Purine nucleoside kinases in human T- and B-lymphoblasts

Biochimica et Bi.physica Acta, 761 (1983) 34-40

34

Elsevier

BBA 21598

PURINE NUCLEOSIDE KINASES IN HUMAN T- AND B - L Y M P H O B L A S T S Y A S U K A Z U Y A M A D A , H A R U K O G O T O and N O B U A K I O G A S A W A R A

Department of Biochemistry, Institute for Developmental Research, A ichi Prefectural Colony. Kasugai. A ichi 480-03 (Japan) (Received April 19th, 1983)

Kev words: Purine nucleoside kinase," Adenosine kinase," Deoxyguanosine kinase, Deoxv~Ttidine kinase: (Human Ivmphoblast)

Purine nucleoside kinases in human B- and T-lymphoblasts were fractionated by DEAE-cellulose chromatography. Human B-lymphoblast cell extracts showed three peaks of nucleoside kinase activities, adenosine kinase (EC 2.7.1.20), deoxyguanosine kinase and deoxycytidine kinase (EC 2.7.1.74). However, T-lymphoblast cell extracts showed a nucleoside kinase activity which phosphorylates deoxycytidine, deoxyadenosine and deoxyguanosine, similar to deoxycytidine kinase, in addition to the three nucleoside kinases. The K m values of T-lymphoblast-specific nucleoside kinase for deoxyadenosine and deoxyguanosine, 15 and 26 ~M, respectively, were smaller than those of deoxycytidine kinase, 150 and 330 I~M, respectively. Deoxyadenosine phosphorylation by deoxycytidine kinase was strongly inhibited by dCTP, but the phosphorylation by T-lymphoblast-specific nucleoside kinase was only weakly inhibited by dCTP. Deoxyadenosine phosphorylating activity in B-lymphoblast extracts was more distinctly inhibited by dCTP than that in T-lymphoblast extracts.

Introduction The phosphorylation of deoxyadenosine and deoxyguanosine is important, in the deficiencies of adenosine deaminase (EC 3.5.4.4) and purine nucleoside phosphorylase (EC 2.4.2.1) associated with immunodeficiency [1]. Patients with adenosine deaminase or purine nucleoside phosphorylase deficiency excrete either deoxyadenosine or deoxyinosine and deoxyguanosine in urine, and their erythrocytes contain elevated concentrations of dATP and dGTP, respectively [2 5]. After addition of deoxyadenosine to T-lymphoblast in the presence of an inhibitor of adenosine deaminase, dATP accumulated in the cells and the levels of dATP correlated well with the cytotoxicity [6,7]. Also the cytotoxicity of exogeneous deoxyguanosine correlated with the intracellular concentration of accumulated d G T P [8,9]. The deficiency of adenosine deaminase is asso0304-4165/83/$03.00 ~:~ 1983 Elsevier Science Publishers B.V.

ciated with severe T-lymphocyte and more variable B-lymphocyte dysfunction [10,11], and purine nucleoside phosphorylase deficiency is associated with disturbed T-cell function and with little, if any, B-cell dysfunction [12,13]. T-lymphoblasts are far more sensitive than B-lymphoblasts to the toxic effects of deoxyadenosine and deoxyguanosine, and dATP or d G T P was more distinctly accumulated in T-lymphoblasts than B-lymphoblasts under the same condition [7,14-18]. However, the differences between T- and B-lymphoblasts in the accumulation rates of dATP or dGTP, were not explained by differences in the total amounts of assayable deoxyadenosine or deoxyguanosine phosphorylating activities in the extract of those cells [14,19]. In mammalian cells, adenosine kinase (EC 2.7.1.20) and deoxycytidine kinase (EC 2.7.1.74) are capable of phosphorylating deoxyadenosine [20-25]. Recent studies [19,26,27] suggest that the

35 intracellular conversion of cytotoxic nucleosides depends on the joint action of adenosine kinase and deoxycytidine kinase. Further, the existence of mitochondrial deoxyguanosine kinase which phosphorylated deoxyguanosine and deoxyadenosine, was suggested [28-30]. If the mitochondrial enzyme is concerned with the intracellular phosphorylation of deoxyribonucleosides, deoxyadenosine is phosphorylated by at least three nucleoside kinases and deoxyguanosine by two enzymes. In this study, we compared T- and B-lymphoblasts in the activities of nucleoside phosphorylation, after fractionation of nucleoside kinases by DEAE cellulose chromatography. We observed the existence of an extra nucleoside kinase similar to deoxycytidine kinase in T-lymphoblasts, but not in B-lymphoblasts, and report some differences between the T-lymphoblast-specific nucleoside kinase and deoxycytidine kinase.

