ERZYMES 9F THE ORNITHINE-UREA CYCLE {3% LYMPHGCYTES IN LOSS-TERM CULTURE Bissertation for the Degree of Ph. D. MiGHiGfiN STAYE UNEVERSITY 16E} BETTY R003 2974 .~ . Fwy-0'"? f" LIBRARY 53.51.3913 State ; .. University This is to certify that the thesis entitled big/7W” 0% 77% QMw’Z/{ou - Clad, (9&4 “é." éWMyf‘fl' 1’." 5:10)}, — Lie/W éqaC/Z‘W presented by has been accepted towards fulfillment of the requirements for 70/2 49 degree in W Wei/WW??? Major professor Date 62/7— 75/ 0-7 639 amgmc 37 ‘ HOAG & SONS' ~ BOOK BINDERY LIBRARY amDERs menu. mam.- of th cytes growt Enzyr ABSTRACT ENZYMES OF THE ORNITHINE-UREA CYCLE IN LYMPHOCYTES IN LONG-TERM CULTURE BY Lou Betty Rood The present study was undertaken to determine which of the enzymes of the ornithine-urea cycle occur in lympho- cytes in long-term culture. Lymphocytes in logarithmic growth phase were used for the enzymatic determinations. Enzyme activity levels were established for ornithine carbamoyl transferase, argininosuccinic acid lyase and arginase. Specific activity for ornithine carbamoyl trans- ferase was .41 umoles citrulline per milligram protein per hour. Specific activity for argininosuccinic acid lyase was 5.25 mu moles urea per milligram protein per hour. Arginase specific activity was 23 mu moles urea per milli- gram protein per hour. Michaelis-Menten constants were established for the substrates of ornithine carbamoyl transferase and arginase. For ornithine carbamoyl trans- ferase the ornithine Km was between is and 17.5 mM; the 1 Lou Betty Rood carbamoyl phosphate Km was 1.5 to 2.2 mM; the Km for arginase was between 2.0 and 2.2 mM. The kinetic studies of ornithine carbamoyl trans- ferase, using ornithine as substrate, and arginase suggest that lymphocytes contain distinct isozymes when compared with rat liver. Lymphocytes in long-term cultures are initiated by Epstein-Barr virus. The possibility is dis- cussed that the isozymes described are products of viral rather than lymphocytic DNA. ENZYMES OF THE ORNITHINE-UREA CYCLE IN LYMPHOCYTES IN LONG-TERM CULTURE BY Lou Betty Rood A DISSERTATION Submitted to Michigan State University in partial fulfillment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Zoology 1974 his cla: of' Of( ACKNOWLEDGMENTS I thank my professor, Dr. James V. Higgins, for his support and encouragement; His enthusiasm and confidence have helped me develop my scientific attitude. Special thanks are due to Dr. Herman M. Slatis, for his pointed comments and critical academic standards have clarified many thinking processes. Dr. Arthur F. Kohrman of the Department of Human Development for his suggestions of directions to try in this research especially merits thanks. I also thank Dr. Richard L. Anderson, of the Bio- chemistry Department,for his time and good advice while serving on my committee. The people of the human genetics group hold a spe- cial place for their help, friendship, encouragement 'through difficult hours and years. They are Michael Abruzzo, Ajovi Scott-Emuakpor, Gary Marsiglia, Robert Pandolfi, Rachael Rich,.Terry Hassold, Dan Friderici, Sally Brian Cullen, Frankie Brown, Cathy Oberg Blight, Richard Rutz, Jerry Purdy, Astrid Mack, Dan Mankoff, and ii especially Carola Cattani Wilson and Ronald Wilson for reasons they know very well. To my parents, Frank W. and Ella L. Rood, who never considered that I might not make it, and to my children, Carl and Roxanne Richardson, who have done without their share of attention for much too long, my love and gratitude extend infinitely. iii TABLE LIST OF TABLES . . . . . LIST OF FIGURES. . . . . . INTRODUCTION . . . . . . LITERATURE REVIEW. . . . . Ammonia Disposal Urea Synthesis . . . . Urea Cycle Errors. . . Carbamate kinase EC 2.7.2.2 (CPS) OF CONTENTS Ornithine carbamoyl transferase EC (OCT)...... Argininosuccinic acid synthetase EC 6.3.4.5 (ASAS). o o o o Argininosuccinic lyase Arginase EC 3.5.3.1. . . . . Inheritance. . . . Study Methods. . . Enzymes of the Urea Cycle in iv EC 4.3.2.1 (ASase). Lymphocytes . . . Page viii ix 10 11 12 13 14 15 17 TABLE OF CONTENTS (cont.) Page MATERIALS AND METHODS. . . . . . . . . . . . . . . . 19 Liver Controls . . . . . . . . . . . . . . . . . 19 Cell Lines . . . . . . . . . . . . . . . . . . . 19 Maintenance. . . . . . . . . . . . . . . . . 19 Harvesting . . . . . . . . . . . . . . . . . 21 Preparation of Cell Lysate and Liver Homogenate. 22 The Colorimetric Assays. . . . . . . . . . . . . 23 The Enzyme Assays. . . . . . . . . . . . . . . . 24 Ornithine carbamoyl transferase. . . . . . . 24 .Argininosuccinic acid synthetase . . . . . . 26 Argininosuccinic acid lyase. . . . ... . . . 27 Arginase . . . . . . . . . . . . . . . . . . 28 RESULTS. . . . . . . . . . . . . . . . . . . . . . . 29 Lymphocytes. . . . . . . . . . . . . . . . . . . 29 Growth Rates . . . . . . . . . . . . . . . . 29 Viability. . . . . . . . . . . . . . . . . . 31 Protein Content. . . . . . . . . . . . . . . 31 Growth on Arginine-Free Medium . . . . . . . 33 Urea and Citrulline Determinations . . . . . . . 34 The Enzymatic Reactions. . . . . . . . . . . . . 34 Ornithine Carbamoyl Transferase. . . . . . . . 36 V TABLE OF CONTENTS (cont.) Page The Enzyme Assays for Ornithine Carbamoyl Transferase. . . . . . . . . . . . . . . . . . 40 Lability at -80° . . . . . . . . . . . . . . 42 Time Dependency. . . . . . . . . . . . . . . 42 Protein Dependency . . . . . . . . . ._. . . 42 Specific Activity. . . . . . . . . . . . . . 45 Location of the Enzyme . . . . . . . . . . . 45 Establishment of Michaelis-Menten Constants (Km) for OCT . . . . . . . . . . . . . . . 47 Ornithine Km for OCT . . . . . . . . . . 47 Carbamoyl Phosphate Km for OCT . . . . . 53 Argininosuccinic Acid Synthetase . . . . . . 59 Urease Effects . . . . . . . . . . . . . 63 Citrulline Concentrations. . . . . . . . 66 Time and Dilution Effects. . . . . . . . 66 Argininosuccinic Acid Lyase. . . . . . . . . 68 Specific Activities. . . . . . . . . . . 69 Arginase . . . . . . . . . . . . . . . . . . 71 Time Dependency. . . . . . . . . . . . . 71 Protein Dependency . . . . . . . . . . . 72 Specific Activity. . . . . . . . . . . . 72 Establishment of Michaelis-Menten Constants. 75 vi TABLE OF CONTENTS (cont.) Page DISCUSSION . . . . . . . . . . . . . . . . . . . . . 83 Lymphocytes. . . . . . . . . . . . . . . . . . . 83 Growth Rates and Viability of Lymphocytes. . 83 Growth on Arginine-Free D' Medium. . . . . . 84 The Enzyme Assays. . . . . . . . . . . . . . . . 85 Ornithine Carbamoyl Transferase. . . . . . . 86 Nonenzymatic Reaction. . . . . . . . . . 86 Ornithine Km for OCT . . . . . . . . . . 89 Isozymes for OCT . . . . ._. . . . . . . 9O Carbamoyl Phosphate Km for OCT .i. . . . 92 Argininosuccinic Acid Lyase. . . ._. . . . . 93 Arginase . . . . . . . . . . . . . . . . . . 94 Arginine Km for Arginase . . .1. . . . . 95 Isozymes for Arginase. . . . . . . . . . 95 SUMMARY. . . . '.' . . . . . . . . . . . . . . . . . 97 APPENDICES . . . . . . . . . . . . . . . . . . . . . 98 RPMI 1640 MEDIUM . . . . . . . . . . . . . . . . 98 D' MEDIUM. . . . . . . . . . . . . . . . . . . . 99 BIBLIOGMPHY O O O O O O '0 O O O O O O O O C O O O O 1 00 vii LIST OF TABLES 1. Lymphocytes harvested from four lines during one eight-week period. . . . . . . . . . . . 2. Viabilities for four lines of lymphocytes harvested during an eight-week period. . . . 3. The production of color by carbamoyl phosphate and ornithine, with and without enzyme . . . 4. Color development from lymphocyte lysate in citrulline determinations. . . . . . . . . . 5. The effects of urease on optical density shifts in the ASAS assay with 1 mM citrulline in assay medium . . . . . . . . . 6. Optical density shifts recorded in ASAS assay after incubation of serial dilutions of liver homogenate in an assay mixture con- taining 1 mM citrulline and urease . . . . 7. Argininosuccinic acid lyase activity . . . . 8. Michaelis-Menten constants for ornithine carbamoyl transferase. . . . . . . . . . . . viii Page 30 32 .38 41 64 67 70 88 Figure 1. 3a. 3b. 4a. 4b. LIST OF FIGURES Page The Krebs-Henseleit ornithine-urea cycle. . . 5 Interrelationship of the ornithine-urea and the tricarboxylic acid cycles . . . . . . . 8 Optical density vs uMoles urea. . . . . . . . 35 Optical density vs uMoles citrulline. . . . . 35 Ornithine 100 mM, carbamoyl phosphate increasing. . . . . . . . . . . . . . . . . 39 Carbamoyl phosphate 50 mM, ornithine increaSing. O O O O O O O O O O O O O O O O 39 Ornithine carbamoyl transferase activities before and after storage at -80°C . . . . . 43 Citrulline production by OCT as a function of time by lymphocytes. . . . . . . . . . . 44 Citrulline production by OCT as a function of lymphocyte protein concentration . . . . 46 Rate of citrulline production by OCT in liver as a function of ornithine concen- tration O O O O O O O 0 O O I. O O O O O O O 48 Rate of citrulline production by OCT in lymphocytes as a function of ornithine concentration C O O O I O O O O O O O O O O 49 ix LIST OF FIGURES (cont.) Figure 10. Lineweaver-Burk plot (1/V vs l/S) of OCT in liver as a function of ornithine concen- tration . . . . . . . . . . . . . . . . . . 11. Lineweaver-Burk plot (l/V vs 1/S) of OCT in lymphocytes as a function of ornithine concentration . . . . . . . . . . . . . . . 12. Hanes plot (S/V vs S) of OCT in liver as a function of ornithine concentration . . . . l3. Hanes plot (S/V vs S) of OCT in lymphocytes as a function of ornithine concentration. . 14. Rate of citrulline production by OCT in liver as a function of carbamoyl phosphate concentration . . . . . . . . . . . . . . . 15. Rate of citrulline production by OCT in lymphocytes as a function of carbamoyl phOSphate concentration . . . . . . . . . . 16. Lineweaver-Burk plot (l/V vs l/S) for OCT in liver as a function of carbamoyl phosphate- concentration . . . . . . . . . . . . . . . l7. Lineweaver-Burk plot (l/V vs l/S) for OCT in lymphocytes as a function of carbamoyl phosphate concentration . . . . . . . . . . 18. Hanes plot (S/V vs S) of OCT in liver as a efunction of carbamoyl phosphate concen- tration O O O O O O I I O O O I O O O O O O 19. Hanes plot (S/V vs S) of OCT in lymphocytes as a function of carbamoyl phosphate concentration . . . . . . . . . . . . . . . 