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Proc. NatL Acad. Sci. USA Vol. 78, No. 11, pp. 6618-6622, November 1981 Purine in the protozoan parasite Eimeria tenella (-- phosphoribosyltransferase/GMP-agarose affinity column/alopurinol) C. C. WANG AND P. M. SIMASHKEVICH* Merck Institute for Therapeutic Research, Rahway, New Jersey 07065 Communicated by P. Roy Vagelos, June 25, 1981

ABSTRACT Crude extracts of the oocysts ofEimeria tenella, cidia Eimenria tenella, which develops inside the caecal epithe- a protozoan parasite of the coccidium family that develops inside lial cells ofchickens, effectively takes up hypoxanthine and pref- the caecal epithelial cells ofinfected chickens, do not incorporate erentially incorporates label from it into nucleic acids when or formate into purine ; this suggests lack of grown in culture (13, 14). in this parasite capability for de novo purine synthesis by the parasite. The ex- is particularly interesting in view of the uricotelic metabolism tracts, however, contain high levels of activity of the purine sal- of chickens and the high levels of hypoxanthine known to ac- vage : hypoxanthine, guanine, xanthine, and cumulate in avian tissues phosphoribosyltransferases and kinase. The absence of (15). AMP deaminase from the parasite indicates thatE. tenella cannot In this study, we have demonstrated that E. tenella cannot convert AMP to GMP; the latter thus has to be supplied by the perform de novo purine synthesis and examined the detailed hypoxanthine, xanthine, or guanine phosphoribosyltransferase of mechanism of purine salvage. A purine phosphoribosyltrans- the parasite. These three activities are associatedwith one ferase that uses hypoxanthine and guanine as well as xanthine (HXGPRTase), which has been purified to near homogeneity in as substrates (HXGPRTase) was identified in the parasite. The high yield (71-80%) in a single step by GMP-agarose affinity col- enzyme was purified and characterized. umn chromatography. The size ofthe enzyme subunit is estimated , an analog of hypoxanthine innocuous for mam- to be 23,000 daltons by NaDodSO4 gel electrophoresis. Kinetic malian cells, is effective in vitro against Leishmania (16), T. studies suggest differences in purine substrate specificity between cruzi (17), and African trypanosomes (18) through sequential E. teneUa HXGPRTase and chicken HGPRTase. Allopurinol conversion to allopurinol and aminopurinol ri- preferentially inhibits the parasite enzyme by competing with hy- bonucleoside mono-, di-, and triphosphates and eventual in- poxanthine; a 1K 22 ,AM. corporation into the RNA of these parasites. It serves as a sub- strate for the HGPRTase ofLeishnmnia donovani promastigotes, All parasitic protozoa examined to date appear to be unable to having K. 68 times that of hypoxanthine (19). In this study, synthesize the purine ring de novo, as reflected by the failure allopurinol was tested on E. tenella HXGPRTase and chicken ofradiolabeled glycine and formate to label purines liver HGPRTase and found to be a preferential inhibitor ofthe of the parasites in minimal defined media. Of particular note parasite enzyme. are studies on Trypanosoma cruzi (1), Leishmania braziliensis (2), Plasmodium lophurae (3), and Trypanosoma megna (4), MATERIALS AND METHODS which provide well-documented cases for lack ofcapability for Materials. Unsporulated oocysts ofE. tenella strain 18 were de novo purine synthesis and, hence, dependence on purine propagated in chickens and harvested and purified as described salvage. Host hypoxanthine, adenine, and adenosine have been (20). Radiolabeled substrates were from New England Nuclear suggested as the three major sources of purines for these par- or Research Products International. GMP-agarose [4%-beaded asites (5), but recent evidence favors the hypothesis that hy- agarose-NH-(CH2)6-NH-8-C--5'-phosphate] was from poxanthine may be the main, if not the only, supply ofpurines P-L Biochemicals. 5-Phosphoribosyl-l-pyrophosphate (PRPP) to Plasmodia (6, 7), Leishmania (2, 8), and Crithidiafasciculata was from Boehringer Mannheim, and 5-aminoimidazole-4-car- (8). Adenosine and adenine are probably converted to hypo- boxamide ribonucleotide (AICAR) and allopurinol were from xanthine by the purine hydrolase (9) and adenine Sigma. 5,10-Methenyltetrahydrofolate was prepared from tet- aminohydrolase (2) ofthe parasites before incorporation into the rahydrofolate (Calbiochem) by a known procedure (21). pool. Enzyme Assays. Purine phosphoribosyl transferase activities Among members of the coccidia family, a group of parasitic were assayed by a modified procedure of Schmidt et al. (22). protozoa developing inside intestinal epithelial or muscle cells Two hundred microliters of 0.10 M Tris-HCl, pH 7.8/7 mM ofinfected animals, Toxoplasma gondii trophozoites were found MgCl2/1.0 mM PRPP containing bovine serum albumin at 50 incapable of incorporating glycine or formate into their DNA ,ug/ml and 4.0 AM [8-'4C]hypoxanthine (52.8 mCi/mmol; 1 Ci (10). They grow normally inside cultured Lesch-Nyhan skin = 3.7 X 10'0 becquerels), [2-'4C]xanthine (48.0. mCi/mmol), fibroblasts, which lack hypoxanthine-guanine phosphoribosyl- [8-'4C]guanine (54.7 mCi/mmol), or [8-_4C]adenine (43.9 mCi/ transferase (HGPRTase), and effectively incorporate exogenous mmol) was incubated at 37°C, and reaction was initiated by add- hypoxanthine and guanine into nucleic acids (11). T. gondii has ing enzyme. After 10 min of incubation, the reaction was ter- no detectable adenine phosphoribosyltransferase (APRTase) minated by the addition ofan equal volume ofan ice-cold 2 mM but has a high level ofadenosine kinase (12). The latter activity unlabeled purine base. The duration ofincubation and substrate is largely lost in a mutant resistant to 1-,B3D-arabinofuranosyl- concentrations were varied in kinetic studies. of adenine (AraA). But wild-type and mutant T. gondii grown in Aliquots the cell culture are equally efficiently labeled by [3H]adenosine cooled reaction mixture were filtered through a glass-fiber filter (12), suggesting that adenosine kinase does not play a major role Abbreviations: H-, X-, G-, and APRTase, hypoxanthine, xanthine, gua- in salvaging purines for the parasite. One other species of coc- nine, and adenine, respectively (and combinations thereof), phos- phoribosyltransferase; PPRP, 5-phosphoribosyl-1-pyrophosphate; Al- The publication costs ofthisarticle were defrayed in partby page charge CAR, 5-aminoimidazole-4-carboxamide ribonucleotide. payment. This article must therefore be hereby marked "advertise- * Present address: Dept. of Pharmaceutical Chemistry, Univ. of Cali- ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. fornia, San Francisco, CA. 6618 Downloaded by guest on September 29, 2021 Biochemistry: Wang and Simashkevich Proc. Nad Acad. Sci. USA 78 (1981) 6619

