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Home , Deoxyadenosine, Deoxycytidine, Deoxyguanosine

Proc. Nati. Acad. Sci. USA Vol. 74, No. 12, pp. 5677-5681, December 1977 Immunology

Lymphospecific toxicity in deaminase deficiency and phosphorylase deficiency: Possible role of nucleoside kinase(s) (immunodeficiency/lymphocyte/purine kinase/purine ) DENNIS A. CARSON*, JONATHAN KAYE*, AND J. E. SEEGMILLERt *Division of Rheumatology, Department of Clinical Research, Scripps Clinic and Research Foundation, La Jolla, California 92037; and t Department of Medicine, University of California, San Diego, La Jolla, California 92037 Contributed by J. Edwin Seegmiller, September 26, 1977

ABSTRACT Inherited deficiencies of the enzymes adeno- deaminase deficiency by enzyme replacement in the form of sine deaminase (adenosine aminohydrolase; EC 3.5.4.4) and purine nucleoside phosphorylase (purine-nucleoside:ortho- erythrocyte transfusions (4). phosphate ribosyltransferase; EC 2.4.2.1) preferentially interfere Three biochemical mechanisms have been proposed to ex- with lymphocyte development while sparing most other organ plain the association of deaminase deficiency with immuno- systems. Previous experiments have shown that through the deficiency disease, i.e., adenosine-induced star- action of specific kinases, can be "trapped" intra- vation (5), deficiency (6), and adenosine-me- cellularly in the form of 5'-phosphates. We therefore measured diated elevations in cyclic AMP concentrations (7). In the ab- the ability of newborn human tissues to phosphorylate adeno- sine and , the substrate of , sence of further information, these hypotheses do not explain and also , deoxyinosine, , and , the preferential impairment of lymphoid development seen the substrates of purine nucleoside phosphorylase. Substantial in both phosphorylase and deaminase deficiency. In the present activities of adenosine kinase were found in all tissues studied, studies, we suggest that lymphospecific toxicity in deaminase while guanosine and inosine kinases were detected in none. and phosphorylase deficiency may result from the selective However, the ability to phosphorylate deoxyadenosine, deoxy- accumulation in inosine, and deoxyguanosine was largely confined to lympho- lymphoid tissues, particularly the thymus, of cytes. Adenosine deaminase, but not purine nucleoside phos- toxic deoxyribonucleotides, mediated by nucleoside phorylase, showed a similar lymphoid predominance. Other kinase(s). experiments showed that deoxyadenosine, deoxyinosine, and deoxyguanosine were toxic to human lymphoid cells. The tox- icity of deoxyadenosine was reversed by the addition of de- MATERIALS AND METHODS oxycytidine, but not , to the culture medium. Based upon Tissue Extracts. Newborn human tissues obtained at these and other experiments, we propose that in adenosine autopsy deaminase and purine nucleoside phosphorylase deficiency, within 24 hr of death were frozen at -20°. Extracts were pre- toxic produced by many tissues are se- pared by mincing the specimens in 10 mM Tris buffer (pH 7.4), lectively trapped in lymphocytes by phosphorylating en- followed by five cycles of freeze-thawing, and ultracentrifu- zyme(s). gation of the particulate material. Peripheral blood lymphocytes and granulocytes were isolated During the past 5 years, Giblett and her colleagues have dem- by dextran sedimentation of heparinized whole blood from an onstrated an association between severe deficiencies of either adult volunteer, followed by centrifugation through Ficoll- adenosine deaminase (adenosine aminohydrolase; EC 3.5.4.4), Hypaque (8). Red cells were lysed with Tris-buffered ammo- or purine nucleoside phosphorylase (purine-nucleoside:ortho- nium chloride and the cells were washed