20. Urea production by arginase as a function of lymphocyte protein concentration. . . . . . Page 51 52 54 55 56 57 58 6O 61 62 73 LIST OF FIGURES (cont.) Figure ‘ . Page 21. Urea production by arginase in liver as a function of arginine concentration. . . . . 74 22. Rate of urea production by arginase in liver as.a function of arginine concentration . . 76 23. Rate of urea production by arginase in lymphocytes as a function of arginine . concentration . . . . . . . . . . . . . . . 77 24. Lineweaver-Burk plot (l/V vs 1/S) of arginase in liver as a function of arginine concen- tration o o o o o o o o 0 L. o o o o o o o o I 78 25. Lineweaver—Burk plot (l/V vs 178) or arginase in lymphocytes as a function of arginine concentration . ... . . . . . . . . . . . . 79 26. Hanes plot (S/V vs S) of arginase in liver as a function of arginine concentration . . 80 27. Hanes plot (S/V vs S) of arginase in lympho- cytes as a function of arginine concentration . . . . . . . . . . . . . . . 81 xi INTRODUCTION A new technique has been established for obtaining apparently permanent cell lines of lymphocytes (Choi and Bloom, 1971). This new development is important as a tool for studying metabolic errors in human beings. This inves- _tigation was undertaken to determine which of the enzymes of the urea cycle could be demonstrated in cultured lympho- cytes. Argininosuccinic acid synthetase and argininosuc- cinic acid lyase are widely found in cultured tissue (Eagle, 1959; Schimke, 1963). From analogy, Spector and Bloom (1973) have shown indirectly that the two enzymes known to be in cultured fibroblasts are present in cultured lymphocytes. Using labelled citrulline, an amino acid not used in protein synthesis, as a substitute for arginine in culture medium, they showed that lymphocytes of normal people, but not those of a patient with citrullinemia (argininosuccinic acid synthetase deficiency),could incor- porate the label into the trichloroacetic acid precipitable fraction. They also reported that Kennaway, using radio-chemical methods, has shown activity of that enzyme in lymphocytes from normal people, but not in those from the citrullinemic patient. Arginase activity had been demonstrated in HeLa strains and in a mouse fibroblast strain (Schimke, 1963) as well as in red blood cells (Tomlinson and Westall, 1964) and freshly drawn "leuko- cytes" (Reynolds et al., 1957). This last activity was highly variable, from 0-270 mg urea nitrogen liberated per 1010 leukocytes. Ornithine transcarbamylase activity has been re- ported in significant amounts only in liverand small intestine (Reichard, 1960). It does not occur in cultured fibroblasts (Schimke, 1963). It is often measured in serum, where it is directly proportional to the amount of liver degeneration occurring, and it is used as an estimate of the extent of liver damage. This study attempts to show that arginase and ornithine transcarbamylase activity are present in lympho- cytes in long-term culture. LITERATURE REVIEW Ammonia Disposal Of the three major caloric components of animal diets, fats and carbohydrates can be completely oxidized to carbon dioxide and water, but proteins provide an addi- tional metabolite, ammonia, which may require additional enzymes for its disposal. Many water dwellers can excrete ammonia through the gills or skin directly into the envi- ronment, but the development of impermeable skins and exploitation of water-poor environments has been made pos- sible by two alternate methods of ammonia disposal. Urea, which is nontoxic and quite water soluble, is made by mammals and some reptiles. Uric acid, which is very in- soluble, and therefore nontoxic even at saturating concen- trations, is formed by birds and by many reptiles to dis- pose of ammonia. Some reptiles make use of both of these methods (Mora et al., 1965). As the reptilian ancestors of mammals and birds were probably both ureotelic and uricotelic, it is not 51'. 3C CO 1.-. surprising that some of the enzymes from each cycle are active in most animals. In mammals, for example, purine and pyrimidine production is dependent upon some of the same enzymes as those found in the uric acid pathway, al- though other enzymes of the pathway are missing. It has also been reported that the first four enzymes active in urea biosynthesis (but not arginase) increase and decrease concordantly on a specific basis, perhaps suggesting a common control mechanism for these enzymes (Mora et al., 1965). In humans the urea cycle normally protects indi- viduals from ammonia intoxication. Because of the occur- rence of inborn errors of metabolism of the urea cycle, the enzyme activities of the cycle are of interest in human genetics. Urea Synthesis Urea synthesis (Figure 1) involves the attachment of two ammonia groups to a carbon atom through a series of five steps (Krebs and Henseleit, 1932; Ratner and Pappas, 1949). Both ammonia molecules are derived from ammonia 6.93 o2?2_£.§:o_o6zujzx 3: .. 2.5... 05.3.50 .5 _ Ban. 1 18w .68 N :2: . ammy «so Eon omc_3§_c_2.< :00u. szw am 1 2% mm x , : -Zuuv :oo :2 ...—£92 . :oOO 0.... D M N . «:2 I at . x :2 mrz 038.5“. residues of amino acids. One is attached to the carbon atom of CO2 upon incorporation into carbamoyl phosphate, while the other is derived from the a-amino group of aspartic acid during a two-step process (Ratner and Pappas, 1949). The manufacture of carbamoyl phosphate from free NH3, ATP, and CO is considered to be the first step in 2 urea synthesis. The next four steps form a cycle, using an ornithine moiety as a backbone to which are attached various other moieties that are subsequently altered and split off, finally resulting in the release of urea and the return of ornithine to its original state. The first stage of the cyclic part of urea syn- thesis is the condensation of carbamoyl phosphate with ornithine to produce citrulline and Pi. Next aspartic acid condenses with the citrulline to form argininosuccinic acid with the cleavage of ATP to AMP and PPi providing the energy. Argininosuccinic acid (ASA) is then cleaved, pro- ducing arginine and fumarate, which is a metabolite of the Krebs carboxylic acid cycle. Arginine may be cleaved to form ornithine and urea or may be used as a building block for protein synthesis. Because of interconnections with other cycles and pathways in the cell, especially the Krebs TCA cycle, the fumarate released by the cleavage enzyme may be reconsti- tuted as either ornithine or aspartic acid (Figure 2). Urea Cycle Errors It is conceivable that errors in any one of the several pathways that connect with the urea cycle may have effects upon an organism's ability to regulate its ammonia disposal. The inborn errors of metabolism that are apparently NOT connected with the urea cycle but that are associated with hyperammonemia, are hyperlysinemia (Colombo et al., 1967), low absorption of basic amino acids (Perkeentupa and Visakorpi, 1965), hyperornithinemia and homocitrul- linuria (Shih et al., 1969), methylmalonic acidemia (Shih and Efron, 1972), and hyperglycinemia associated with an isoleucine metabolic defect (Keating et al., 1972). At present there are no known interrelationships among these diseases except for the associated hyperammonemia. 6.9x» 28 2.5.38... 2.. 18 332...... o... to ale-8.328.... .N at it»... u on» 6:98.98 n (<0 81.5.8 u 25 £8.55 a «:2 20.2.. a ..(2 2225. u .22 86%;... 3.55... n (no 2.2.5:...w u 30 6.183.. 2.858 u a? 33.6% 84.8 «NCO _ . 5.3.1 .338 ...u 25:...» u :u 228%.. u a? i .8183??? u <2 , . 2.39.. a 0.2 633.8348 n 9.... 33.6%; There are many other conditions that affect blood ammonia levels, liver failure being the major clinical cause of hyperammonemia. Among the less commonly seen conditions are several rare metabolic disorders which result in hyperammonemia. Five of these conditions would be directly predictable from the urea cycle as it is now understood (Figure 1). In the following review, the established interna- tional nomenclature and numerical designation are used to identify each enzyme, followed by the few-letter abbrevia- tion that shall be used (Levin, 1971). Carbamate kinase EC 2.7.2.2 (CPS) The formation of carbamoyl phosphate from ammonia, bicarbonate, and ATP is the first step in urea synthesis. Carbamoyl phosphate synthetase (carbamate kinase) is the mediating enzyme. A defect in its function causes the predictable hyperammonemia, and also is related to unusual amino acid levels in blood or urine. In one case of partial CPS deficiency, there was increased excretion of ornithine and proline (Kirkman and Kiesel, 1969). In 10 another case, in which the primary error was very low 993 activity accompanied by 20% CPS activity, the excretion of glutamine was greatly elevated (Levin and Russell, 1967). CPS deficiency is very rare, only two primary cases having been reported in addition to the one immediately above (Freeman et al., 1964; Hommes et al., 1969; Kirkman and Kiesel, 1969). Ornithine carbamoyl transferase EC 2.1.3.3 (OCT) Ornithine transcarbamylase mediated condensation of carbamoyl phosphate with ornithine makes citrulline. Its deficiency is the second most commonly reported defect for the urea cycle, some twenty cases having been reported to date (Russell et al., 1962; Levin and Russell, 1967; Corbeel et al., 1968; Hopkins et al., 1969; Levin et al., 1969a; Schneider et al., 1970; Campbell et al., 1971; Matusuda et al., 1971; Sunshine et al., 1972; Campbell et al., 1973; Short et al., 1973). Of the few males de- scribed, only two, with unusually mild forms of the disease, have survived early infancy (Levin et al., 1969b; 11 MacLeod et al., 1972). The age of onset and the severity of symptoms varies widely among females, ranging from habitual protein avoidance, to severe episodes of lethargy, seizures, retarded mental and physical development, and early death. Argininosuccinic acid synthetase EC 6.3.4.5 (ASAS) ASAS is the enzyme responsible for the condensation of aspartate with citrulline to make argininosuccinic acid, releasing pyrophosphate. Lack of the enzyme leads to citrullinemia, as well as to the expected hyperammonemia with high protein dietary stress (Scott-Emuakpor et al., 1972; McMurray, 1963). Two of three surviving cases are associated with severe mental retardation. The one re- ported adult is mildly retarded. Three of the four cases of this disorder had normal blood urea nitrogen and normal urea output. One case, which was fatal in infancy, showed. no activity of the enzyme in the liver but some low level activity of ASAS in the kidney (Vidailhet et al., 1971). One could hypothesize either an isozymal system or 12 organ-specific control mechanisms to Account for this observation. Morrow et al. (1967) reported a patient in whom urea production was low; Tedesco and Mellman (1967) showed that the citrulline Km of the enzyme in fibroblasts from that patient was many times normal, meaning that the affinity of the enzyme for citrulline was very low. One possible case of citrullinemia was reported to be asso- ciated with cystinuria, but the patient died before fur- ther studies could be initiated (Visakorpi, 1962). Argininosuccinate lyase EC 4.3.2.1 (ASase) Immediately upon the manufacture of argininosuc- cinate, the splitting enzyme, ASase, catalyses the reaction of ASA to arginine + fumarate. The enzyme catalyzing this reversible step, hypothesized by Ratner and Pappas in 1949, was proven to be correct by the first description of a person affected with ASase deficiency in 1958 (Allan et a1” 1958). The patient showed massive excretion of ASA in the urine and elevations of ASA in serum.