Purification and characterization of the adenine phosphoribosyltransferase and hypoxanthine-guanine...

Molecular and Biochemical Parasitology, 33 (1989) 273-282 273 Elsevier

MBP 01112

Purification and characterization of the adenine phosphoribosyltransferase and hypoxanthine-guanine

phosphoribosyltransferase activities from Leishmania donovani

Thomas Allen, Eugene V. Henschel, Terry Coons, Laura Cross, Joseph Conley and B u d d y U l l m a n

Department of Biochemistry, The Oregon Health Sciences University, Portland, OR, U.S.A.

(Received 26 August 1988; accepted 28 October 1988)

The adenine phosphoribosyltransferase (APRTase) and hypoxanthine-guanine phosphoribosyltransferase (HGPRTase) activities from promastigotes of Leishmania donovani have been purified to homogeneity using ammonium sulfate precipitation, DEAE- cellulose exclusion, and either AMP-agarose (APRTase) or GTP-agarose (HGPRTase) affinity chromatography. The specific activities of the affinity-purified APRTase and HGPRTase fractions were 326-fold and 1341-fold greater than those in the 40-80% ammonium sulfate precipitate, respectively. The purified APRTase migrated as a single band on sodium dodecyl sulfate (SDS) polyacrylamide gels with a size of 29 kDa, while HGPRTase was also determined to be homogeneous by SDS gel electrophoresis with a size of 24 kDa. In addition, a mutant cell line, APPB2, partially deficient in APRTase activity, still contained quantities of purifiable APRTase protein, while a clonal secondary derivative of the APPB2 cell line that is completely deficient in APRTase activity, APPB2-640A3, failed to express purifiable APRTase protein. The homogeneous enzymes possessed apparent K m values for their nucleobase substrates between 2.0 and 5.0 txM, and both enzymes were inhibited by their immediate or ultimate reaction endproducts, APRTase by AMP and PPi and HGPRTase by GMP, GTP, and PP~. The generation of homogeneous preparations of APRTase and HGPRTase protein will serve as a prerequisite for the generation of immunological and molecular biological probes to analyze the leishmanial phosphoribosyltransferases.

Key words: Adenine phosphoribosyltransferase; Hypoxanthine-guanine phosphoribosyltransferase; Purine

Introduction

Most of the major m~tabolic pathways in parasites are similar to those in humans with one ex- ception. Leishmania [1], as well as all the parasitic protozoa that have been investigated to date [2-10], are incapable of synthesizing the purine ring de novo and are therefore auxotrophic for purines. In order to meet their purine require-

Correspondence address: B. Ullman, Dept. of Biochemistry, The Oregon Health Sciences University, Portland, OR 97201, U.S.A.

Abbreviations: APRTase, adenine phosphoribosyltransferase; HGPRTase, hypoxanthine-guanine phosphoribosyltransferase; DME-L, Dulbecco's modified Eagle-Leishmania; PRPP, phosphoribosylpyrophosphate; SDS, sodium dodecyl- sulfate.

ments, each genus of parasites has evolved a unique series of purine salvage enzymes which enable them to scavenge host purines [2-11]. These unique purine salvage enzymes offer a ra- tional approach to chemotherapeutic manipula- tion of parasitic diseases. For instance, the pyra- zolopyrimidines, 4-hydroxypyrazolo[3,4-d]pyrimidine (allopurinol), 4-hydroxypyrazolo[3,4-d]- pyrimidine riboside (allopurinol riboside), 4-thiopyrazolo[3,4-d]pyrimidine (4- thiopurinol),4- thiopyrazolo[3,4-d]pyrimidine riboside (4-thiopurinol riboside), and 7-hydroxy-3-[3-D-ribofura- nosylpyrazolo[4,3-d]pyrimidine (formycin B) are compounds that are selectively metabolized by and cytotoxic toward Leishrnania and other pathogenic hemoflagellates [9,10].