and frozen (9). phosphate ribosyltransferase; EC 2.4.2.1) and inherited forms The protein content of all tissue extracts was determined by of human immunodeficiency disease (1, 2). Although the Lowry's method, with bovine serum albumin as a standard clinical pictures overlap, children with adenosine deaminase (10). deficiency usually suffer from a combined immunodeficiency Enzyme Assays. Kinase activities in cell extracts and column syndrome, with impairment of T cell development and in most fractions were determined by a modification of the method of cases of B cell function as well, while those with purine nucle- Ives et al. (11). For the measurement of deoxyguanosine, gua- oside phosphorylase deficiency have primarily a deficit in T nosine, deoxyinosine, and inosine kinases, the final concentra- cell development and the associated cellular immune functions. tions of the reactants were: 50mM Tris (final pH 7.4), 10 mM Both diseases are accompanied by severe lymphopenia. Al- ATP, 10 mM MgCI2, 15 mM NaF, 1 mg of protein per ml, and though enzyme is virtually absent from all tissues examined, 0.4-2 ,uCi of substrate at a concentration of 300 ,M in a total apparently only the growth and development of the lymphoid volume of 100 X. Adenosine and deoxyadenosine kinases were system is severely retarded (3). It is therefore likely that the similarly measured, except that the ATP and magnesium immune defect in deaminase and phosphorylase deficiency is concentrations were 5 and 2.5 mM, respectively. In addition, not due to a generalized disorder of growth, but rather to a to each sample was added the deaminase inhibitor erythro- primary lymphocyte abnormality and/or circulating toxins that 9-(2-hydroxy-3-nonyl) hydrochloride (EHNA) to a final are specifically lymphocytoxic. The latter concept is in accord concentration of 5 ,M. with the reversal of the immunodeficient state accompanying The reactions were initiated by the addition of labeled sub- strate. After 30 min at 370 in a shaking water bath, the samples The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked Abbreviations: deaminase, adenosine deaminase; phosphorylase, purine "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate nucleoside phosphorylase; EHNA, erythro-9-(2 hydroxy-3-nonyl)- this fact. adenine hydrochloride. 5677 Downloaded by guest on October 2, 2021 5678 Immunology: Carson et al. Proc. Natl. Acad. Sci. USA 74 (1977)

Table 1. Enzyme activities in human tissues Purine Adenosine Deoxyadenosine Deoxyguanosine Deoxyinosine Adenosine nucleoside kinase kinase kinase kinase deaminase phosphorylase I II I II I II I II II II Thymus 0.79 0.86 1.35 0.78 1.92 1.39 0.34 0.31 982.8 23.3 Spleen NA 0.53 NA 0.20 NA 0.33 NA 0.07 12.4 54.0 Brain NA 1.01 NA 0.14 NA 0.16 NA 0.05 5.0 10.3 Kidney NA 1.15 NA 0.07 NA 0.08 NA 0.10 1.8 100.0 Liver 0.81 2.26 0.12 0.07 0.04 0.07 0.05 0.04 1.1 36.2 Lung 1.32 0.81 0.11 0.06 0.03 0.08 0.03 0.02 0.8 38.0 Small intestine 0.41 0.52 0.13 0.08 0.03 0.11 0.09 0.07 14.2 63.9 Heart 0.48 0.51 0.13 0.08 0.03 0.11 0.07 0.06 2.1 32.2 Peripheral lymphocytes 1.00 0.32 0.21 0.09 20.7 114.7 Peripheral granulocytes 0.83 0.05 <0.02 0.03 11.9 121.4 Human tissues were obtained from two babies (I and II) who died during parturition, while peripheral lymphocytes and granulocytes were isolated from the blood of a normal adult. Activities are expressed as nmol of product per min/mg of protein at a substrate concentration of 300 PM. NA, not available for study. were boiled for 2 min and insoluble material was centrifuged Reagents. [8-14C[Adenosine and [8- 4C[deoxyadenosine at 4°. Control experiments with [14C]inosine monophosphate were purchased from New England Nuclear (Boston, MA) and showed no breakdown under these conditions. used to