‘ This is the most commonly reported error of the urea cycle,with twenty-three 13 known cases as of 1972 (Shih and Efron, 1972). The effects of the disease are highly variable. Some of the children are severely retarded; some have close to normal intelli- gence. The symptoms often include friable, patchy hair. The severity of effect may be unrelated to amount of defi- ciency. One child, diagnosed in early infancy, completely lacked ASase activity in cultured fibroblasts, but was apparently normal mentally after two years on a low protein diet (Shih, 1972). Another patient with very low, but mea- surable, activity died at six days of age (Kint and Carton, 1968). Arginase EC 3.5.3.1 The final enzyme in the series, arginase, has been reported to be deficient in only two families (Peralta- Serrano, 1965; Terheggen et al., 1969). Terheggen's family was the product of a consanguineous mating, and included two female patients with very low or absent levels of arginase activity in their red blood cells, the patients' two sisters and the parents with low levels of arginase, and one sister with normal levels. 14 Inheritance The genes for ASase deficiency and arginase defi- ciency would seem to be autosomal recessive because of sibling involvement and reduced enzyme activity in both parents when studied. There are too few cases to judge CPS deficiency inheritance. Although there are no reported sibships involved in ASAS deficiency, the fact that both males and females have been reported with this disease would lead one to speculate that this disease is caused by an autosomal recessive gene. OCT deficiency may be caused by a sex-linked gene (Short et al., 1973). Several families were described with this defect. In one of the families, a partial defect was transmitted from females to females with apparently no affected males. In another family, three male infants of a woman with a partial defect died shortly after birth with a total deficiency of OCT. Lyon (1961) has proposed that in females, one or the other of the X chromosomes in each cell becomes in- active in early embryogenesis. All descendants of each cell will have the same active X and the same inactive one as the original cell. The descendants of an embryonic liver cell in which the X bearing a defective gene for 15 OCT was active would form a clone of cells deficient for OCT. As the original inactivation process is random and early in development, one would expect a wide range of effect on the carriers of one normal and one defective gene for OCT. The first reported family with this defect included a set of identical female twins and their female cousin. The mothers of the patients were sisters. The mothers appeared unaffected. One twin died in childhood, the other had no symptoms until the age of nine (Russell et al., 1962; Levin and Russell, 1967). Lyonization offers an explanation for the wide variability. Study Methods Among the methods of studying the genetic and metabolic bases for variation in humans are: l) observing anomalous findings in the body fluids, and speculating from the biochemical literature the metabolic pathways which might be involved; 2) enzyme analysis of biopsy and autopsy materials; 3) stressing the supposedly defective pathway by loading the patient with a substrate of the suspect enzyme; 4) administering substrates labelled with 16 radioisotopes to patients with metabolic errors to check for rates and products of degradation; and 5) performing the enzyme analysis, stressing the suspect pathways, and using radioisotopes on human tissues grown in culture. The most common type of tissue culture is that of fibroblasts; however, the usefulness in errors of the urea cycle is limited by the fact that one of the enzymes is missing in the fibroblast. ASAS and ASase are present in these cells, as is arginase, but OCT is not. Recently, Choi and Bloom (1970) described a tech- nique for maintaining human lymphocytes in apparently permanent culture. At the time of the initiation of the present research the enzymes of the urea cycle had not been thoroughly studied in lymphocytes. Many genetic diseases may be diagnosed by enzyme analysis on freshly drawn lymphocytes (Hsia, 1972), and others on lymphocytes that have been induced to divide in temporary culture (Nadler and Egan, 1970; Hirschhorn et al., 1969). The technique of Choi and Bloom (1970) has provided a way to gain a population of white blood cells with an apparently infinite life span in culture. As fibroblasts have a finite number of divisions in culture, 17 lymphocytes potentially overcome the limitations of time that hamper the analysis of fibroblast cultures. Enzymes of the Urea Cycle in Lymphocytes Spector and Bloom (1973) have shown indirectly that ASAS and ASase are probably present in normal lympho- cytoblastoid cells in culture. They found that normal cells would grow with citrulline as an arginine source while those from a patient with citrullinemia would not substitute citrulline for arginine. Arginase has been reported to be present in freshly drawn white blood cells, but the activity varied so exten- sively that it has not been utilized as a technique for measuring arginase activities (0-270 mg urea nitrogen liberated per 1310 leukocytes) (Reynolds et al., 1957). .Ornithine carbamoyl transferase has not been re- .ported to be present in white cells. Its presence in serum is related to the degeneration of liver cells, and serum OCT levels are used in testing for liver failure. 5 Up to the present time, all studies on peOple heterozygous for OCT deficiency have been done by liver biopsy. 18 Tests for urea cycle enzymes would be useful in establishing whether the enzymes are active in cultured lymphocytes, and would provide an alternate system with which to study inborn errors of the urea cycle. The discovery of useful colorimetric tests for such assays would put diagnostic procedures within the reach of most laboratories. MATERIALS AND METHODS Liver Controls Adult Sprague-Dawley rats were maintained on a standard laboratory diet. They were guillotined and their livers were chilled and divided into small sections, weighed, wrapped individually in aluminum foil and stored at -80°C. The liver was used to set activity standards for assays, to establish lower limits of detectable ac- tivities, and to check Km's of the crude liver homogenate against those of the cell lysates. Cell Lines Maintenance The lymphocytic cell lines UM 43 and 61, from males; and 54 and 56, from females, were supplied by Dr. Arthur Bloom's laboratory at the University of Mich- igan. They had been established by the method of Choi 19 20 and Bloom (1970), in which a lysate from a previously established line was used to stimulate growth of a new line. The donor was of sex opposite to that of the estab- lished line. They were maintained in RPMI 1640 (Grand Island Biological Company), enriched with 20% heat- inactivated fetal calf serum, and contained 60,000 units penicillin G and 60 mg streptomycin per liter of RPMI 1640. They were incubated in a 5% CO humidified atmosphere at 2 37°C. The flasks used were disposable plastic 25 ml Falcon tissue culture flasks containing 10 ml of cell suspension and medium or 260 cc Greiner tissue culture flasks with 30 m1 of cell suspension and medium. The lymphocytes grew clumped together in semi-suspension, settling to the bottom of the flasks, but easily suspended by shaking the flask gently. The medium was changed when its pH dropped below 7 or when it was desired to have the cells in logarithmic growth phase within two or three days. To change the medium, nine-tenths of the culture medium was drawn off and the cells were suspended in the remainder. Fresh medium at 37°C was added under sterile conditions. No pipetting by mouth was done, as the cells were presumably 21 infected with an Epstein-Barr virus (Gerber, 1973), and were treated as pathogenic. Harvesting Harvesting was done with the cells in logarithmic growth phase, one or two days after changing medium. Two- thirds of the visible colonies were drawn off with a sterile pipette from the bottom of the flask. The cells and accompanying medium were put into screw-top test tubes and centrifuged at 1,000 RPM for 10 minutes. All cells harvested from a single line were vigorously resuspended in 10 ml of medium. Two-tenths ml of the resuspended cells were added to 0.3 m1 of 1% Trypan blue and mixed vigorously. The suspension was then sampled and counted on a hemocytometer. Total cells harvested were calculated by standard methods. Viability was determined by dye exclusion. The suspension was recentrifuged, the medium dis- carded into a beaker, and the cells were washed in an isotonic