Materials and Methods

Materials. [8-14C]Adenosine (59 mCi/mmol) and [8-3H]deoxyguanosine (1.7 Ci/mmol) were purchased from Amersham, and [8-14C]deoxyadenosine (53.8 Ci/mol) and [2-14C]deoxycytidine (25.2 Ci/mol) were from New England Nuclear. Nucleosides, Nucleotides, phosphoenolpyruvate and pyruvate kinase were obtained from Sigma Chemical Co. and Boehringer Mannheim. DEAEcellulose (DE-52) was obtained from Whatman. Erythro-9-(2-hydroxy-3-nonyl)adenine (EHNA), was a gift from Burroughs Welcome Corp. (Research Triangle Park, NC, USA). Other reagents were commercial preparation of the highest purity available. Cell lines and cell culture. Three human Tlymphoblast cell lines, MOLT 4F; CCRF CEM; RPMI 8402, and two B-cell lines, BALL 1; EBV (Epstein-Barr virus) transformed B-lymphoblast, were studied. All cell lines were grown in suspension culture using 1640 medium supplemented with 15% fetal bovine serum. Enzyme assay and protein determination. Adenosine kinase activity was assayed at pH 7.5 (50 mM Tris-HC1) with 1 #M [8-14C]adenosine as previously described [25]. Deoxyguanosine kinase activ-

ity was assayed at pH 6.0 (50 mM sodium cacodylate) with 10 #M [8-3H]deoxyguanosine by the assay method for mitochondrial deoxyguanosine kinase [30]. Deoxycytidine kinase and deoxyadenosine kinase activities were usually assayed at pH 8.0 (50 mM Tris-HCl) with 10 ttM [2-14C]deoxycytidine and 20 #M [8-14C]deoxyadenosine, respectively, by a method [30] similar to that for adenosine kinase and deoxyguanosine kinase. For the assay of deoxyadenosine kinase activity in the cell extracts, 10 #M EHNA was added to the reaction mixture to inhibit the deamination by adenosine deaminase. 1 unit of the each enzyme activity is defined as the amount catalyzing the phosphorylation of 1 pmol of nucleoside for 1 min. Specific activity was defined as units/mg protein. Protein concentration was determined by the method of Lowry et al. [31] using bovine serum albumin as a standard. Preparation of cell extracts and DEAE-cellulose chromatography. Cells were collected by centrifugation and resuspended in saline solution (0.154 M NaC1). After washing 3 times with saline, the cell pellet was stored at -70°C. The frozen cells were thawed and suspended in buffer (10 mM Tris-HC1 (pH 7.5), 1 mM dithiothreitol and 10% glycerol) at about 108 cells/ml, and were lysed by ultrasonication (twice at 20 s using an ARTEX sonic dismembrator, model 150). The lysate was centrifuged for 20 min at 20 000 × g to remove cellular debris. The supernatant was assayed immediately or stored at 4°C. The extract (about 1 ml) was applied to a column of DEAE-cellulose (0.4 cm2 × 5 cm) equilibrated with buffer A, and was washed with 10 ml buffer, to elute all unbound protein. The column was further eluted with a linear gradient of 0-0.25 M KC1 in buffer A (10 ml each), and followed by buffer A containing 0.5 M KC1. To stabilize the enzyme, bovine serum albumin was added to the all fractions at the final concentration of 0.2 mg/ml. Four nucleoside kinase activities in the each fraction were assayed immediately. Human tissues obtained at autopsy and stored - 7 0 ° C were homogenized in 5 vol. buffer A and centrifuged at 20000 × g for 20 min. The supernatant was assayed and applied to the DEAE-cellulose chromatography.