Purine salvage enzymes have been identified in crude cell extracts of Leishmania promastigotes,

0166-6851/89/$03.50 (~) 1989 Elsevier Science Publishers B.V. (Biomedical Division)

274

although relatively little information is available concerning the functional roles of these enzymes in the intact parasite [11-16]. The major purine source for Leishmania is likely to be the host adenylate nucleotide pool, since ATP is the most abundant nucleotide in mammalian cells. Aden- ylate nucleotides can be dephosphorylated at the cell surface of the parasite by nucleotidase activities [17-19], and the adenosine can then per- meate the plasma membrane of the parasite via the adenosine-pyrimidine nucleoside transporter [20,21[, a process which makes the nucleoside available to the leishmanial metabolic machinery. Once inside the parasite, adenosine can either be phosphorylated directly by adenosine kinase or be cleaved to adenine by adenosine nucleosidase and/or phosphorylase [15]. Adenine can then be phosphoribosylated directly via adenine phosphoribosyltransferase (APRTase) to AMP or can be deaminated by adenine deaminase to hypoxanthine [22], which is then converted to the nucleotide level by hypoxanthine-guanine phosphoribosyltransferase (HGPRTase). Mutational analysis of APRTase function has demonstrated, however, that the predominant pathway for adenine metabolism in Leishmania donovani promastigotes is via deamination and HGPRTase catalyzed phosphoribosylation of hypoxanthine [16]. Looker et al., however, have biochemical evidence that leishmanial amastigotes lack the adenine deaminase activity [ll], suggesting that APRTase may be functional, if not essential, in the infective form of these parasites. Thus, both APRTase and HGPRTase may play critical roles, perhaps in a developmentally regulated fashion, in the purine salvage process of these organisms.

To clarify the roles of these phosphoribosyltransferase activities in purine salvage in these pathogenic hemoflagellates, we have initiated a biochemical, immunological, and molecular analysis of both enzymes in Leishmania. We now report the purification to homogeneity of both APRTase and HGPRTase from L. donovani promastigotes.

Materials and Methods

Chemicals and reagents. [U-14C]Adenine (274 mCi mmo1-1) was bought from the Amersham Cor-

poration (Arlington Heights, IL), while [8- 14C]hypoxanthine (49 mCi mmol-1), [3H]adenin e (50 Ci mmol-1), [3H]hypoxanthine (35 Ci mmol-1), and [3H]guanine (11 Ci mmol l) were purchased from Moravek Biochemicals (Brea, CA). Agarose-hexane-adenosine 5'-phosphate Sepharose 4B (AGAMP Type 2) in which the nucleotide was linked to the spacer by its 6-amino group and agarose-hexane-guanosine triphos- phate (AGGTP Type 4) in which the nucleotide was bound through the 2'- hydroxyl of the ribose ring were obtained from Pharmacia P-L Bio- chemicals Inc. (Piscataway, N J). All other chemicals, materials, and reagents were of the highest quality commercially available.

Cell culture conditions. L. donovani promastigotes originally obtained from Dr. Edna Kane- shiro (University of Cincinnati Medical Center) were routinely cultivated in continuous culture at 25°C in a humidified 10% CO 2 incubator. Wild- type and mutant cells were propagated in the completely defined Dulbecco's modified Eagle- Leishmania (DME-L) medium initially described by Iovannisci and Ullman [23]. When large scale cultures of parasites were required for enzyme preparation, a dense stock culture of promastigotes in DME-L medium was inoculated into a 2 1 Erlenmeyer flask containing 1200 ml of freshly sterilized brain-heart infusion medium (Difco Laboratories) containing 5% fetal calf serum, and supplemented with 5 mg 1-1 hemin and 0.1 mM xanthine. The Erlenmeyer flasks were tightly capped and incubated in a model G-25 shaker incubator (Brunswick Scientific) at 27°C and shaken at 60 rpm until they reached a density of 2-4 x 107 organisms ml-1, a late stage of exponential growth.

The DI700 cell line is a wild type clone of the Sudanese 1S strain of L. donovani that was isolated on soft agar by the single cell cloning tech- nique developed by Iovannisci and Ullman [23]. The APPB2 and APPB2-640A3 cell lines were derived after mutagenesis of DI700 cells as described by Iovannisci et al. [16]. APPB2 and APPB2-640A3 cells are respectively 50% and 100% deficient in APRTase activity as measured by direct enzymatic assay [16]. The growth phen- otypes and properties of the mutant cell lines have been described in detail previously [16].