salt solution followed by centrifugation. The 22 salt solution was also discarded into the beaker, and the button was stored dry in the test tube at -80°C until use. All materials and medium that came in contact with the cells were autoclaved before cleaning or discarding as a precaution against infection. Preparation of Cell Lysate and Liver Homogenate At the time of assay, the cells were placed in suspensions of 2 X 107 cells per ml, so that differences in activities between lines and between cell lines and liver could be compared. Preparation of the lysates was accomplished by freezing and thawing ten times in alcohol and dry ice alternating with a 37°C water bath. Schimke (1963) has shown that freezing and thawing is an acceptable method for preparing lysates from HeLa and fibroblast cultures for determinations of arginase, argininosuccinic acid synthetase and argininosuccinic acid lyase. The freezing and thawing were done in the test tube used for storage, which maximized the amount of recoverable lysate and min- imized the possible exposure to the E-B virus. .23 Liver was prepared by homogenizing in ice cold water with a Potter glass homogenizer. The final dilution was 1 g of liver in 200 ml of homogenate. The Colorimetric Assays The four enzymes assayed were those involving the ornithine moeity. Citrulline determinations were done for ornithine carbamoyl transferase and argininosuccinic acid synthetase by the method of Archibald (1944) as modified by Ratner (1955). One half ml of deproteininzed reaction mixture was mixed with 0.2 ml of .75% diacetyl monoxime and one ml of an acid mixture. The acid was composed of commercial grade H2804, 85% H3PO4, boiling in the dark for 15 minutes and cooling in the dark and water in proportions of 1:3:6. After for 15 minutes, the samples were read at 290 nm on a Hitachi spectrophotometer or a Bausch and Lomb colorimeter. Urea determinations were done for ASase and argi- nase assays. One-tenth m1 of 1.6% a-isonitroso prOprio- phenone in 100% Ethanol was added to 0.5 ml of deproteinized reaction mixture. One ml of H2804, H3PO4, and water (1:3:5) 24 was added, the mixture was boiled in the dark for one hour, cooled for 15 minutes. Optical densities were read at 540 nm. Standard curves were established each time an assay was run; Activities were expressed in uMoles mg liver hours. The Enzyme Assays The enzymes ornithine carbamoyl transferase, argininosuccinic acid synthetase, argininosuCcinic acid Alyase, and arginase were tested for activity. Each assay was performed in triplicate and~repeated at least once. Ornithine Carbamoyl Transferase -The reaction produces citrulline from ornithine and carbamoyl phosphate, and is measured as the rate of appearance of citrulline. The assay medium contained 20 mM dilithium carbamoyl phosphate and 15 mM ornithine in a 50 mM glycylglycine buffer at pH 8.3. To 0.2 ml of 25 30 mM ornithine, pH 8.3, was added 50 ul of liver homog- enate or 50 ul of cell lysate. The ornithine with the liver or cell preparation was brought to the incubation temperature of 37°C. Carbamoyl phosphate was added to 0.1 M glycylglycine buffer (pH 8.3, 37°C), mixed quickly and .2 ml was added to the ornithine mixture to start the reaction. The incubation period was 15 minutes at 37°C. The reaction was stopped with 0.4 ml of 15% perchloric acid and centrifuged to remove the protein. One half ml was taken for color development. Controls for this assay were particularly important as the reaction proceeds nonenzymatically. ~Therefore, each time period and/or substrate level was checked for develop- ment of background color with no enzyme. In addition, carbamoyl phosphate reacts with the glycylglycine buffer‘ to produce background color in the reaction. Because of the fact that the non-enzymatic reaction is not stopped by the perchloric acid treatment, no delays between completion of the reaction and determination of product were allowable. 26 , Argininosuccinic Acid Synthetase The production of argininosuccinic acid from citrulline, aspartate and ATP is measured by detection of a depletion of the amount of citrulline in the reaction mixture. As urea produces color development in the citrul- line assay, urea interfered with the determination of citrulline depletion. Therefore urease was added to the reaction mixture to eliminate the urea (Wixom et ale, 1971L The assay medium was made up in 5 X final strength: 0.5 M Tris buffer, pH 7.5, 25 mM aspartate, 50 mM MgSO 5 mM 4, citrulline, and 25 mM ATP. Ten 01 ASase, 1 mg arginase and 0.5 mg grade II urease (Sigma) were added per ml final volume. To 50 ul of the assay medium was added 200 pl of the liver homogenate or the cell lysate, and the mixture was incubated for one hour at 37°C. The cell lysate was prepared in 0.01 M Tris buffer, pH 7.5, as was the liver homogenate. The reaction was stopped in a boiling water bath for five minutes. Protein was removed by centrifugation for 10 minutes at 2,000 RPM. Fifty ul of supernatant was taken for color development. 27 Argininosuccinic Acid Lyase Argininosuccinic acid is cleaved by argininosuc- cinic acid lyase to produce arginine and fumarate. The reaction is measured as the production of urea in the presence of excess arginase. The method is that of Schimke (1962) modified for our conditions. One half ml of 10 mM barium argininosuccinic acid (Sigma) in 50 mM potassium phosphate buffer, pH 8.3, was added to 50 ul of cell lysate of liver homogenate. Lysate in this case was about 4 X 108 cells per m1, prepared by suspending the button in a minimal amount of medium. Liver homogenate was 1 g: 20 ml water. One half ml of 30% perchloricacid containing .1 M NaZSO4 tion and to precipitate the barium ion before color devel- i . was added to stop the reactions after incuba- opment. The zero time control was pre-treated with the acid before adding the reaction mixture. Protein was re- moved by centrifugation at 2,000 RPM for 15 minutes. For color development 0.5 ml of the supernatant was taken. 28 Arginase The cleavage of arginine to ornithine and urea is measured by the rate of appearance of urea. The assay medium was .250 M arginine in .001 M MnSO4, pH 9.7. To one half ml of the assay medium was added 50 ul of pre- treated lysate or liver homogenate. The pretreatment was incubation in .05 M MnSO4 for five minutes at 55°C to activate the enzyme. The final concentration of cells in the lysate was 2 X 107 cells/m1. Liver was lg:800 ml H20. Incubation was for one hour at 37°C. The reaction was stopped by 1/2 ml of 15% perchloric acid. After centrifu- gation to remove protein, 0.5 ml was taken for color development. RESULTS Lymphocytes The enzyme assays required constant supplies of fresh lymphocytes, which were maintained on RPMI 1640 medium (Appendix I). These were cell lines derived from normal people (U.M. lines 43, 54, 56, and 61). Growth Rates The lines could be harvested once or twice a week, depending upon the rate of growth of the lymphocytes or the density of the cultures remaining after harvesting. Table 1 shows that over an eight-week period, the amount of harvested cells per flask varied with the line of cells. Line 43 had the lowest production rate, less than 106 lymphocytes per flask per week; while line 61 had the highest, or nearly 107 cells per flask per week. There- fore, line 43 was expanded to seven flasks in order to supply comparable numbers of cells from each line. 29 30 TABLE l.--Lymphocytes harvested from four lines during one eight-week period. . Bottle- Total cells Cells per Line weeks harvested week per x 106 flask 43 50 45 .91 54 32 83.6 2.6 56 32 64.3 2.1 61 32 280 8.76 31 Viability Viability checks were done at each harvest. The Viabilities ranged from 65 to 93%. When viability was below 75%, the cells from that harvest were not used in the experiments. Table 2 shows the Viabilities for the harvests during a representative eight-week period for each line. Ten per cent of all harvests were below the level of acceptability; 7.5 per cent were above 90% via- bility. Protein Content A protein determination (Lowry et al., 1951) was done to establish the amount of protein for a given number of cells. Line 61, the rapidly growing lymphocyte line, was chosen for this determination (see Table 1). There were 1.3 mg protein per 106 lymphocytes. This estimate was used throughout to establish specific enzyme activ- ities. 32 TABLE 2.--Viabilities for four lines of lymphocytes harvested during an eight-week period. Line Date . % Viability 43' 6/29 81 7/4 85 7/9 87 7/17 76 7/27 75 7/30 73 8/6 82 8/19 78 54 6/29 82 7/14 82 7/17 86 8/3 65 8/19 85 56 7/14 85 7/17‘ 86 8/3 69 8/14 82 8/19 89 61 6/29 76 7/4 88 7/5 77 7/9 84 7/11 78 7/14 90 7/17‘ 93 8/3 87 8/14 83 33 Growth on Arginine-Free Medium In order to determine if the entire cycle was present in the lymphocytes, ornithine was substituted for arginine in the growth medium. It was decided to use arginine-free‘D' medium with ornithine added in place of arginine. D' medium (see Appendix II) enriched with 5% fetal calf serum was available in both arginine-free and arginine-containing forms, and was selected to be used for this experiment. Lymphocytes from line 43 were centrifuged at 50 X G, the RPMI 1640 medium was removed, the lympho- cytes were washed with arginine—free D' medium, resuspended in 10 ml of D' medium, and distributed in 2.5 ml aliquots to four culture flasks, two with arginine, two with orni- thine. No increase in numbers of cells was observed in either medium, and after four weeks, there were no observ- able clusters of cells on the bottom of the flasks. The lymphocytes did not seem to be able to adapt to the D' medium. 34 Urea and Citrulline Determinations Urea was determined by the method of Archibald (1944). Citrulline was determined by the:method of Archi- . bald as modified by Ratner (1955). Optical density vs umoles of citrulline and/or urea were established for the appropriate substances at the time of each experiment. Typical curves are shown in Figure 3a for urea, and Figure 3b for citrulline. Both of the assays followed Beer's law between .02 uMoles and 1.0 uMoles. The Enzymatic Reactions Three of the four enzymatic reactions of the ornithine-urea cycle were demonstrated colorimetrically in the lymphocytes. They were: OCT, Asase, and arginase, but not ASAS. 35 o3b > H '8 C 8 2 o E U 15 O. C’ .l- .0] .05 .10 uMoles Urea Fig. 3a.