36

Results and Discussion 50- A (1000)

Nucleoside kinase activities in each cell extract of human T- and B-lymphoblasts are summarized in Table I. There was no distinct difference in deoxyadenosine phosphorylating activity between T- and B-lymphoblasts. The activity of deoxyadenosine phosphorylation was assayed in the presence of EHNA, an inhibitor of adenosine deaminase. Without EHNA in the reaction mixture, the activity was decreased to 20-30%, but the ability to phosphorylate adenosine was constant whether EHNA was added or not. The phosphorylating activities of adenosine and deoxyguanosine were slightly higher in B-cell lines (BALL 1 and EBV-transformed B-lymphoblast) than T-cell lines (MILT 4F, CCRF CEM and RPMI 8402). EBV-transformed B-lymphoblast had less deoxycytidine kinase activity (about 50%) than other cell lines. Thus, the differences among Tand B-cell lines of the activities of deoxyribonucleoside phosphorylation in cell extracts cannot explain the differences between T-cell and B-cell dysfunction in adenosine deaminase and purine nucleoside phosphorylase deficiencies, as reported previously [14,19,32]. Deoxyadenosine and deoxyguanosine, nucleosides associated with cytotoxicity in these deficiencies, were phosphorylated by the joint action of various nucleoside kinases. If deoxyguanosine kinase which was strongly inhibited by a low concentration of dGTP would be present much more in B-lymphocytes than in Tlymphocytes, the differences between T-cell and B-cell dysfunction in purine nucleoside phosphorylase deficiency might be clearly explained.

y

-O5

i

2:-1i

o

--

i

lO

20 Fraction

30 No

40

50

Fig. 1. DEAE-cellulose chromatography of nucleoside kinases in human B- and T-lymphoblast cell extracts. About 1 ml of the cell extracts of BALL 1 (A) and MOLT 4F (B) were applied to a column of DE-52 cellulose and eluted, as described under Methods. The activities to phosphorylate nucleosides, adenosine (11 II), deoxyadenosine (O O), deoxyguanosine (O O), and deoxycytidine (A zx), were assayed under standard conditions. Adenosine kinase activity is shown in parentheses. (. . . . . . ), shows KCI concentration.

Thus, the nucleoside kinases in the cell extract were fractionated by DEAE-cellulose chromatography (Figs. 1 and 2). There are no differences in the amount of respective enzymes as expected, but there is a specific nucleoside kinase only in the extracts of T-lymphoblasts. In BALL 1 (Blymphoblast), adenosine kinase was eluted at 0.08

TABLE I P U R I N E N U C L E O S I D E KINASE ACTIVITIES IN CELL EXTRACTS The enzyme activities were assayed under standard conditions described under Methods. Results ( p m o l / m i n per mg protein) are shown with the mean_+ S.D. obtained from 4-8 series of experiments. Cell lines

Adenosine

Deoxyadenosine"

Deoxyguanosine

Deoxycytidine

MOLT 4F CCRF CEM RPMI 8402 BALL 1 EBV-transformed B

1850 + 193 1890_+235 1830 + 161 2140 ___115 2560_+ 127

79.0 + 10.2 62.5 + 14.0 44.9 _ 4.6 57.0 _+12.2 34.3 + 6.8

3.85 _+0.69 4.32+_0.97 4.21 _ 0.48 6.40 + 1.04 5.62 + 0.84

83.1 +_16.0 84.6+ 18.5 96.2 + 10.7 90.9 + 11.3 41.1 + 14.3

10/zM EHNA was added to the reaction mixture.

37

l

aI '~25 A

~

b, cI

_Id

!

-I 0.5

o

~, 25-

[......... 05

0

v

0 ee~ ~ O" ._c •~ 25- C

........... 05

Q

10

20

Fraction

30

40

No.

Fig. 2. DEAE-cellulose chromatography of nucleoside kinases in the cell extracts of C C R F CEM (A), RPMI 8402 (B), and EBV-transformed B-lymphoblast (C). Chromatographic conditions were described under Methods. Only the activity to phosphorylate deoxycytidine (e e) is shown. The peaks of four nucleoside kinases, T-lymphoblast-specific nucleoside kinase (a), adenosine kinase (b), deoxyguanosine kinase (c), and deoxycytidine kinase (d), are shown at the top of the figure. ( . . . . . . ), shows KCI concentration.