evenly coated with 32 mg of polyethyleneimine-cellulose are incorporated into the nucleotide fraction after 20 min ofin- (Merck) preequilibrated with 1.0 mM NH4OAc, pH 5.0. The cubation with chicken liver extract. loaded filter was washed with three 5-ml portions of the same To verify that the assays reflected de novo purine synthesis, solution and then soaked in Aquasol 2 (New England Nuclear). 2.0-ml aliquots of the reaction mixtures were mixed with 0.1 Levels of radioactivity were determined with a Beckman LS ml of 4.2 M perchloric acid after the incubation. The samples 8000 liquid scintillation spectrometer. were then chilled in an ice bath for 30 min, neutralized with Purine kinase and phosphotransferase activ- 0.1 ml of 4.42 M KOH, centrifuged briefly, and concentrated ities were assayed by methods similar to those of Nelson et al. to 0.1 vol by lyophilization. Aliquots (10 1.l) ofeach extract were (23) using 1.3 mM [8-'4C] (5.2 mCi/mmol), [2-'4C]-xan- analyzed by high-pressure liquid chromatography. The results thosine (5.0 mCi/mmol), [8-14C]guanosine (5.3 mCi/mmol), or show that both [14C]glycine and ['4C]formate remain largely [8-'4C]adenosine (5.5 mCi/mmol) as substrate. Reactions were unchanged after incubation with E. tenella extracts but chicken terminated by filtering the mixtures through polyethyleneim- liver extracts convert some ofthe label in ['4C]glycine to a major ine-cellulose filters, and radioactivities were assayed as described. fraction that has a retention time corresponding to the inter- Adenosine deaminase was assayed according to Hoagland and mediate in de novo purine synthesis (27) and Fisher (24). AMP deaminase activity was assayed by the method a smaller fraction corresponding to IMP (Fig. 1). [I4C]Formate ofAshby and Frieden (25); the reaction was followed at 265 nm was partly incorporated into IMP, with much less into the in- in a Beckman ACTA III spectrophotometer. Protein concen- termediate ribonucleotide formyl AICAR, the immediate pre- trations were determined by the method ofBradford (26) unless cursor to IMP. otherwise stated. Thus, this incubation system serves as a specific assay for de High-Pressure Liquid Chromatography. Purine bases, nu- novo purine synthesis. The lack of incorporation of glycine or cleosides, and nucleotides were separated and identified by formate into purine nucleotides in extracts ofE. tenella unspo- paired-ion reverse-phase chromatography as described by rulated oocysts indicates that these extracts are incapable ofde Rowe et al. (27). A Partisil 10-ODS-2 (Reeve Angel) column (25 novo purine synthesis. cm X 4.6 mm) equilibrated with 5.0 mM tetrabutylammonium Purine Salvage Enzymes in Extracts ofE. tenella Oocysts. hydroxide, adjusted to pH 6.0 with phosphoric acid/10% (vol/ The extracts of E. tenella oocysts and chicken were also vol) methanol was washed with the same buffer for 10 min at assayed for HPRTase activity. The results (Table 1) show a a flow rate of1.0 ml/min following sample injection. Then, con- HPRTase specific activity in E. tenella 9 to 10 times that in centration of methanol was increased linearly to 50% (vol/vol) chicken liver. When the assay mixtures were extracted with over 15 min at the same flow rate and maintained at.50% for perchloric acid, and the contents were analyzed by HPLC, most another 15 min. The eluate was collected in 0.5-ml fractions, of the label in ['4C]hypoxanthine was incorporated by the E. and the radioactivity was determined as described above. tenella crude extract into a fraction corresponding to IMP (Fig. Affinity Column Chromatography. A supernatant