--0ptical density vs uMoies urea. -.3 > H '3 '.2 I: O O '3 U .5 -0] O. O .01 205 .10 uMoies Citrulline Fig. 3b.--0ptical density vs uMoies Citrulline. 36 Ornithine Carbamoyl Transferase Other than the liver homogenate or lymphocyte lysate,* the ingredients in the OCT assay were ornithine, carbamoyl phosphate, and glycylglycine buffer._ The assay was stopped with 15% perchloric acid. It was noted that preboiled blanks in the OCT experiments produced color. Experiments were run to determine whether color was developed by the various ingredients in the assay mixture independently of the amount of citrulline. varying amounts of carbamoyl phos- phate (0.0, 2.5, 5.0, 7.5, 10 uMoles) were tested and no trace of color was produced in the color reaction experi- ment. Citrulline'added to a solution of 5 uMoles of carbamoyl phosphate in amounts of .00, .01, .02, .03, and .04 uMoles gave the same results as citrulline added to the color reagents in water. Perchloric acid, which was used to stOp the reaction, gave no color development. However, the total mixture, but without enzyme, incubated 15 minutes, produced color. When 50 ul samples of an enzyme-free system were used for color development, optical density readings were in the 0.01 O.D. range. When 500 01 *because of the method of preparation the suspension of liver in water or buffer is called homogenate, and the lymphocyte suspension is called lysate. 37 were used, a typical O.D. reading was .250. This was nearly half of the color development shown by the lympho- cyte assays. Table 3 shows a sample series of Optical density readings, and the net production of citrulline by the enzyme. At time zero, an O.D. reading of .250 would be equivalent to .11 uM of citrulline. I Figure 4a is a graph showing the increased color production in a near-linear fashion when glycylglycine buffer and carbamoyl phosphate were held constant, and ornithine was increased. It should be noted that color was produced even at zero levels of ornithine in the pres— ence of high levels (.1 M) of carbamoyl phosphate. 'Al— though carbamoyl phosphate did not produce color in a .previously described experiment, the fifteen minute incu- bation time probably allowed a color-producing reaction of carbamoyl phosphate with glycylglycine buffer. The addi- tion of perchloric acid to the reaction mixture did not prevent the continued nonenzymatic color production. Figure 4b shows similar results when ornithine is held at.a constant .1 M concentration and carbamoyl phos- phate is increased. It should be noted that between zero and 1 mM concentrations of carbamoyl phosPhate, there was 38 TABLE 3.--The production of color by carbamoyl phosphate and ornithine, with and without enzyme. Number of Optical densities . Net production of lymphocytes (mean of three b enz e uMoles X 105 values) y ym ' 0 .249 -- 1.25 .369 .06 2.5 .435 .09 5.0 .575 '.16 10. .723 .23 39 i 5 10 g 20 30 40 50 Fig. 4a.--Carbamoyi phosphate (mM) (ornithine at 100 mM). A 0'5'10 sh I'oo . " - 500 .Fig. 4b.--0rnithine (mM) (carbamoyl phosphate at 50 mM) Fig. h.--Non-enzymatic color development by the OCT assay ' mixture.. 40 no discernible color development, confirming the prior con- clusion that ornithine alone or with glycylglycine buffer produces no color reaction. ' Because of the nature of the nonenzymatic color development, the experiments with OCT were run with reagent blanks as controls and the reactions were started by the. addition of carbamoyl phosphate in buffer to previously equilibrated test tubes. The appropriate subtractions for background color were then made. Endogenous citrulline or interference from other constituents of the cell lysate could have contributed to the determination. Table 4 shows that verysmall amounts of color were present, approximately .01 O.D. units for about 6 X 105 lymphocytes. Therefore, no preboiled con- trols were used. The Enzyme Assays for Ornithine Carbamoyl Transferase Activity of OCT was demonstrated in lymphocyte lines 43, 54, and 61. Lines 43 and 61 had the highest activities: 106 lymphocytes produced .8 uMoles citrulline in a 15 minute incubation. -41 TABLE 4.--Color development from lymphocyte lysate in citrulline determinations. r Numbers of . lymphocytes Optical density uM citrulline x 105 3 .005 .001 6 .012 .002 9 .017 .0025 1.2 .021 .003 1.5 .026 .005 42 Lability at -80°C It is often stated that OCT loses much of its ac— tivity when stored overnight at ~15°C (Levin, 1971). Lability was tested on samples stored overnight at -80°C. Figure 5 shows that the activity of a crude homogenate prepared from a button stored overnight was greater than that of a fresh sample. Since line 43 had the higher ac- tivity after being frozen, subsequent experiments were done on this line. Time Dependency The production of citrulline was shown to be time dependent (Figure 6). At times of 0, 5, 10, and 20 minutes 0, .26, .51, and 1.1 uMoles of citrulline were produced per 106 lymphocytes. Protein Dependency To establish that a reaction is enzymatic it is necessary to show that the rate of product formation is 43 .lo .9- .3- 1. 52’ \. '7' m 0 H 3 .6. O .c E >. ._ .5_ \D 2 \ «‘5’ oh- '3 .5 .3L ‘3 Z =- .2 ~ 0]- A B A B Cell Line 43 Cell Line 6] A Activity when tested immediately after harvest. B Activity when tested after 20 hours at -80°C. Fig. 5.--0rnithine carbamoyl transferase activities before and after storage at -80°C. W: 1‘ V .4 44 in l.25 " 3 >~ U 2 100 E; , ‘00 - 75 - \ 0 E E .50 - o u . '6 m 3 025 I- 2 1 o l k _1 5 lo 20 Time in minutes Fig. 6.--Citrulline production by OCT as a function of time by~lymphocytes. 45 linearly dependent upon the amount of protein in the system. Figure 7 shows that for OCT the rate of citrulline produc- tion does increase linearly with increasing amounts of protein, expressed in numbers of lymphocytes (2.5, 5, and 10 X 105). Specific Activity Specific activity for the lysate was .41 uMoles citrulline per milligram protein per hour. In the same experiment, the specific activity of liver was 107 uMoles citrulline per milligram protein per hour.» Therefore, the activity of the lymphocytes was about 1/260 that of the activity on the liver. Location of the Enzyme When the lysate was centrifuged before assay, twice the activity was found in the supernatant as in the button resuspended in an equal amount of water. This may indicate that some cell organelles were not completely ruptured dur- ing the lysing process and that OCT is located within these organelles. 46 .5 .4 L 3 .8 \ -3 0 .E E o .1'.’ ° .2 m .3.’ 1? :1 O .l 2 ~ —I# 2.5 5 l0 Numbers of lymphocytes X l05 ‘Fig. 7.--Citrulllne production by OCT as a function of lymphocyte protein concentration. ' I‘V 3...; ‘n-‘H '. lb 47 Establishment of Michaelis-Menten Constants (Km) for OCT Michaelis-Menten constants were derived for both substrates on the whole lysate and compared with laboratory values for crude liver homogenate. The experiments were done in triplicate and repeated at least once for each Km. Graphs are shown from one representative experiment in each group. Ornithine Km for OCT The velocity vs substrate curve for liver homog- enate shows a linear rate to 2 mM and a flattening above 5 mM of ornithine (Figure 8).‘ (This indicates substrate inhibition.) The velocity vs substrate curve for the lymphocyte lysate increases as substrate concentration rises up to 0.1 M ornithine (Figure 9). 'Not included on the graph are concentrations of ornithine at 500 and 1000 mM. These values are less than the value at 100 mM, also indicating substrate inhibition with the lymphocyte enzyme. These Km's were run at 50 mM carbamoyl phosphate. 48 mMoles citrulline/mg liver protein/hour + —0—1|—o Ornithine concentration (mM) Fig. 8.--Rate of citrulline production by OCT in liver as a functiop of ornithine concentration. . {T7— _~—.— .... 49 lO - 9 . I- 3 .2 st 8 - C 0 H 2 7 Q. 3 6 >~ U .2 O. E 5 >~ E’ 4 73 C 3 3 2 H U h l 9 E J 1 l0 50 lOO Ornithine concentration (mM) Fig. 9.--Rate of citrulline production by OCT in lymphocytes as a function of ornithine concentration. 50 Lineweaver-Burk Plots Lineweaver-Burk plots (l/V vs l/S) were done for both crude liver homogenate and lymphocyte lysate (Figures 10 and 11). The Michaelis-Menten constant (Km) for liver was 3 mM ornithine (Figure 10). The plot shows that as the concentration increases, there is substrate inhibition, because the theoretical maximum velocity of the reaction (V max) is greater than the actual figure. Repetition again produced 3 mM ornithine as the Km. The value at 1 mM has the greatest likelihood of error. It is the minimum I substrate level read at the lowest optical density. The inversion of this figure results in small errors being greatly magnified. The Km for cells was estimated at 15 mM ornithine from a Lineweaver-Burk plot (Figure 11). The value at 1 mM has the greatest likelihood of error. It is the minimum substrate level read at the lowest optical density. The inversion of this figure results in small errors being greatly magnified. Repetition of the experiment produced a Km of 17.5 mM ornithine. 51 llO . 100 90 ’5 80 2 B .s 70» g 60 r '3 E 50’ 13-. 1:0- > > 30' 20' 10 ' J A l 2 3 A l0 l/Ornithine concentration (mM) Fig. l0.--Llneweaver-Burk plot (l/V vs l/S) of OCT in liver as a function of ornithine concentration. 52 1.1. 1.0 * -l/m--.06 ’g '9' ‘Km-Is mM 2 . B .8r r.E E -7* H '3 .6 ‘ ti) .3 :2 .5.” 3 I. >.. f.4" :‘ ...3_ .2- .l - o A l/Ornithlne concentration (mM) Fig. ll.--Lineweaver-Burk plot (l/V vs 1/5) of OCT in lymphocytes as a function of ornithine concentration. 