M KC1 concentration and deoxyguanosine kinase at 0.15 M by a linear gradient of 0-0.25 M KCI, and then deoxycytidine kinase was eluted with the buffer containing 0.5 M KC1. However, in MOLT 4F (T-lymphoblast), a peak of nucleoside kinase not bound to DEAE-celluose was observed in addition to the three nucleoside kinase peaks. This nucleoside kinase phosphorylated deoxycitidine, deoxyadenosine and deoxyguanosine, similar to deoxycytidine kinase [20-22]. T-cell lines (CCRF CEM and RPMI 8402) possess a nucleoside kinase which is not bound to DEAE-cellulose (we have called it T-lymphoblastspecific nucleoside kinase), with varying amounts, but B-cell line (EBV-transformed B-lymphoblast) did not (Fig. 2). DEAE-cellulose chromatography was also carried out using extracts of human tissues, such as thymus, spleen, placenta and liver, but all these tissues did not contain the Tlymphoblast-specific nucleoside kinase. The porperties of the T-lymphoblast-specific nucleoside kinase and deoxycytidine kinase were studied using respective DEAE-cellulose fractions.

Each enzyme fraction was adenosine deaminasefree, since the activity to phosphorylate deoxyadenosine was not decreased in the absence of EHNA. The specific activities of Fractions I and II were 238 and 156 pmol/min per mg protein, respectively, when deoxycytidine was used as the substrate. Their kinetic properties are compared in Table II. Both enzymes showed similar K m values for deoxycytidine. However, the K m values of Tlymphoblast-specific nucleoside kinase for deoxyadenosine and deoxyguanosine, 15 and 26/xM, respectively, were much smaller (about 1/10) than those of deoxycytidine kinase. The K m values for deoxyadenosine and deoxyguanosine, and the ratio of Vmax for respective substrates of deoxycytidine kinase were similar to those reported in previous studies [20-22] The profiles of pH optimum of T-lymphoblastspecific nucleoside kinase and deoxycytidine kinase are shown in Fig. 3. Both enzymes had a pH optimum at pH 8.0, but the profile of T-lymphoblast-specific nucleoside kinase was broader than that of deoxycytidine kinase. At pH 7.3, deoxyadenosine phosphorylating activity by deoxycytidine kinase was decreased to 40% of the optimum, but the T-lymphoblast-specific nucleoside kinase showed a similar activity as optimum. Similar pH

T A B L E II K I N E T I C "PROPERTIES OF T-LYMPHOBLAST-SPECIFIC NUCLEOSIDE KINASE AND DEOXYCYTIDINE KINASE The enzyme activities were assayed at p H 8.0, in the presence of 0.5-100 g M deoxycytidine, 1-500 # M deoxyadenosine or 1 - 1 0 0 0 g M deoxyguanosine. Two series of experiments were carried out and very similar g m and Vm~ values were obtained; the respective differences were less than 10%. Fraction I contains T-lymphoblast-specific nucleoside kinase and Fraction II contains deoxycytidine kinase.

Km (p.M) Deoxycytidine Deoxyadenosine Deoxyguanosine Vm~ (Relative activity) Deoxycytidine Deoxyadenosine Deoxyguanosine

Fraction I

Fraction II

8 15 26

5 150 330

1.0 6.0 2.3

1.0 4.2 2.2

38

~o

0

.

5

.

.

6

.

7 pH

.

.

8

9

Fig. 3. Effects of pH on deoxyadenosine phosphorylation by T-lymphoblast-specific nucleoside kinase (O O), and deoxycytidine kinase ( O O). Cacodylate buffers at pH 4.9-6.8, and Tris-HCl buffers at pH 7.3-9.0 were used.

optimum profiles were also observed by other substrates. Effects of other nucleosides on the phosphorylation of deoxycytidine and deoxyadenosine were tested for both enzymes (Table III). Deoxycytidine and arabinocytidine which were good substrates of deoxycytidine kinase [21], strongly inhibited the phosphorylation of deoxyadenosine by deoxycytidine kinase, but they weakly inhibited the T - l y m p h o b l a s t - s p e c i f i c n u c l e o s i d e kinase. A r a b i n o a d e n o s i n e inhibited T - l y m p h o b l a s t specific nucleoside kinase, but it did not (or only weakly) inhibit deoxycytidine kinase. Effects of other nucleosides on the phosphorylation of deoxycytidine were not significantly different between the two enzymes.