synthesize inosine and deoxyinosine by treatment with were separated from the nucleosides and bases calf deaminase (Calbiochem, San Diego, CA). [8-14C]Guanosine by chromatography on PEI-cellulose thin-layer plates (E. was also obtained from New England Nuclear, while [8-3H]- Merck, Darmstadt) affixed with a paper wick and developed deoxyguanosine came from Amersham/Searle (Arlington overnight in methanol/water (1:1) (12). In representative ex- Heights, IL). All isotopes were tested for purity by thin-layer periments the nucleotides that remained at the origin were chromatography and appropriately diluted with unlabeled further fractionated into the mono-, di-, and triphosphates by nucleoside before use. EHNA was kindly provided by the a second development in sodium formate (pH 3.4) (13) or by Burroughs Wellcome Co. (Research Triangle Park, NC). All two-dimensional chromatography with a discontinuous buffer other reagents were of the highest grade obtainable from system (14). commercial sources. When tritiated isotopes were used, the nucleotide spots were cut out and extracted with 1 ml of 1 M TrisIHCl/0.7 M MgCl2 RESULTS at pH 7.4 before the addition of scintillation fluid (11). When Kinase Activities in Human Tissues. Table 1 shows the 14C isotopes were used, extraction of the product was unrtec- activities of adenosine kinase, deoxyadenosine kinase, deoxy- essary. inosine kinase, and deoxyguanosine kinase in newborn human Deaminase and phosphorylase activities were also deter- tissues as well as in adult human Jymphocytes and granulocytes. mined radiochemically, as previously described (8, 15). Guanosine and inosine kinase activities were undetectable (less Ion-Exchange Chromatography. Twenty milligrams of than 0.02 nmol/min per mg of protein) in any tissue, and are thymic cell extracts was dialyzed against 5 mM sodium phos- not shown. As can be seen, the ability to phosphorylate adeno- phate (pH 7.9) and applied to a 1-ml column of DE52-cellulose sine was widespread among human organs. On the contrary the (Whatman Ltd., Maidstone, Kent) equilibrated at 40 with the ability to phosphorylate deoxyadenosine and deoxyguanosine same buffer. After unbound material was washed with the was largely confined to the thymus and peripheral blood above buffer, the column was eluted with a linear gradient lymphocytes. Deoxyinosine kinase also showed greatest activity made from 6 ml of the above buffer and 6 ml of the same buffer in lymphoid cells, although not as marked as that of deoxy- with 0.3 M NaCl. One-milliliter fractions were collected and adenosine and deoxyguanosine kinases. assayed for enzymatic activity as described above. From 25-75% of the phosphorylated deoxyadenosine and Lymphocyte Growth Studies. Normal human peripheral deoxyguanosine in thymic extracts was recovered as the de- blood lymphocytes were cultured as described in RPMI 1640 oxyribonucleoside triphosphate. The product of deoxyinosine medium supplemented with 2 mM glutamine, 10% heat-in- kinase, on the other hand, was exclusively deoxyinosine activated human AB Rh-positive serum, and 10 ,tg of phyto- monophosphate. hemagglutinin-M per ml (DIFCO, Detroit, MI) as well as var- Deaminase and Phosphorylase Levels in Human Tissues. ious nucleosides (8). After 3 days, blastogenesis was measured Confirming the results of Adams and Harkness, we found that by the linear uptake of tritiated leucine into protein over a 4-hr deaminase, like deoxyadenosine kinase, was present in higher period. uptake was not measured, insofar as pre- concentration in lymphoid tissues than in other organs (Table vious experiments showed that nucleosides can increase or 1) (17). Phosphorylase, however, showed no lymphoid pre- decrease thymidine uptake independent of any effects on