53 Hanes Plots Hanes plots (S/V vs S) were done for liver (Figure 12) and lymphocytes (Figure 13). The Hanes plot attempts to correct for errors at low concentrations by plotting the substrate levels directly. Km's estimated from each of these plots were the same as those estimated from the Lineweaver-Burk plots: 3 mM for liver, and 15 and 17.5 mM for lymphocytes. Carbamoyl Phosphate Km for OCT The velocity vs substrate curve for liver shows nearly linear increases to 5 mM and inhibition above 10 mM (Figure 14). The same curve for lymphocytes (Figure 15) shows a linear increase to 7.5 mM and a slower rate of increase after 20 mM. LineWeaver-Burk and Hanes Plots A Lineweaver-Burk plot of l/V vs 1/8 for liver gives a Km estimate of 3.3 mM (Figure 16), while a Hanes plot on the same data gives an estimate of 2.4 (Figure 18). . . .:o_umcucoocou oc_;u_cco mo co_uoc:w m mm to>m_ c. boo mo Am m> >\mv uo_a mocmzun.~_ .m_m Nzev co_uucucoucou oc_;u_cco m N _ _. . mu 54 4 a « .co_umLucoocou oc_cu_cco mo co_uuc:w m mm mou>005ae>_ c_ kuo mo Am m> >\mv uo_a mocmzuu.m_ .m_u Azsv :o_umcucoocoo oc_;u_cco 00. cm . o. m _ o_a 55 1 di 1 Hi I I \ a .o. . mo. . 0.. oc_;u_cto :2 m. u ex . ~_. oc_;u_cto :2 m_- u ex- . 56 .6 ‘ .5 » $— 3 .2 \ .5 .3 o .4 ' L D. L 0 .2 3' 3 ‘ \ 0 .E E 3:3 .2 - U W .2 2 ,- E .l '- l {o 20 30 no so Carbamoyl phosphate concentration (mM) Fig. lh.--Rate of citrulline production by OCT in liver as a ' function of carbamoyl phosphate concentration. ' 5'7 Inti- .2so - ' L— 3 2 E .200 i- O '3 H o I— a. o H >- 8 .5- .150 ' E 3? E’ - o .E ;: .IOO ’ a L H '5 m .3 :2 .50 ’ .4 j 5 10 20 30 50 IOO Carbamoyl phosphate concentration (mM) Fig. 15.--Rate of citrulline production by OCT in lymphocytes as a function of carbamoyl phosphate concentration. l‘Lh. ‘ 58 zoo. - ’C 8 150 . .C In. 0 .2 8’ \ ‘3 3 100 ' Z :3 > > so 12 5 - l0 l/Carbamoyl phosphate concentration (mM) Fig. l6.--Lineweaver-Burk plot (l/V vs l/S) for OCT in liver as a function of carbamoyl phosphate concentration. 7". S9 Repetition produced a value of 3.3 mM for liver on both the Lineweaver-Burk plot and the Hanes plot. The Lineweaver-Burk plot for the lymphocytes givesa. Km estimate of 2.2 mM carbamoyl phosphate (Figure 17). Rep- etition produced a value of 1.5 mM. A Hanes plot for the same data gives an estimate of 2.5 mM for one experiment and 1.5 mM for the repeat (Figure 19). Argininosuccinic Acid Synthetase Argininosuccinic acid is produced from citrulline, aspartic acid, and ATP. The assay was based upon the deple- tion of the substrate citrulline. The assays of Brown and Coehn (1959) and Schimke (1961) were run with a 1:40 dilu- tion of liver homogenate. The enzymes ASase and arginase were added to prevent product inhibition by argininosuccinic acid. Citrulline determination was by the method of Ratner (1955). An assay mixture containing 5 mM citrulline and the 1:40 dilution of liver homogenate produced optical density shifts of 0.1 O.D. units after incubation for one hour. To obtain comparable results with the lymphocytes in theASAS assay, it would have been necessary to use approximately mH-IT‘TtfiEA -_ k .; 6O 6O - - i5; ' ' “5 Km=22 50 ' T? 3 o .C \ I, .5 110' '5 I. H '3 3 30' :9 3 > h 20- IO - /, . l/Carbamoyl phosphate (mM) "te Fig. l7.--Llneweaver-Burk plot (l/V vs l/S) for OCT in lympho- cytes as a function of carbamoyl phosphate concentration. v. . "alum-1 61 7, 6- 5- S/V 4 - 3. 2 . :- l 5 IO Carbamoyl phosphate concentration (mM) Fig. l8.--Hanes plot (S/V vs S) of OCT ln liVer as a function of carbamoyl phosphate concentration. ..M'w I 62 lO -Km=l.5 ... g 8 E 13: 6 5 SN 1. 2 i (mM carbamoyl phosphate) Fig. l9.--Hanesfipiot (S/V vs S) of OCT in lymphocytes as a function of carbamoyl phosphate concentration. 63 4 X 109 lymphocytes or 5.2 g protein per ml, and therefore could not be performed. Urea is produced in the reaction mixture described above. The ureido group (~g-NH2), which is free in both citrulline and urea, produces color in the citrulline de- termination method. Therefore, urea interferes with the determination of the citrulline concentration.) The addi- tion of urease to the reaction mixture to eliminate the urea has been reported to increase the sensitivity some threefold (Wixom et al., 1972). Urease Effects To confirm that the addition of urease WOuld in- crease the sensitivity, three replicate experiments of the reaction were performed. The summary of these exper- iments is seen in Table 5. The experiment included the following treatments, each step of which was terminated by boiling for five minutes: A. preboiled enzyme incubated with the assay mixture without.urease. 1L. 64 TABLE 5.--The effects of urease on optical density shifts in the ASAS assay with 1 mM citrulline in assay medium. Optical Density Readings Time r71 A B C D A-B C-D- B-D 5 .282 .272 .270 .242 .010 .028 .030 j 10 .271 .267 .253 .230 .004 .023 .037 iii 20 .251 .249 .245 .219 .002 .026 .030 30 .243 .223 .221 .180 .020 .041 .043 60 .231 ' .220 .173 .118 .011 .055 .102 Treatments: A. Preboiled enzyme, boiled after incubation, no urease B. Aliquot of A. Urease added, incubated 10 minutes, stopped by boiling. C. Fresh homogenate, boiled after incubation, no urease. D. Aliquot of C. Urease added, incubated 10 minutes, stopped by boiling. 65 B. an aliquot of A to which urease was added and incubated for ten minutes. C. freshly prepared homogenate incubated with the assay mixture without urease. D. an aliquot of C incubated with urease for ten minutes. Treatment A controlled the effect of time on color development. Treatment B controlled the level of endogenous urea. Treatment C showed the effects of urea in the assay. Treatment D removed the endogenous and enzyme-produced urea. The change in optical density seen in the preboiled control indicates that some free ureido group present in the assay mixture deteriorates with incubation time in the ab- sence of active enzyme. Therefore, preboiled incubated controls must be run with each experiment. The results of the experiment indicated that.l8rnuMoles of citrulline per mg per hour was converted. By adding urease to the reac- tion mixture, it was possible to increase the sensitivity of the assay fourfold. This small increase in sensitivity was not sufficient to make the lymphocyte assay possible. 66 Citrulline Concentrations Because it was desirable to use some 20% of the citrulline to register appropriate optical density shifts. (Brown and Cohen, 1959), various concentrations of citrul- line were attempted. With a concentration of'1 mM citrulline and 20 minutes incubation, an O.D. shift of 0.2 O.D. units was obtained. The lower citrulline level thus increased the sensitivity sixfold. Time and Dilution Effects To test the effects of time and dilution on the homogenate, replicate experiments were performed using dilutions of 1:20, 1:60, 1:100, 1:500, and 1:1000, and incubations of 1 hour and 2 hours. The results are shown in Table 6. In these experiments, the 1/20 and 1/60 dilu- tions of liver homogenate produced similar O.D. shifts, while the 1:100 and the 1:180 dilutions resulted in less citrulline reduction. The 1/500 and 1/1000 dilutions pro- duced lower rates of citrulline utilization. The rates of utilization were not linear over the range. The effect may be due to the presence of ATPases ih the homogenate 67 TABLE 6.--0ptical density shifts recbrded in ASAS assay after incubation of serial dilutions of liver homogenate in an assay mixture containing 1 mM citrulline and urease. ‘— ,_.V k Experiment Dilution of Optical density number Time homogenate shift 1 1 hr 1:20 .110 1:60 .097 1:180 V .068 2 1 hr 1:20 .132 1:60 .134 1:180 , .075 3 1 hr 1:100 .070 1:500 .049 1:1000 .050 2 hr 1:100 .101 1:500 .058 1:1000 .058 68 and have caused ATP to become rate limiting at the higher concentrations of homogenate. The detection of activity in a 1:500 dilution of liver homogenate is a 250 fold increase in the sensitivity of the assay as reported commonly for colorimetric assays of ASAS. With this level of sensitivity, activity should be detectable with about 107 lymphocytes per tube. The experiments were done repeatedly with lymphocytes, using liver homogenate as a control. However, no activity was detectable in the lymphocytes. Argininosuccinic Acid Lyase This enzyme catalyzes the splitting of ASA into arginine and fumarate, and was measured as urea production in the presence of excess arginase. Urea was measured by the method of Archibald (1944). Activity for this second-least—active enzyme of the urea cycle was detectable when there were 4 X 107 lympho- cytes per m1 of reaction medium. The experiment was run with reagent blanks and controls of preboiled enzyme. The reagent blanks showed no color development, indicating that 69 spontaneous conversion did not occur significantly and that the commercial arginase was not contaminated with ASase. The preboiled controls showed endogenous urea to be present in both liver and lymphocytes. The assay mixture with pre- boiled enzyme showed 5 muM endogenous urea per ml for the liver assay, which was 5.6 muM endogenous urea per mg pro- tein. The lymphocytes had 100 muM endogenous urea per ml, or 1.78 muM endogenous urea per mg protein (Table 7). Specific Activities After ten minutes, the liver homogenate showed production of urea at 2000 muMoles urea per mg protein per hour. After 60 minutes, the rate of production was 1440 muMoles urea per mg protein per hour, indicating that the enzyme activity is reduced with time. The lymphocytes showed 5.25 muMoles urea per mg protein per hour. The lymphocytes had 1/275 of the ASase activity of the liver in this experiment. 70 TABLE 7.--Argininosuccinic acid lyase activity. W Enzyme Source muMoles urea per hour Time per mg protein Liver O 5.6 10 min 2000 60 min 1440 Lymphocytes 0 1.78 60 min 5.25 71 Arginase Arginase is the enzyme which catalyzes the split- ting of arginine to urea and ornithine. Its activity was measured by the appearance of urea in a solution of argi- nine. Urea determinations were by the method of Archibald (1944). As it had been found that when samples of 500 pl were taken for color development for OCT determinations, significant amounts of citrulline were discovered, zero enzyme controls were tested for color development. When 1.6% isonitrosopropriophenone was used for the color- producing reagent, no background color appeared. Reagent blanks were used for controls for the Km studies, but no significant color development occurred even at very high concentrations of arginine (1 M). Arginase activity was established in all four lymphocyte lines. Line 61 had the highest activity. Time Dependency Experiments with time periods of one half, one, and two hours showed the production of urea to be time 7.2, dependent (Figure 21). The production of urea was not linear over the time period of two hours. Therefore, it appeared that the enzyme activity diminished with time, with about 75% activity after one hour. Protein Dependency The production of urea was shown to be linearly dependent upon protein concentration (Figure 20): 1 X 106 lymphocytes produced 400 muM urea per hour, 2 X 106 lympho- 6 cytes produced 860 mpM per hour, and 3 X 10 lymphocytes produced 1200 muM urea per hour. Specific Activity The arginase activity of liver was 9,340 muM per mg protein per hour. The activity of the lymphocytes was 23 mph per mg protein per hour. Therefore, in this exper- iment the specific activity of the lymphocytes was about l/400 of the specific activity of the liver. 