275

Preparation of cell-free extracts. Two to 16 1 of late exponential phase (2-3 x 107 organisms m1-1) L. donovani were harvested by centrifugation at 3000 x g for 10 min and washed thoroughly with phosphate buffered saline. Pellets were either frozen at -20°C until required for enzyme purification or were extracted immediately. To lyse the promastigotes, two volumes of 50 mM Tris pH 7.4 containing 5 mM MgC12, 1 mM dithiothreitol, and 1 mM phosphoribosylpyrophosphate (PRPP) were added to the Leishmania, and the parasites were then lysed by sonication using a Vibra Cell (Son- ics and Materials, Danbury, CT) fitted with a mi- cro tip after three 15 s bursts at 40% output, each burst separated by a 30 s cooling period on ice. No intact parasites, as monitored by trypan blue exclusion on a hemocytometer , remained after sonication. The sonicated cell extracts were then centrifuged at 45 000 × g 0°C for 60 min, the cell debris discarded, and the supernatant decanted for enzyme purification.

Enzyme purification. Both phosphoribosyltransferase activities were purified from the 45 000 x g cellular supernatants by a modification of the procedure described by Tuttle and Krenitsky [12]. Briefly, the cell extract was brought to 40% sat- uration with a neutralized saturated ammonium sulfate solution at 4°C. After 1 h, the precipitate was removed by centrifugation at 45 000 x g for 30 min, and the supernatant brought to 80% sat- uration with solid ammonium sulfate and stirred an additional 12-20 h at 4°C. The 40-80% ammonium sulfate fraction could be stored at -80°C for 6 months without appreciable loss of either APRTase or HGPRTase . Further purification steps were carried out with either freshly pre- pared or frozen salt precipitates as follows. The 40-80% ammonium sulfate precipitate was sedi- mented by centrifugation and redissolved in a small amount of 50 mM Tris pH 7.4 containing 5 mM MgCI 2 and 1 mM dithiothreitol (buffer A) and gel sieved over a Sephadex G-25 (1 x 25 cm) column to remove excess salt. The desalted extract was passaged over a DEAE-cellulose (1 x 25 cm) column and the void volume collected. The ion exchange column was then washed with 2 column volumes of buffer A and the excluded material pooled with the void volume. The excluded protein fractions were passaged over either a 5 ml

AMP-agarose or a 5 ml GTP-agarose column, either in series or in parallel, at a flow rate of ap- proximately 5 ml h -1. Both affinity columns resins were washed with 2 column volumes of buffer A, followed by two more column volumes of buffer A containing 0.5 M KCI. APRTase was eluted from the AMP-agarose column and HGPRTase was eluted from the GTP-agarose column with 15 ml of buffer A containing 1 mM PRPP, a substrate of both purine salvage enzymes. The PRPP-containing eluates were collected either in 1.0 ml fractions or in a single fraction. Samples were concentrated by Amicon filtration when necessary.

Enzyme assays. APRTase and HGPTase activities were assayed by the method described by Io- vannisci et al. [16].

Km determinations. The Km values of each purified enzyme for its naturally occurring nucleobase substrates were determined with either 0.5 IxM [3H]adenine (50 Ci mmol - l ) for APRTase and 0.5 ~M [3H]hypoxanthine (35 Ci mmo1-1) or 1.0 IxM [3H]guanine (11 Ci mmo1-1) for HGPRTase and increasing concentrations of nonradiolabeled nucleobase substrate using the same assay conditions described. Isotopic re- placement of the nucleobase substrate in the standard assay mixture was necessary to ensure linearity of the K m value determination assays at low substrate concentrations.

Protein determinations. Protein concentrations were determined by the method of Bradford [25].

Polyacrylamide gel electrophoresis. Sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis was performed under reducing conditions in 12% acrylamide according to the method of Laemmli [26]. Samples were boiled for 4 min in lysis buffer and electrophoresed at 30 mA in a Hoeffer model 600 vertical slab gel unit at 15°C. Molecular weight standard lanes contained bovine serum albumin (66 kDa), egg albumin (45 kDa), and carbonic anhydrase (29 kDa). The proteins on the gel were observed visually sub- sequent to silver staining by the method of Wray et al. [27].