fraction 2A). Similar results were obtained from the chicken liver extract of the crude extract of E. tenella unsporulated oocysts was ad- except that less label was found in the IMP fraction (Fig. 2B). justed to 0.10 M Tris HCI, pH 7.0/0.10 M MgClJO.50 M KC1 The observation of the lack of de novo purine synthesis and (TMK), and a 15.6-ml aliquot containing 86.3 mg ofprotein was the high HPRTase activity in E. tenella was followed up by com- loaded onto a 3 ml GMP-agarose column previously equili- parison of the profile of purine salvage enzymes in E. tenella brated with TMK buffer at 0-4°C. The loaded column was oocysts with that in chicken liver. The results showed significant eluted with 250 ml of TMK buffer at a flow rate of30 ml/hr and then with 40 ml ofTMK buffer/10 mM PRPP at 10 The mVhr. Table 1. Incorporation of radiolabeled substrates into purine effluent was collected in 5-ml fractions and assayed for enzyme nucleotides activities and protein content at 280 nm. NaDodSOJPolyacrylamide Gel Electrophoresis. The pro- Incorporation, cedure is that of Laemmli using 1 cm of 30% stacking gel atop (nmol/20 min)/5 9 to 10 cm of 12.5% running gel (28). mg of protein staining has the sensitivity to detect 0.05 ,ug of protein in the Chicken gel. Protein standards were from Boehringer Mannheim. Substrate Reaction mixture liver E. tenella RESULTS [14C]Glycine Complete 333 2.2 Lacking PRPP 7.7 0.5 Lack of de Novo Purine Synthesis in Extracts of E. tenella Containing boiled 0 1.0 Oocysts. Freshly purified unsporulated oocysts of E. tenella extract were broken at 0-4°C by a briefsonication in 50 mM Tris-HCl, [14C]Formate Complete 139 0 pH 7.0, at a density of 1.2 X 108 oocysts per ml. The crude Containing AICAR 365 0 homogenate was centrifuged at 10,000 X g for 30 min, and the Containing boiled 0 1.7 supernatant was collected and centrifuged-at 100,000 X g for extract 1 hr. The supernatant, containing 5.4 to 5.5 mg ofprotein/ml, [14C]Hypoxanthine Complete 44 402 was immediately assayed for de novo purine synthesis as de- Lacking-PRPP 0 0 scribed Rowe et al. Freshly cell-free extracts by (27). prepared Each assay was carried out in 2.0 ml of a mixture of 4.0 ,umol of L- (25.5 mg of protein per ml) of livers obtained from 2-week-old aspartate, 4.0 panol of L-glutamine, 5.0 ,mol of ATP, 10 p.mol of White Rock chickens fasted for 24 hr before sacrifice were as- MgCl2, 20.0 /Lnol of KHCO3, 100 pmol each of KCl and Tris HCl, pH sayed in the same way. The results indicate no appreciable in- 7.8, 3.0 pmol each of PRPP and 5,10-methenyltetrahydrofolate, 20 corporation ofradiolabel from either ['4C]glycine or [14C]formate pmol of phosphoenolpyruvate, 28.6 units of pyruvate kinase, cell-free into the polyethyleneimine-cellulose-adsorbable fraction by extracts containing 5.0 mg of protein, and 4.0 ymol of AICAR when crude extracts ofE. tenella 1). Chicken liver extracts cat- specified (27). After addition of 1.2 panol of [14Clglycine (0.83 mCi/ (Table mmol), 4.0 pmol of [14C~formate (0.25 mCi/mmol), or 3.3 ,umol of alyze both PRPP-dependent incorporation of ['4C]glycine and [14Clhypoxanthine (52.8 mCi/mmol), incubation was carried out at AICAR-stimulated incorporation of['4C]formate into the prob- 37TC for20 min. Purine nucleotides in 50 Al ofincubation mixture were able purine nucleotide fraction. Under optimal conditions, trapped on PEI-cellulose filters and washed, and radioactivity was -28% ofthe total [14C]glycine and 9% ofthe total ['4C]formate assayed. Downloaded by guest on September 29, 2021 6620 Biochemistry: Wang and Simashkevich Proc. Nad Am& Sci. USA 78 (1981)