When the effects of added nucleoside triphosphate were tested on the phosphorylation (Table IV), a marked difference was observed in the effects of dCTP. The phosphorylation of deoxyadenosine by deoxycytidine kinase was completely inhibited by 1 mM dCTP as previously described [20,21], but that of the T-lymphoblast-specific nucleoside kinase was inhibited only 30% by 1 mM dCTP. Furthermore, with deoxyadenosine as substrate, deoxycytidine kinase was inhibited to 50% by less than 10 /~M concentration of dCTP. But T-lymphoblast-specific nucleoside kinase was inhibited to 50% at much higher concentration, in the mM range (Table V). At a lower concentration of deoxyadenosine (1 ~M), dCTP slighly activated rather than inhibited the T-lymphoblast-specific nucleoside kinase. Observed activation is not due to the enzyme stabilization by dCTP, since the enzyme is stable under the assay condition. Using extracts of MOLT 4F and BALL 1 cells, the effects of various dCTP concentration on phosphorylation of deoxyadenosine were examined. The extracts of MOLT 4F cells having T-lymphoblast-specific nucleoside kinase and deoxycytidine kinase were less affected by dCTP that those of BALL 1 cells having only deoxycytidine kinase; 1 mM dCTP decreased the phosphorylating activities in MOLT 4F and BALL 1 cell extracts to 50 and 15%, respectively. In BALL 1 cell extract, the residual activities to phosphorylate deoxyadenosine in the presence of 1 mM dCTP, seemed to depend upon the phosphorylating activ-

T A B L E Ill EFFECTS OF A D D E D N U C L E O S I D E ON D E O X Y C Y T I D I N E O R D E O X Y A D E N O S I N E P H O S P H O R Y L A T I N G ACTIVITY The enzyme activities were assayed with 10 # M deoxycytidine or 20/~M deoxyadenosine at pH 8.0, plus other nucleosides. Fraction 1 contains T-lymphoblast-specific nucleoside kinase and Fraction 11 contains deoxycytidine kinase. Values represent relative activity. Nucleoside

Deoxycytidine phosphorylation

Deoxyadenosine phosphorylation

(100/xM)

Fraction 1

Fraction 11

Fraction 1

Fraction 1I

None Adenosine Deoxyadenosine Guanosine Deoxyguanosine Cytidine Deoxycytidine Arabinoadenosine Arabinocytidine

100 101 51 80 74 105

100 100 77 81 55 81

100 100

100 97

54 78

80 51

92 101 98 40 58 70

97 75 80 0 94 3

39 T A B L E IV EFFECTS OF A D D E D N T P ON D E O X Y C Y T I D I N E OR D E O X Y A D E N O S I N E P H O S P H O R Y L A T I N G ACTIVITY The enzyme activities were assayed with 10 m M ATP and 10 ~M deoxycytidine or 20 ~tM deoxyadenosine at p H 8.0, plus other nucleoside triphosphates. Fraction I contains T-lymphoblast-specific nucleoside kinase and Fraction II contains deoxycytidine kinase. Values represent relative activity. NTP (1 mM)

Deoxycytidine phosphorylation

Deoxyadenosine phosphorylation

Fraction 1

Fraction II

Fraction I

Fraction II

None GTP CTP UTP dATP dGTP dCTP dTTP

100 70 21 48 40 54 12 47

100 73 19 40 42 68 0 83

100 85 70 71 14 69 68 44

100 104 44 97 46 64 0 78

ity by adenosine kinase and deoxyguanosine kinase. As in the case of deoxycytidine kinase, the T-lymphoblast-specific nucleoside kinase could phosphorylate deoxycytidine, deoxyadenosine and deoxyguanosine. In B-lymphoblast, deoxyadenosine was phosphorylated by the joint actions of three nucleoside kinases. The three nucleoside kinases, adenosine kinase, deoxycytidine kinase and deoxyguanosine kinase, were regulated by respective strong inhibitors, adenosine, dCTP and