ponderance. growth (8). Chromatographic Properties of Nucleoside Kinases. The A clone (Cl-35) of the human lymphoblastoid cell line WIL-2, chromatography of a human thymic extract on DE52-cellulose deficient in hypoxanthine phosphoribosyltransferase (EC clearly separated adenosine and deoxyadenosine kinase ac- 2.4.2.8), was selected as described and grown in minimum es- tivities (Fig. 1). Deoxyguanosine and deoxyinosine kinase ac- sential medium with 10% dialyzed fetal calf serum (16). tivities cochromatographed with deoxyadenosine kinase (results Downloaded by guest on October 2, 2021 Immunology: Carson et al. Proc. Natl. Acad. Sci. USA 74 (1977) 5679

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0.1 Am L.OAM 10sM lOO1M 1mM Nucleoside Concentration FIG. 2. Potentiation of deoxyadenosine toxicity by the deaminase Fraction Number inhibitor EHNA. Phytohemagglutinin-stimulated human peripheral FIG. 1. Chromatography of nucleoside kinases on DE52-cellulose. lymphocytes were cultured with varying concentrations of deoxy- A human thymic-cell extract was fractionated on DE52-cellulose with adenosine, either (0) with or (0) without 5,uM EHNA. After 3 days, an NaCl gradient in phosphate buffer (pH 7.9). Individual fractions uptake of tritiated leucine into acid-precipitable material was de- were assayed for (0) A28o, (0) adenosine kinase, and (-) deoxyade- termined. Percent control leucine uptake = (cpm with nucleoside/cpm nosine kinase activity. Deoxyguanosine kinase and deoxyinosine ki- without nucleoside) X 100. Each point represents the mean percent nase, which were also assayed, cochromatographed with the major uptake ±SEM of six replicate cultures. In the presence of 5 ,M EHNA deoxyadenosine kinase peak. alone, leucine uptake was 82 + 4% of control values.

not shown). The approximate Km of the enzyme purified on osides can be acted upon by specific kinases and "trapped" DE52-cellulose for all the purine deoxyribonucleosides was 400 intracellularly in the form of 5'-phosphates which do not readily ,uM at 37°C. This value represents an upper limit, since the traverse the plasma membrane. Thus the uptake of nucleosides preparation was contaminated with phosphorylase. The relative by tissues is limited by the availability of specific phosphor- V'max of deoxyinosine phosphorylation was only one-third that ylating enzymes. of deoxyadenosine and deoxyguanosine. There was no evidence Because human adenosine kinase is distributed among many of substrate inhibition at deoxyribonucleoside concentrations organs, plasma adenosine is not likely to be selectively taken up up to 1 mM. by lymphoid tissues. In fact, under physiologic conditions most Lymphocyte Growth Studies. The addition of deoxyade- adenosine produced intracellularly via nucleotidases and nosine to peripheral lymphocyte cultures depressed their re- phosphatases may be rephosphorylated by the highly avid sponse to phytohemagglutinin (Fig. 2). The toxicity of deoxy- adenosine kinase without reaching the extracellular environ- adenosine was potentiated 1000-fold by inhibition of deaminase ment, since at low substrate concentrations the metabolism of with EHNA. adenosine is a function of the relative Km of adenosine kinase Not having a potent phosphorylase inhibitor or a phospho- (3 AM) and deaminase (25-40 AM) for adenosine (14, 21, 22). rylase-deficient cell line, we could not study in isolation the There is no good evidence that deaminase functions normally effects of deoxyguanosine and deoxyinosine on human lym- as a major pathway in the production of uric acid. In support phocytes. However, the growth of the hypoxanthine phos- of this interpretation, deaminase-deficient children have normal phoribosyltransferase-deficient human lymphoblastoid cell line uric