73 uHoles urea/hour i 2 3 Numbers of lymphocytes x i06 Fig. 20.--Uree production by arginase as a function of lympho- cyte protein concentration. ‘05.?“ 74 1.0 b /‘ / 8' - / / m. o u >~ U o ‘5. E >. .6 \D O ‘\ fl! 0 L 3 m .4 0 =2 .2 , ' a I '0. 1/2 l 2 Time in hours Fig. 2|.--Urea production by arginase in liver as a function of time. 75 Establishment of Michaelis-Menten Constants (Km) Michaelis-Menten constants were measured and com-. pared between liver and lymphocytes. Figure 22 shows the velocity as a function of the substrate level for the liver en2yme. The velocity was linear with respect to substrate to about 10 mM and then gradually leveled off. The sub- strate vs velocity curve for the lymphocytes (Figure 23) is linear to about 5 mM, and then approaches a 1eVel." Lineweaver-Burk Plots Lineweaver-Burk plots (l/V vs l/S)were done for liver and lymphocytes. From the Lineweaver-Burk plot for the liver, the Km is estimated as 10 mM (Figure 24). The same plot for lymphocytes results in a Km estimate of 2.0 mM (Figure 25). When the experiments were repeated, the Km estimates were 11.1 mM for liver and 2.2 mM for lymphocytes. '-5 K‘ I? r" r "'alls ... 76 ' hMoles urea/mg liver protein/hour j A I I 10 '20 30 ’ 40 50 Arginine concentration (mM) Fig. 22.-~Rate of urea production by arginase in liver as a function of arginine concentration. 77 i. .08 - . . 4.: _ amt-.54....” ~.Irlnuj “EE 1.- .- ‘ . a.“ n. UMoles urea/mg lymphocyte protein/hour .( ' L 1 —l i L . 3 II 5 lo 3 25 ' so 100 Arginine concentration (mM). “_Fig. 23.--Rate of urea production by arginase in lymphocytes as a function of arginine concentration. 78 i/V 1 k .4 i' i/Arginase concentration (mM) Fig. Zh.--Lineweaver-Burk plot (i/V vs i/S) of arginase in liver as a function of arginine concentration. “1'7““ 79 50 40 30 l/V 20 iO -l l 2 l/Arginine concentration (mM) Fig. 25.-rhineweaver-Burk plot (l/V vs l/S) of arginase in lymphocytes as a fuhction of arginine concentration. 80 Hanes Plots Hanes plots (S/V vs S) were done on the same data as the Lineweaver-Burk plots. Both experiments for liver gave Km estimates of 11.1 mM arginine (Figure 26). .One experiment gave a Km estimate of 2.0 mM arginine for lym- phocytes (Figure 27) and the second gave a Km estimate of 2.2 mM arginine. [...-"45 an. inland! 3&qu Hi ‘li ‘ 81 50 T 40 - 30 ' S/V l0 ’ 50 i00 5 (mM arginine) Fig. 26.--Hanes plot (S/V vs S) of arginase in liver as a function of arginine concentration. 300 250 200 S/V l50 100 50 82 A 5 l0 20 25 5 (mM arginine) Fig. 27.--Hanes plot (S/V vs S) of arginase in lymphocytes as a function of arginine concentration. DISCUSSION Lymphocytes 'The advent of long-term lymphocyte cultures and the laboratory interest in errors of the urea cycle led to the study of the enzymes of the ornithine-urea cycle in these lymphocytes. Growth Rates and Viability of Lymphocytes Streeter et a1. (1973) in studies on optimal growth conditibns'for lymphocytes reported maximum levels of 1.6 to 1.8 X 106 lymphocytes per ml with 30% nonviable cells. In the present study, line 43 had .075 X 106 lymphocytes per ml at harvest, with 20% nonviable cells. Line 61 had .75 X 106 lymphocytes per m1 at harvest, about half of that reported by Streeter et a1. However, the viability of the present study was greater, with 85% of the lymphocytes viable. BeCause the desire was to have maximum viability 83 n . .WTJIT Y- }I"-} 5 84 and logarithmic growth rates fer the enzyme assays, our population densities were reduced. Growth on Arginine-Free D' Medium ffiz; i Spector and Bloom (1973) have reported that cul- tured lymphocytes from normal people could utilize citrulline, in arginine-free medium, to form the needed g arginine for normal cell growth. If the complete ornithine- urea cycle were present in cultured lymphocytes, then orni- thine, as well as citrulline, could serve as an arginine source. Arginine-free RPMI 1640 (Appendix I) was not readily available: therefore, an attempt was made to adapt the lymphocytes to D' medium (Appendix II). This medium was available in both arginine-free and arginine-added forms. This medium has high concentrations of all other amino acids except glutamic acid, plus high concentrations of vitamins and additional glucose; however, less fetal calf serum had been added than was used with RPMI 1640. The lymphocytes did not grow in D' whether arginine was present or was substituted by ornithine. It is possible that such high concentrations of the ingredients have an 85 inhibiting effect on the growth of the lymphocytes, or that the change in available nutrients did not allow adaptation to take place. Chu (personal communication) is successful with D' medium in plating experiments with lymphocytes. The reasons for the difference in the reaction of the lymphocytes in culture and in plating experiments is pres- ently unknown. Since the lymphocytes in this laboratory are preadapted to RPMI 1640, culturing the cells in arginine-free 1640 medium with ornithine substituted for arginine, would establish whether the enzyme activities described in this study are sufficient to produce arginine for growth. The Enzyme Assays The four enzymes studied were ornithine carbamoyl transferase (OCT), argininosuccinic acid synthetase (ASAS), argininosuccinic acid lyase (ASase), and arginase. By colorimetric methods, all but ASAS were determined to be present. ASAS activity has been demonstrated by radio— chemical methods in another laboratory (Kennaway, as re- ported by Spector and Bloom, 1973). 86 Ornithine Carbamoyl Transferase Nonenzymatic Reaction Brown and Cohen (1959) stated that "Some nonenzy- matic transcarbamylation accompanies the enzymatic trans- carbamylation" (p. 1771). Schimke (1961) noted the instability of carbamoyl phosphate, but did not suggest the extent of the spontaneous reaction. Levin (1971) noted that besides this reaction, the carbamoyl phosphate also reacts with the glycylglycine buffer. Because most assays use the high activities of liver, the relative differences between the reagent blanks and the experimental tubes are large. However, studies done on OCT levels in serum indi- cated that the nonenzymatic conversion of carbamoyl phos- phate and ornithine to citrulline in blood probably equalled the amount converted enzymatically (Snodgrass and Parry, 1969). In the present study the nonenzymatic conversion was of the same order of magnitude as the con- version by 106 lymphocytes, but at low substrate levels the enzymatic conversion was much greater in proportion. In studies of lymphocytes, in which specific activity is low, reagent blanks become essential for each assay level. 87 OCT activity had not previously been reported in lymphocytes. In the present study, specific activity was shown to be .41 pmoles of citrulline per milligram protein per hour. Subsequently, the Michaelis-Menten constants ..- were established fer each substrate. ~ 3:". ',»-.-. ffl i: ran. . d e -)I .i Potentially, showing OCT activity in lymphocytes in long-term culture could solve the problem of identifying carriers of OCT deficiency. If, as Short et a1. (1973) if ‘ have suggested, the gene is sex-linked, then two popula- tions of lymphocytes are possible: one with the normal gene being active, the other with only the abnormal gene. The detection of the presence of enzymatic activity in one clone and the absence in another from the same person would be strong evidence for carrier status. Identifying hetero- zygotes at present is unsuccessful because the activity levels of OCT activity in them overlap normal levels when determined on liver samples obtained by needle or surgical biopsy. Table 8 lists the KM's established for OCT from several sources, including the present study. 88 .. r .e Fini.’ [I il‘lfi “‘ y. mumommonm HMQEmoumo m0 mowsuwono ll E O scuba ucmmmum m.m ~.~um.H m.eanma mmusooaessa amass spasm ucmmuum m.m m.m .. o.m u0>na umm mama .muumm pom mmmnmoocm m.h mm.|bm. mmo. Eamon omens Huma ..Hm um cosmos: mm.m mm.a Aucmusev uo>HH spasm Huma ..Hm um mosmumz m hm.aumv.a nv.anmm.a Ho>HH spasm mmmH ..Hm um omomob m.m h.H v.H Ho>fia x0 nmma .cmnoo ocm uuocusm o.m ~.H o.m =uo>wa oowmauomg hmma .oumooflom m.h av. ¢.H uo>wa pom mmma .Hamnmumz one conoo m.m n.m .m manomH .m mmma .Haao>oz one muomom m.m mma. m.& waoo .m moomnomom mm AZEV 8% mo AZEV EM zmo monsom .mmMHQHmGMHU HNOEMQHMO QGHSDHGHO .HOM mflGmeGOO CG¥G02ImHH®M£OH2ilom Mgmflh. 