TABLE V EFFECTS OF dCTP ON D E O X Y A D E N O S 1 N E PHOSPHORYLATION BY T - L Y M P H O B L A S T - S P E C I F I C N U C L E O S I D E K I N A S E OR D E O X Y C Y T I D I N E KINASE. The enzyme activities were assayed with 1 /~M and 20 /~M deoxyadenosine at pH 8.0, plus various concentrations of dCTP. Fraction I contains T-lymphoblast-specific nucleoside kinase and Fraction II contains deoxycytidine kinase. Values represent relative activity. dCTP Fraction I (mM) Deoxyadenosine: 1 # M

20/~M

1 /~M

20 # M

0 0.001 0.01 0.1 1.0

100 100 98 95 72

7.98 5.08 1.58 0.23 0.03

100 75.9 21.7 3.06 0.36

14.2 16.5 17.0 18.5 20.4

Fraction II

dGTP. However, in T-lymphoblasts, a specific nucleoside kinase was present in addition to the three nucleoside kinases. This kinase has low K m values for deoxyadenosine (15 /~M) and deoxyguanosine (26/~M). The value for deoxyguanosine was higher than that of mitochondrial deoxyguanosine kinase, 2.5 #M [30], but lower than that of cytoplasmic deoxycytidine kinase, 330/~M. And the value for deoxyadenosine was much lower than that of deoxycytidine kinase, 150 /~M, adenosine kinase, 480 ~tM [25], or deoxyguanosine kinase, 630 #M [30]. Furthermore, no strong inhibitor for T-lymphoblast-specific nucleoside kinase has been found. These data indicate that the differences between T-cell and B-ceU dysfunction might be explained by the difference of phopshorylation of nucleosides. The mechanism of deoxyadenosine phosphorylation was studied recently, using mutant CEM cell lines, adenosine kinase-deficient, deoxycytidine kinase-deficient and both two deficient double mutant cell lines [19,27]. Most of deoxycytidine kinase deficient cell lines and double mutant cell lines completely lost the activity of deoxycytidine phosphorylation. One of the double mutant cell lines, RC3b, had an altered deoxycytidine kinase [19], but it was far different from Tlymphoblast-specific nucleoside kinase in this study. It is of interest to know whether those mutant cell lines lacking deoxycytidine kinase also lack T-lymphoblast-specific nucleoside kinase.

40

From the MOLT 4F cell extracts, T-lymphoblast-specific nucleoside kinase was tried to purify by ammonium sulfate fractionation, chromatographies on DEAE and phosphocellulose, and gel filtration. But the recovery of the enzyme activity at each step was less than 30%, since the enzyme is unstable at low protein concentrations. Tlymphoblast-specific nucleoside kinase may be a modified deoxycytidine kinase, since the K m value for deoxycytidine and the molecular weight determined by Sephadex G-75 gel filtration were similar to those of deoxycytidine kinase. In order to make clear those questions, further studies are necessary to purify and characterize T-lymphoblast-specific nucleoside kinase, and to compare with purified deoxycytidine kinase.

Acknowledgments We wish to thank Dr. Yutaro Nishida, Tokyo University, and Dr. Ryuzo Ueda, Aichi Cancer Center Institute, for providing cell lines.

References 1 Carson, D.A., Kaye, J. and Seegmiller, J.E. (1977) Prec. Natl. Acad. Sci. U.S.A. 74, 5677-5681 2 Cohen, A., Hirsch_horn, R., Horowitz, S.D., Rubinstein, A., Polmar, S.H., Hong, R., and Martin, D.W., Jr. (1978) Prec. Natl. Acad. Sci. U.S.A. 75, 472-476 3 Cohen. A., Doyle, D., Martin, D.W., Jr. and Ammann, A.J. (1976) N. Engl. J. Med. 295, 1449-1454 4 Coleman, M.S., Donofrio, J., Hutton, J.J., Hahn, L., Daoud, A., Lampkin, B. and Dyminski, J. (1978) J. Biol. Chem. 253, 1619-1626 5 Cohen, A., Gudas, L.J., Ammann, A.J., Staal, G.E.J. and Martin, D.W., Jr. (1978) J. Clin. Invest. 61, 1405-1409 6 Ullman, B., Gudas, L.J., Cohen, A. and Martin, D.W. Jr. (1978) Cell 14, 356-375 7 Mitchell, B.S., Mejias, E., Daddona, P.E. and Kelley, W.H. (1978) Prec. Natl. Acad. Sci. U.S.A. 75, 5011-5014 8 Chan T.-S. (1978) Cell 14, 523-530 9 Gudas, L.J., UUman, B., Cohen, A. and Martin, D.W., Jr. (1978) Cell 14, 531-538 10 Giblett, E.R., Anderson, J.E., Cohen, F., Pollara, B. and Meuwissen, H.J. (1972) Lancet ii, 1067-1069