acid levels in plasma and urine (23). C1-35, although unaffected by either guanosine or inosine, was The ability to phosphorylate and trap deoxyadenosine, unlike inhibited by deoxyguanosine and deoxyinosine (Table 2). Other adenosine, is largely confined to lymphoid tissues, whether or studies showed that this cell line, as expected, had the ability not they are actively dividing, as is the newborn thymus, or are to phosphorylate the purine deoxvribonucleosides but not the in a quiescent state, as is the adult peripheral blood lymphocyte. guanosine or inosine. The toxicity of deoxyadenosine alone or deoxyadenosine plus Table 2. Effect of nucleosides upon the growth of a human EHNA toward phytohemagglutinin-stimulated lymphocytes lymphoblastoid cell line deficient in hypoxanthine was reversed by the addition of but not uridine phosphoribosyltransferase to the culture medium (Table 3), consistent with previous ex- Cells/ml X 10-4 periments by Reichard et al. (18) and Klenow (19). The in- Nucleoside 0 hr 24 hr 48 hr 72 hr hibitory effects of deoxyguanosine and deoxyinosine on the growth of human lymphoblastoid cell lines, however, were not None 5 9 27 72 reversed by deoxycytidine. Inosine, 2 mM 5 7 20 52 Inosine, 1 mM 5 10 25 65 DISCUSSION Deoxyinosine, 2 mM 5 5 7 8 Deoxyinosine, 1 mM 5 5 10 19 In the studies reported here, we have tried to explain not the Guanosine, 1 mM 5 7 16 40 detailed biochemical mechanisms by which deficiencies of Guanosine, 0.5 mM 5 8 38 94 adenosine deaminase and purine nucleoside phosphorylase Deoxyguanosine, 1 mM 5 4 5 8 produce immunodeficiency, but rather why both defects might Deoxyguanosine, 0.5 mM 5 3 12 25 preferentially impair lymphocyte function. Patterson has Cells were grown in minimum essential medium with 10% dialyzed summarized evidence which suggests that may be ex- fetal calf serum. Nucleosides at the concentrations indicated were changed between cells and tissues at the nucleoside level, via added at time zero. Comparable concentrations of hypoxanthine or a process of facilitated diffusion (20). Subsequently the nucle- 0.1 mM had no effect on cell growth (32). Downloaded by guest on October 2, 2021 5680 Immunology: Carson et al. Proc. Natl. Acad. Sci. USA 74 (1977) Table 3. Deoxycytidine reversal of deoxyadenosine toxicity during phytohemagglutinin-induced human lymphocyte transformation % control leucine Inhibitor Reversor, 100,uM uptake i SEM* Pt Deoxyadenosine, 1 mM None .35 i 1 Uridine 36 + 2 NS Deoxycytidine 59 + :3 <0.05 Deoxyadenosine, 10 AM + EHNA, 5 AM None 19 ± :3 Uridine 26 i 2 NS Deoxycytidine 70 + 5 <0.05 No phytohemagglutinin 5 + 1 * Human peripheral blood lymphocytes were stimulated with phytohemagglutinin in the presence or absence of the indicated nucleosides. After 3 days the percent control leucine uptake into acid-precipitable material was determined as described in the legend ot Fig. 2. Six cultures were examined in all cases. t P values compare the means of the cultures with uridine or deoxycytidine with those containing deoxyadenosine alone by Students' t test. NS, not significant.

However, the Km of 400 gAM for deoxyadenosine phosphoryl- the calf thymus enzyme and showed that it could also phos- ation far exceeds the reported Km of 7 AiM for deamination (22). phorylate deoxyadenosine and deoxyguanosine (27). Krygier Hence, when deaminase is present, deoxyadenosine is likely to and Momparler, on the other hand, purified an activity from be deaminated rather than phosphorylated at low substrate calf thymus that phosphorylated deoxyadenosine and deoxy- concentrations. A low rate of conversion of deoxyadenosine to guanosine but not deoxycytidine (28). Recently, Krenitsky et adenine has also been reported (14). That the al. have studied in detail the