 0 w N ~ .... 70 I- <t w :::> _J <( 60 > 0~ 20 TIME (min) 40 60 80 100 120 h5 a FIGURS 7o MECR~IITSU 'JF Ai.IINO A.CID El"FZCT Inhibited by amino acids and perhaps by adenylic acid l Phosphatases {2) Biotin 1 > XC ,.,p ----~ Non-urea X- P + co 2 - - - - - -XC il\. (1) ATP x + NH3 products XCN l 1 !IP04= (3) XCN-P XCN""P +ATP Arginine Balic + ADP Urea (1) X represents a compound w'nich when phosphorylated, would r eact Yrith co2 to form the pritnarrJ fj_xation product XC. (2) Am.ino acids by inhibiting the phosphatases breaking dorm XC- P to XC would stimulate the r emoval of XC to non-urea productso The ~reate::.· effectiveness of adenyli..c acid over Nfi? might be a con sequence of its inhibi tlon of this enzyme. (3) The requirements for inor::::mic phosphate would be explained if the system generated its ovm high energy phosphate for use in the reaction ornithine to citruJ.line.