11 Meuwissen, H.J., Pollara, B. and Pickering, R.J. (1975) J. Pediatr. 86, 169-181 12 Giblett, E.R., Ammann, A.J., Wara, D.W., Sandman, R. and Diamond, L.K. (1975) Lancet i, 1010-1013 13 Stoop, J.W., Zegers, B.J.M., Hendrickx, G.F.M., van Heukelom, L.H.S., Staal, G.E.J., de Bree, P.K., Wadman, S.K. and Ballieux, R.E. (1977) N. Engl. J. Med. 296, 651-655 14 Carson, D.A., Kaye, J. and Seegmiller, J.E. (1978) J. Immunol. 121, 1726-1731 15 Gelfand, E.W., Lee, J.J. and Dosch, H.-M. (1979) Proc. Natl. Acad. Sci. U.S.A. 76, 1998-2002 16 Carson, D.A., Kaye, J., Matsumoto, S., Seegmiller, J.E. and Thompson, L. (1979) Prec. Natl. Acad. Sci. U.S.A. 76, 2430-2433 17 Wortmann, R.L., Mitchell, B.S., Edwards, N.L. and Fox, I.H. (1979) Prec. Natl. Acad. Sci. U.S.A. 76, 2434, 2437 18 Carson, D.A., Kaye, J. and Wasson, D.B. (1980) J. Immunol. 124, 8-12 19 Hershfield, M.S., Fetter, J.E., Small, W.C., Bagnara, A.S., Williams, S.R., Ullman, B., Martin, D.W., Jr., Wasson, D.B. and Carson, D.A. (1982) J. Biol. Chem. 257, 6380-6386 20 Momparler, R.L. and Fischer, G.A. (1968) J. Biol. Chem. 243, 4298-4304 21 Durham, J.P. and Ives, D.H. (1970) J. Biol. Chem. 245, 2276-2284 22 Krenitsky, T.A., Tuttle, J.V., Koszalka, G.W., Chen, I.S. Beacham, L.M., III, Ride.out, J.L. and Elion, G.B. (1976) J. Biol. Chem. 251, 4055-4061 23 Miller, R.L., Adamczyk, D.L., Miller, W.H., Koszalka, G.W., Rideout, J.L., Beacham, L.M., III, Chao, E.Y., Haggerty, J.J., Krenitsky, T.A. and Elion, G.B. (1979) J. Biol. Chem. 254, 2346-2352 24 Andres, C.M. and Fox, I.H. (1979) J. Biol. Chem. 254, 11388-11393 25 Yamada, Y., Goto, H. and Ogasawara, N. (1981) Biochim. Biophys. Acta 660, 36-43 26 Ullman, B., Levinson, B.B., Hershfield, M.S. and Martin, D.W., Jr. (1981) J. Biol. Chem. 256, 848-852 27 Verhoef, V., Sarup, J. and Fridland, A. (1981) Cancer Res. 41, 4478-4483 28 Gower, W.R., Jr., Carr, M.C. and Ives, D.H. (1979) J. Biol. Chem. 254, 2180-2183 29 Barker, J. and Lewis, R.A. (1981) Biechim. Biophys. Acta 658, 111-123 30 Yamada, Y., Goto, H. and Ogasawara, N. (1982) Biochim. Biophys. Acta 709, 265-272 31 Lowry, O.H., Rosebrough, N.J., Farr, A.L. and Randall, R.J. (1951) J. Biol. Chem. 193, 265-275 32 North, M.E., Nowton, C.A. and Webster, A.D.B. (1980) Clin. Exp. Immunol. 42, 523-529