substrate specificity of a partially tissue distribution of deaminase in general parallels that of purified calf deoxycvtidine kinase that could phosphorylate deoxyadenosine kinase may indicate a role for deaminase in the deoxyadenosine, deoxyinosine, and deoxyguanosine as well as prevention of deoxyadenosine phosphorylation. In deaminase many synthetic substrates (29). Not yet having purified the deficiency, on the other hand, deoxyadenosine may be excreted human enzyme to homogeneity, we cannot conclusively de- by many tissues impaired in their ability to deaminate or termine its substrate specificity. However, in preliminary ex- phosphorylate the nucleoside, only to become trapped in periments we found that human deoxycytidine phosphorylating lymphoid tissues in spite of a low plasma concentration. activity cochromatographed on DE52-cellulose with deoxy- Polmar et al. have reported a 10-fold increase in ATP content adenosine kinase. of peripheral lymphocytes from a deaminase-deficient patient The mechanisms by which trapped deoxyribonucleotides as compared to those of normal controls, and that transfusion might inhibit lymphocyte growth are unclear. Under certain of frozen irradiated erythrocytes decreased ATP concentrations conditions triphosphates can be inhibitors (4). This result conflicts with our hypothesis, which would not of reductase (30, 31). Our studies showed that predict the selective accumulation of adenine ribonucleotides deoxyadenosine and deoxyguanosine were indeed converted by lymphocytes. However, the enzymatic methods used by by lymphocyte extracts to the respective triphosphates. How- Polmar et al. would not distinguish ATP from dATP. It is ever, deoxyinosine, which was converted only to the mono- therefore possible that, in fact, deoxyadenosine nucleotides and phosphate form, still inhibited cell growth, albeit at higher not adenine ribonucleotides are elevated in deaminase-deficient concentrations than did deoxyguanosine. lymphocytes. Further investigations with more specific methods Before enzyme replacement in the form of erthyrocyte should resolve this question. transfusion was used, the immunodeficient state associated with Patients with phosphorylase deficiency show elevated con- deaminase deficiency had been successfully treated with bone centrations of inosine, deoxyinosine, guanosine, and deoxy- marrow or fetal liver transplantation (4, 33). When these guanosine in their plasma and urine, thus providing further preferential methods fail or are unavailable, an attempt might evidence for the absence of guanosine kinase or inosine kinase be made to modify the disease state at the molecular and cel- activity in human tissues (24). Consequently, phosphorylase lular level. As we have shown, under certain conditions in vitro, (and hypoxanthine phosphoribosyltransferase), may well be deoxycytidine can partially reverse deoxyadenosine toxicity, required for the normal recycling of inosine and guanosine, a perhaps through competitive inhibition of deoxyadenosine fact that is reflected in the necessarily widespread tissue dis- phosphorylation and "trapping". Whether or not deoxycytidine tribution of phosphorylase and the reported overproduction of is of value in the treatment of adenosine deaminase or purine purines by phosphorylase-deficient patients (24). nucleoside phosphorylase deficiency disease remains to be es- Deoxyguanosine and deoxyinosine, unlike their ribonucle- tablished. In any case, further investigations on the mechanisms oside analogues, can be directly phosphorylated and trapped of deoxypurine toxicity seems warranted. As was true by lymphocytes enriched in the appropriate kinase. This study was supported by National Institutes of Health Grants for deoxyadenosine, here too