276

Results

Purification of APRTase and HGPRTase. APRTase and HGPRTase were purified 326-fold and 1341-fold, respectively, from the 40-80% ammonium sulfate fractions by exclusion from an ion exchange column followed by affinity chromatography. APRTase was purified on an AMP- agarose column, while HGPRTase was purified from a GTP-agarose matrix. The final specific activities of APRTase and HGPRTase were 8.6 and 27.5 txmol min -1 (mg protein) -~, respectively. Virtually the entire purification of both enzymes was a consequence of the affinity chromatography. Neither the affinity purified APRTase nor the affinity purified HGPRTase preparations contained detectable amounts of the other phosphoribosyltransferase enzyme as measured by direct assay. Interestingly, none of the GMP-containing affinity resins which have been exploited to purify the HGPRTase activities from Giardia lambIia [28], Schistosoma mansoni [29], and mammalian [30] sources were capable of retain- ing the leishmanial HGPRTase activity. Both enzymes were extraordinarily sensitive to inactiva- tion by storage at 25°C, 4°C, -20°C, or -80°C. Over 90% of the activity of either purified enzyme was lost after only 72 h storage at - 80°C. The shelf life of detectable enzyme was not pro- longed by EDTA, divalent cations, glycerol, eth- ylene glycol, bovine serum albumin, or sulfydryl reducing agents. Thus, all enzyme assays and kinetic analyses were performed on the same day as the enzymes were purified. Therefore, the ex- tents of purification of both APRTase and HGPRTase represent minimal estimates due to the inherent instabilities of both enzyme activities in highly purified preparations.

As monitored by silver staining, each affinity purified preparations appeared homogeneous. The PRPP-eluted APRTase preparation from the AMP agarose column contained a single band that migrated with a molecular weight of 29 000 (Fig. 1, lane A), while the PRPP-eluted HGPRTase fraction also contained only a single silver staining protein with a molecular weight of 24 500 (Fig. 1, lane B). To corroborate that the observable protein bands on SDS polyacrylamide gels cor- related with enzyme activity, fractions of the

A B

Fig. 1. SDS polyacrylamide gels of purified phosphoribosyltransferases from L. donovani. SDS polyacrylamide gels of AMP-agarose purified APRTase (lane A) and GTP-agarose HGPRTase (lane B) were run as described in Materials and Methods and silver stained according to the procedure described by Wray et al. [21]. The arrow corresponds to the point

of migration of the 29 kDa protein standard.

PRPP-eluates from the AMP-agarose and GTP- agarose columns were collected and monitored for both protein and enzyme activity. As shown in Fig. 2 and 3, the intensity of the silver-stained band in each purified fraction from the AMP- agarose (Fig. 2) and GTP-agarose (Fig. 3) affinity columns was directly proportional to the amount of measurable enzyme activity in that fraction. These data, therefore, strongly substantiate the hypotheses that the affinity-purified proteins that migrate on SDS gels with molecular weights of 29 000 and 24 500 corresponded to the leishmanial APRTase and HGPRTase activities, respectively.

Determination of K m values. The apparent K m value of the homogeneous APRTase enzyme for adenine was resolved to be 2.5 txM by Line- weaver-Burk analysis (Fig. 4A). The apparent K m values of HGPRTase for hypoxanthine and guan-

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Fig. 2. Correlation of APRTase activity with APRTase protein in PRPP-eluted fractions off an AMP-agarose column. 4 ml fractions of eluate from the AMP-agarose column were collected from a DEAE-cellulose excluded APRTase preparation and monitored for APRTase activity. PRPP was added to the mobile phase prior to the collection of fraction 28. The remaining PRPP-containing APRTase fractions were dried by lyophilization, resuspended in Laemmli buffer, and subjected to SDS polyacrylamide gel electrophoresis as described in Materials and Methods. The arrows correspond to the 45 and 29 kDa standards. This is one of several typical experiments

each of which gave similar results.

Fig. 3. Correlation of HGPRTase activity with HGPRTase protein amount in eluted fractions from a GTP-agarose column. 4 ml fractions of eluate from the GTP-agarose column were collected from a DEAE-cellulose excluded HGPRTase preparation and monitored for HGPRTase activity. PRPP was added to the mobile phase prior to the collection of fraction 29. The remaining PRPP-eluted HGPRTase fractions were lyophilized, resuspended in Laemmli buffer, electrophoresed in SDS, and silver stained as described in Materials and Methods. Fraction 29 was not subjected to SDS gel electrophoresis. The arrow corresponds to the 29 kDa marker. This is one of multiple experiments each of which gave similar re-

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ine were similarly de t e rmined to be 2.0 and 1.0, respectively (Fig. 4B). No a t t empt was made to ascertain the K m value for P R P P of e i ther enzyme, since 1 m M P R P P was presen t in the purified enzyme preparations. At tempts to dialyze the P R P P away f rom the affinity purif ied enzymes re- sul ted in a comple te loss of bo th A P R T a s e and H G P R T a s e activities.