89 Ornithine Km for OCT It can be seen that the ornithine Km's established for human lymphocytes are an order of magnitude different from those established for human liver (Matsuda.et al., 1971). It is not clear from the table what effect the range of pH has on the reCOrded Km. Though it is not in- cluded in the table, most of the assays were buffered by TRIS rather than by glycylglycine buffer. Therefore, the conditions of the assay are not directly comparable. Even so, the magnitude of the Km difference found is not easily explained. The low concentration of glycylglycine buffer (.05 M) used in these experiments must be considered. This concentration is used to minimize the reaction of glycyl- glycine with carbamoyl phosphate, but may not be suffi- cient to maintain the pH throughout the experimental period (Levin, 1971). The large amount of protein has its own buffering effect, perhaps overwhelming the effect of the glycyl- glycine buffer and producing a pH very different from the one with which the experiment was initiated. There are two methods to overcome this difficulty. Enzyme purifica- tion (Burnett and Cohen, 1957) on large amounts of ‘57:- 1_ 90 lymphocytes could produce higher specific activities, and reduce the level of extraneous protein. The second method would be to use radioisotopes. Using l4C-labeled carbamoyl phosphate would allow the use of a higher concentration of glycylglycine buffer, as citrulline would be separated from the other constituents in the assay mixture before determination (Schimke, 1963). By this separation, the spontaneous reaction of carbamoyl phosphate with glycyl- glycine buffer would not contribute to spurious product formation. Isozymes for OCT There may be isozymes of OCT present in the lympho- cytes that have not been detected in the liver. Isozymes are proteins with similar catalytic properties which differ from each other in one or more ways. They may be as similar as Hemoglobin A is to Hemoglobin S, differing by only one amino acid (See MoKusick, 1972, for a recent list of all known hemoglobin differences in humans dis- covered to date), or they may be different combinations of polypeptide chains, such as occurs with lactate dehydro- genase (Harris, 1971). Lactate dehydrogenase consists of ' fl m ; Arr—‘1'. .: mum ‘m: .1, A i 91 all possible tetrameric combinations of subunits A and B, eaCh with its own isoelectric point. Skeletal muscle LDH consists almost entirely of A4, while heart muscle LDH con- sists mostly of B4. Other tissues have other combinations. There are at least two isozymes of human OCT: liver and serum. If human serum OCT has a Km of 1/50 Of that of human liver OCT for the substrate ornithine (.069Imn vs 1.26 mM), another Km of 10 times the liver Km in another -tissue may not be surprising. Different species usually have different molecular forms of the same enzyme. The more widely divergent the species, the more the isozymes differ. The lymphocytes which are in permanent culture presumably are infected with an Epstein-Barr virus (Gerber, 1973). Viral-induced enzymes generally differ from enzymes present in uninfected cells in kin- etic, chromatographic, and immunological . properties. Frequently, these altered char- acteristics have been observed even after extensive enzyme purifications . . . if the properties of the new enzyme differ from those of the normal host enzyme, then it is unlikely that a normally functioning host gene is "de- repressed" or induced to synthesize increased amounts of the same enzyme species (Kit and Dubbs, 1969, p. 55). Although it cannot be proven at present, the viral infection may have induced the enzymatic activity observed 92 in the lymphocytes. The first steps in solving this problem could involve establishing that there is, indeed, an isozyme of OCT in cultured lymphocytes that is not in liver; or, short-term cultured lymphocytes (without virus) ‘could be tested for enzymatic activity of OCT. If the short-term cultured lymphocytes do not have OCT activity, and the enzyme in the lymphocytes in long-term culture is ‘3' e o~ m 0'. summit-“xx v "u“?! u ' _‘ - ' - an isozyme of OCT, it is more likely that the virus is responsible for the enzymatic activity than the host lymphocyte. If lymphocytes in short-term culture show OCT activity then most likely the isozyme is the product of the lymphocytic DNA. If lymphocytes from patients who have altered OCT characteristics in their liver could be induced to grow, the activities of those cell lines would establish whether liver changes were concurrent with changes in cultured lymphocytes. Concurrent changes of lymphocytic and liver enzymes would suggest common genetic control. Carbamoyl phosphate Km for OCT The Km estimates as listed in Table 8 for carba- moyl phosphate for OCT in lymphocytes are very near to 93 that established for human liver (Matsuda et al., 1971). They are slightly different from that mentioned for human serum OCT (Snodgrass and Parry, 1969): 1.5-2.2 mM vs .97-.95 mM. However, all Km's of carbamoyl phosphate in OCT are similar except for that of E. coli (Rogers and Novelli, 1962), and for rat liver (Reichard, 1957). The former was .196 mM, the latter .41 mM. In the present study the rat liver Km (3.3 mM) differs significantly from that of Reichard (1957), but it is only double that for "purified liver" (Burnett and Cohen, 1957), ox liver (Joseph et al., 1963), and human liver (Matsuda et al., 1971). Either differences in strains of laboratory rats or differences in experimental conditions may have affected the Km determination slightly. But it is difficult to ex- plain the large Km differences between the liver-value given by Reichard and that determined in the present study. Argininosuccinic Acid Lyase ASase had been shown indirectly in the lymphocytes by Spector and Bloom (1973), but the enzyme activity had never been measured. Spector and Bloom (1973) showed that wen-arms. ‘ F a 94 the cultured lymphocytes could incorporate the label from l4C-labeled citrulline into the TCA precipitable fraction. This was interpreted as the lymphocytes having changed citrulline into arginine, through ASAS and ASaSe. While tests for OCT and arginase required 2 X 106 cells per ml of assay medium, the ASase assay required 4 X 107 cells .~ 6" U‘. 5‘! Ma. -:.F7ii-w-7 I per ml of assay medium, a 20-fold increase. In addition, the small amount of background color from the-lysate that was present in the OCT assays became a significant factor in the ASase assays. Therefore, preboiled lymphocyte lysate was an essential control. As the ASA did not break down spontaneously nor contribute color to the assay, no allowance for extraneous color was necessary. Approxi- mately 600 X 106 lymphocytes would have been required for a Km determination on ASase. Three months would have been required to accumulate the cells for one Km determination: cost of media precluded this assay. Arginase Although arginase activity had been shown in freshly drawn "leukocytes" by Reynolds et a1. (1957), it had not 95 been previously shown in lymphocytes in long-term culture. Therefore, when the presence of arginase activity was established, it was decided to determine the Km for the lymphocyte arginase. Arginine Km for Arginase The published Km for bovine liver arginase is 11.6 mM arginine (Hunter and Downs, 1945). The experiments herein described produced a Km for rat liver of 11.1 mM arginine, virtually the same es for bovine liver. The Km for the lymphoCytes was 2.1 mM arginine in these experi- ments. . Isozymes for Arginase There may be isozymes of arginase in the lympho— cytes that have not been detected in liver. However, it is not known that isozymes of arginase exist within the same organism. The genetic evidence is that arginase activity is produced by the same protein in liver and in red blood cells, as‘the elimination of arginase activity in liver is accompanied by a similar loss in RBC's J ~- .rs .131”..ka ”flee-fl 96 (Terheggen et al., 1969). The difference in the Km's of the liver and lymphocytes in these experiments requires consideration of isozymal activities as previously de- scribed for OCT. ‘mrrr SUMMARY The present study was undertaken to determine which‘ of the enzymes of the ornithine-urea cycle occur in lym- phocytes in long-term culture. Three enzymes: ornithine carbamoyl transferase, argininosuccinic acid lyase, and arginase were demonstrated colorimetrically in the lympho- cytes. Kinetic studies of ornithine carbamoyl transferase, using ornithine as substrate, and arginase indicated that the isozymes found in the lymphocytes were distinct from those in rat liver. It is highly probable that the lym- phocytes in long-term culture were infected with Epstein- Barr virus: therefore, it cannot be-stated whether the isozymes are the product of lymphocytic or viral DNA. 97 APPENDICES COMPONENT CaiNO3)2-4HZO Glucose . . . MgSO4-7H20. . KCL . . . . . Nazi-$04 01:20. NaCl. . . . . L-Arginine. . (free base) L-Asparagine. L—Aspartic Acid L-Cystine . . L-Glutamic acid L-Glutamine . Glutathione . (reduced) Glycine . . . L-Histidine (free base) L-Hydroxyproline. . . . L-Isoleucine (Allo free). L-Leucine (Methionine free) L-Lysine HCL. LeMethionine. . . . . . . APPENDIX I mg/l 15 20 30 20 20 20 .2 .005 .25 RPMI-1640 mg/ L COMPONENT 100 L-Phenylalanine. . . . . 2000 L-Proline. . . . . . . . 100 ggzgjoxy L-Proline 400 L-Serine . . . . . . . . 1512 L-Threonine (Allo free). 6000 L-Tryptophane. . . . . . 20? L-Tyrosine .6. . . . . . 50 L-Valine . . . . . . . . 20 Biotin . . . . . . . . . 50 Vitamins B12 . . . . . . 20 D-Ca pantothenate. . . . 300 Choline C1 . . . . . . . 1 Folic Acid . . . . . . . i-Insitol. . . . . . 10 Nicotinamide . . . . . . 15 Para Aminobenzoic Acid . 20 Pyridoxine HCl . . . . . 50 Riboflavin . . . . . . SO Thiamine HCL . 40 Phenol red . . . . . . . 15 NaHCO3 . . . . . . . . .2000 98 APPENDIX II D' MEDIUM COMPONENT mg/L COMPONENT mg/L NaCl. . . . . . . . . 6800 L-Methionine . . . . . 22.5 XCl . . . . . . . . . 400 L-Phenylalanine. . . . 48 NaH2P04-H20 . . . . . 140 L-Proline. . . . . . . 230 119504-7320. . . . . . ' 200 L-Serine . . . . . . . 210 CaC12 (anhyd.). . . . 200 L-Threonine. . . . . . 72 Glucose . . . . . . . 3333 L-Tryptophan . . . . . 15 L-Alanine . . . . . . 178 L-Tyrosine . g . . . . 54 L-Arginine. . . . . . 36 L-Valine . . . . . . . 69 LrAsparagine'Hzo. . . 300 Choline C. . . . . . . 1.5 L-Aspartic acid . . . 266 Folic acid . . . . . . 1.5 L-Cystine . . . . . . 36 i-Inositol . . . . . . 3.0 L-Glutamic acid . . . 294 Nicotinamide . . . . . 1.5 L-Glutamine . . . . . 292 D-Ca pantothenate. . . 1.5 Glycine . . . . . . . 150 , Pyridoxal HCl. . . . . 1.5 L-Histidine . . . . . 46.5 Riboflavin . . . . . . 15 L-Isoleucine. . . . . 78.75 1 ' Thiamine HCl . . . . . 1.5 L-Leucine . . . . . .. 78.60 NaHCO3 . . . . . . . . 2200 L—Lysine. . . . . . . 87 Na Pyruvate. . . . . . .36 99 BIBLIOGRAPHY ‘37.... BIBLIOGRAPHY Allan, J. D., D. C. Cusworth, C. E. Dent, and V. D. Wilson. 1958. 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