the K111 of 400,M for phospho- GM 23200, GM 17702, and AM 13622, grants from the Kroc Foun- rylation compared to approximately 50 ,uM for phosphorolysis dation, National Foundation, and by Special Grant 797 from the (25) suggests that at physiologic substrate concentrations California Division of the American Cancer Society. D.A.C. is a Special phosphorylation is unlikely to be significant. In phosphorylase Fellow of the Leukemia Society of America. and deficiency on the other hand, deoxyguanosine deoxyinosine 1. Giblett, E. R., Anderson, J. E., Cohen, F., Pollara, B. & Meuwissen, should be taken up by lymphocytes at an increased rate as well H. J. (1972) Lancet ii, 1067-1069. as excreted into the urine unchanged. 2. Giblett; E. R., Amman, A.J., Sandman, R., Wara, D. W. & Dia- Durham and Ives have previously shown that the murine mond, L. K. (1975) Lancet ii, 1010-1013. enzyme is confined to lymphoid tissues, 3. Hirschhorn, R., Levvtska, V., Pollara, B. & Meuwissen, H. J. especially the thymus (26). These authors subsequently purified (1973) Nature New Biol. 246, 200-202. Downloaded by guest on October 2, 2021 Immunology: Carson et al. Proc. Natl. Acad. Sci. USA 74 (1977) 5681

4. Polmar, S. H., Wetzler, E. M., Stern, R. C. & Hirschhorn, R. C. 20. Patterson, A. R. P., Kim, S. C., Barnard, 0. & Cass, C. E. (1975) (1975) Lancet ii, 743-746. Ann. N.Y. Acad. Sci. 255,402-410. 5. Green, H. & Chan, T.-S. (1973) Science 182, 836-837. 21. Schnebli, H. P., Hill, D. L. & Bennet, L. L., Jr. (1967) 1. Biol. 6. Benke, P. J. & Dittman, D. (1976) Pediatr. Res. 10, 642-646. Chem. 242, 1997-2004. 7. Wolberg, G., Zimmerman, T. P., Hiemstra, K., Winston, M. & 22. Agrawal, R. P., Sagar, S. M. & Parks, R. E., Jr. (1975) Biochem. Chu, L.-C. (1975) Science 187, 957-959. Pharrmacol. 24, 693-701. 23. Mills, G. C., Schmalsteig, F. C., Trimmer, K. B., Goldman, A. S. 8. Carson, D. A. & Seegmiller, J. E. (1976) J. Clin. Invest. 57, & Golblum, R. M. (1976) Proc. Natl. Acad. Sci. USA 73, 274-282. 2867-2871. 9. Boyle, W. (1968) Transplantation 6, 761-772. 24. Cohen, A., Doyle, D., Martin, D. W., Jr. & Amman, A. J. (1976) 10. Lowry, 0. H., Rosebrough, N. J., Farr, A. L. & Randall, R. J. N. Engl. J. Med. 95, 1449-1454. (1951) J. Biol. Chem. 193,265-271. 25. Kim, B. K., Chu, S. & Parks, R. E., Jr. (1968) J. Biol. Chem. 243, 11. Ives, D. H., Durham, J. P. & Tucker, V. J. (1969) Anal. Biochem. 1771-1776. 28, 192-205. 26. Durham, J. P. & Ives, D. H. (1969) Mol. Pharmacol. 5, 358- 12. Crabtree, G. W. & Henderson, J. F. (1971) Cancer Res. 31, 375. 985-991. 27. Durham, J. P. & Ives, D. H. (1970) J. Biol. Chem. 245, 2276- 13. Randerath, K. & Randerath, E. (1964) J. Chromatogr. 16, 2284. 111-125. 28. Krygier, V. & Momparler, R. L. (1971) J. Biol. Chem. 246, 14. Snyder, F. F. & Henderson, J. F. (1973) J. Biol. Chem. 248, 2745-2751. 5899-5904. 29. Krenitsky, T. A., Tuttle, J. V., Koszalka, G. W., Chen, I. S., 15. Snyder, F. F., Mendelsohn, J. & Seegmiller, J. E. (1976) J. Clin. Beacham, L. M., III, Rideout, J. L. & Elion, G. B. (1976) J. Biol. Invest. 58, 654-666. Chem. 251, 4055-4061. 16. Lever, J. E., Nuki, G. & Seegmiller, J. (1974) Proc. Natl. Acad. 30. Reichard, P. (1968) Eur. J. Biochem. 3, 259-266. Sci. USA 71, 2679-2683. 31. Moore, E. C. & Hurlbert, R. B. (1966) J. Biol. Chem. 241, 17. Adams, A. & Harkness, R. A. (1976) Clin. Exp. Immunol. 26, 4802-4809. 647-649. 32. Brenton, D. P., Astrin, K. H., Cruikshank, M. K. & Seegmiller, 18. Reichard, P., Canellakis, Z. N. & Canellakis, E. S. (1960) Biochim. J. E. (1977) Biochem. Med. 17,231-247. Biophys. Acta 41, 558-565. 33. Keightley, R. G., Lawton, A. R., Cooper, M. D. & Yunis, E. J. 19. Klenow, H. (1962) Biochim. Biophys. Acta 61, 885-890. (1975) Lancet ii, 850-883. Downloaded by guest on October 2, 2021