Inhibitor studies. The data in Table I indicate that bo th affinity purif ied enzymes were inhib i ted by a variety of an ionic metabol i tes . A P R T a s e was

subject to feedback inhib i t ion by its two endproducts , inorganic pyrophospha te inhibi t ing the enzyme by 36% and A M P by 57%, while H G P R T a s e was inhibi ted 34%, 39%, 72%, and 61%, by pyrophosphate , IMP, G M P , and G T P , respectively. To ascertain the na tu re of the b ind- ing in terac t ion of each enzyme to the l igand cov- alently b o u n d to the respective affinity resin, the extent of nucleot ide inh ib i t ion of enzyme activity was mon i to red at several nuc leobase substrate concent ra t ions . The inhib i t ion of bo th A P R T a s e by A M P and H G P R T a s e by G T P was deter-

278

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Fig. 4. Lineweaver-Burk analyses of purified APRTase and HGPRTase activities from L. donovani. Affinity purified enzyme preparations of APRTase (A) and HGPRTase (B) were incubated with 1.0 to 20 ixM concentrations of radiolabeled nucleobase

substrate and 1 mM PRPP as described in Materials and Methods.

m i n e d to be n o n c o m p e t i t i v e in n a t u r e ( d a t a no t

shown) . W h e t h e r t h e e n d p r o d u c t i n h i b i t i o n ob-

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we l l as on o t h e r e n v i r o n m e n t a l f ac to rs .

T h e resu l t s p r e s e n t e d in T a b l e I a lso c o n f i r m e d

t h e s u b s t r a t e spec i f ic i t i es o f t h e t h r e e l e i s h m a n i a l

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th ine and aden ine did no t inhibit H G P R T a s e , and

x a n t h i n e , h y p o x a n t h i n e , and g u a n i n e d id no t in-

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d a t a c o n f i r m tha t t h e l e i s h m a n i a l A P R T a s e ,

H G P R T a s e , and x a n t h i n e p h o s p h o r i b o s y l t r a n s -

f e r a se e n z y m e s a re b i o c h e m i c a l l y d i s t i ngu i shab l e

[121.

TABLE I

Effects of inhibitors of [14C]adenine and [~4C]hypoxanthine phosphoribosylation of affinity purified enzymes

Addition % Activity

APRTase HGPRTase

None 100 100

Adenine 0 100 + 33 Hypoxanthine 105 + 6 0 Guanine 95 4 + 3 Xanthine 110 + 4 102 + 12

AMP 64 + 1 131 + 12 ATP 123 + 16 101 + 14 IMP 109 + 11 61 + 10 GMP 86 28 + 13 GTP 112 39 + 17 Phosphate 99 + 10 101 + 27 Pyrophosphate 43 + 4 66 + 11

The effects of 1 mM concentrations of various compounds on the phosphoribosylation of 20 ixM []4C]adenine and 20 IxM []4C]hypoxanthine were carried out using either AMP-agarose purified APRTase or GTP-agarose purified HGPRTase. The av- erages and standard deviations of three determinations for APRTase and five determinations for HGPRTase are presented except when indicated.

A B C

Fig. 5. SDS polyacrylamide gel electrophoretograms of AMP- agarose purified APRTase from wild type and APRTase-deficient organisms. Extracts of wild type (DI700), partially APRTase-deficient (APPB2), and totally APRTase-deficient (APPB2-640A3) cells were subjected to the salt precipitation, ion exchange, and affinity chromatographic purification scheme described in Materials and Methods. PRPP-eluted material from wild type (lane A), APPB2 (lane B), and APPB2-640A3 (lane C) cells was concentrated by lyophilization and run on SDS gels. The arrow corresponds to the position of a 29 kDa

protein standard after electrophoresis.

Mutational analysis of APRTase. Iovannisci et al. have previously isolated and characterized mutant L. donovani that are partially or totally deficient in APRTase activity [16]. APPB2 cells contain 50% of the wild type APRTase levels, while APPB2-640A3 cells are completely deficient in enzyme activity. In order to determine whether mutant cells genetically deficient in APRTase activity express APRTase protein, extracts from both mutant cell lines were subjected to the same purification procedures described for the wild type cell extracts. As demonstrated in Fig. 5, the APPB2 cell line contained observable but lower amounts of the 29 kDa protein moiety, while the APPB2-640A3 cells totally deficient in enzyme activity did not appear to contain any affinity purifiable 29 kDa protein. These data pro- vide powerful genetic evidence that the protein band that migrated with a molecular weight of 29 000 in SDS gels was the leishmanial APRTase.

Discussion

Tuttle and Krenitsky reported the existence of three biochemically distinct purine phosphoribosyltransferase activities in extracts of L. donovani promastigotes; APRTase, HGPRTase, and xanthine phosphoribosyltransferase [12]. Using affin-

279

ity chromatography, Tuttle and Krenitsky purified the leishmanial APRTase and HGPRTase 46- fold and ll0-fold, respectively. In this report, the published purification schemes for APRTase and HGPRTase [12] have been modified to purify both phosphoribosyltransferases to homogeneity as assessed by silver staining of SDS polyacrylamide gel electrophoretograms. Two modifications employed in these studies were essential to the isolation of the homogeneous proteins. Both the ion exchange column and the 0.5 M KC1 washes of the affinity resins were indispensible to ensure homogeneity. Omission of either step precluded the successful homogeneous purification of either protein (data not shown). Determinations of the kinetic parameters of both APRTase and HGPRTase indicated that the K m values of APRTase for adenine and HGPRTase for guanine and hypoxanthine were very similar to the Km values reported by Tuttle and Krenitsky for the partially purified enzyme preparations [12]. Since detailed kinetic studies on the partially purified preparations of both phosphoribosyltransferases have been carefully performed previously by Tut- tie and Krenitsky, further requirements for thor- ough and detailed kinetic analyses of the completely purified enzymes were obviated.

The molecular weights determined for the purified APRTase and HGPRTase enzymes from L. donovani under reducing conditions in SDS gels were 29000 and 24500, respectively. As previously determined by Tuttle and Krenitisky on Sephadex G-100 columns, the particle molecular weight for the leishmanial APRTase is 25 000 in the absence and 54000 in the presence of 1 mM PRPP, while the particle molecular weight for HGPRTase is 110000 and 60000 in the absence and presence of PRPP, respectively [12]. This suggests that the native HGPRTase enzyme might naturally exist either as a dimer or tetramer, while native APRTase could exist in either monomeric or dimeric forms. The apparent subunit molecular weight of the leishmanial HGPRTase is slightly lower than that determined by SDS gel electrophoresis for the HGPRTase from G. lamblia [28], much lower than the HGPRTase from S. mansoni [29], but virtually identical to that of the human HGPRTase as determined by protein purification and cDNA sequencing [30,31]. The

27 Wray, W., Boulikas, T., Wray, V.P. and Hancock, R. (1981) Silver staining of proteins in polyacrylamide gels. Anal. Biochem. 197-203.

28 Aldritt, S.M. and Wang, C.C. (1986) Purification and characterization of guanine phosphoribosyltransferase from Giardia larnblia. J. Biol. Chem. 261, 8528-8533.

29 Dovey, H.F., McKerrow, J.H., Aldritt, S.M. and Wang, C.C. (1986) Purification and characterization of hypoxanthine-guanine phosphoribosyltransferase from Schisto- soma mansoni. A potential target for chemotherapy. J. Biol. Chem. 261,944-948.

30 Holden, J.A. and Kelley, W.N. (1978) Human hypoxanthine-guanine phosphoribosyltransferase. Evidence for te- trameric structure. J. Biol. Chem. 253, 4459-4463.

31 Jolly, D.J., Okayama, H., Berg, P., Esty, A.C., Filpula,

32

33

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D., Bohlen, P., Johnson, G.G., Shively, J.E., Hunkapil- lar, T. and Freidmann, T. (1983) Isolation and characterization of a full-length expressible cDNA for human hypoxanthine phosphoribosyltransferase. Proc. Natl. Acad. Sci. USA 80, 477-481. Holden, J.A., Meredith, G.S. and Kelley, W.N. (1979) Human adenine phosphoribosyltransferase. Affinity purification, subunit structure, amino acid composition, and peptide mapping. J. Biol. Chem. 254, 6951-6955. Broderick, Schaff, D.A., Bertino, A.M., Dush, M.K., Tischfield, J.A. and Stambrook, P.J. (1987) Comparative anatomy of the human APRT gene and enzyme: nucleotide sequence divergence and conservation of a nonran- dom CpG dinucleotide arrangement. Proc. Natl. Acad. Sci. USA 84, 3349-3353.

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