1.0 0 24 1.5 |1l EB 5 I' o24

C ~D 00 E 16

15 ±2 1 2

8 1 .0 ID7~

0 .L.-...... t-..... ---- ...... ,h!\-J---*-- 0 ...... ::~b ..... 0 10 20 30 10 20 30 40 0 10 20 30 40 50 Minutes Minutes

FIG. 1. High-pressure liquid chromatography analysis after in- FIG. 2. High-pressure liquid chromatography analysis of hypo- cubation of crude extracts with labeled substrates. (A) E. tenelka xanthine incorporation into nucleotides in E. tenella oocyst (A) and oocysts and [14C]gl~ycine. (B) Chicken livers and [14C]glycine. (C) E. chicken liver (B) extracts. tenella oocysts,[ 4C]formate, and AICAR. (D) Chicken livers, [14C]formate, and AICAR. agarose affinity column chromatography. The elution profile indicates that most of the proteins pass through the column in levels of HPRTase, APRTase, GPRTase, XPRTase, and aden- TMK buffer (Fig. 4). Most of the HPRTase, XPRTase, and in E. osine kinase activities tenella (Table 2). The products of GPRTase activities were recovered in a sharp peak after addi- all enzyme reactions were identified as the corresponding ri- tion of PRPP.to the elution buffer. The total HXGPRTase ac- bonucleotides by HPLC analysis (data not shown). There was tivity (fractions 50-58) represents 71-0% yield. These frac- no kinase, detectable inosine kinase, guanosine ki- tions were pooled, concentrated to 1.0 ml by using a Millipore nase, purine. nucleoside phosphotransferases, adenosine de- immersible CX unit, and dialyzed against 1 liter of2.0 mM so- aminase, or AMP deaminase activity in the parasite. The ab- dium phosphate buffer, pH 7.2, at 0-40C with three changes sence enzymes not ofthe last two strongly suggests that AMP is of buffer in 16 hr. The dialyzed sample was lyophilized and converted to GMP in E. tenella. dissolved in 40 /.l ofwater. The protein content ofthis sample The chicken liver extract has a similar profile of purine sal- was determined by the fluorescamine spectrofluorometry vage enzymes with lower specific activities, except that it has method (29) and found to be 100 pug, which leads to an estimated very low XPRTase activity but significant amounts ofadenosine purification of HXGPRTase of 610 to 688-fold. deaminase and AMP deaminase. The chickens are apparently The same concentrated sample was examined by NaDodSO4 capable of converting AMP to GMP. polyacrylamide gel electrophoresis, and only a single protein E. Characterization and Purification of HXGPRTase from band was found in the sample (Fig. 5). No other protein bands E. tenela. The profile ofpurine metabolism enzymes in tenella are visible when the gel is overloaded with 40 ug of protein. suggests that the only way for the parasite to make GMP is by It is thus concluded that, assuming that the protein band rep- its HPRTase, XPRTase, or GPRTase activity. To determine whether the three purine phosphoribosyl- transferase activities of E. tenella are in one, two, or three in- Table 2. Purine salvage enzymes in crude extracts of chicken dividual enzymes, heat inactivation experiments were carried liver and E. tenella unsporulated oocysts out. The crude extract of E. tenella unsporulated oocysts was Specific activity, (nmol/min)/ incubated at 370C, and samples were taken after different pe- mg of protein riods of time assays various activ- incubation for of transferase Enzyme Chicken liver E. tenelka ities. The results show that, although APR.Tase activity re- mained relatively constant during the 3-hr incubation, HPRTase, HPRTase 0.48 ± 0.11* 6.43 ± 0.80t XPRTase, and GPRTase were all inactivated with a similar time GPRTase 0.77 ± 0.04* 4.90 ± 0.78t course, suggesting that these three activities reflect one enzyme XPRTase <0.1* 2.34 + 0.53t (Fig. 3). Supporting evidence was obtained from a study of the APRTase 0.66 + 0.19* 1.93 ± 0.06t + ± optimal pH values for different enzyme activities; APRTase has Adenosine kinase 4.18 1.40* 3.32 0.74* Xanthosine kinase <0.1* <0.1* a rather broad pH optimum and retains full activity at pH 9.0, Guanosine kinase <0.1* <0.1* whereas the other three activities share a curve in bell-shaped Inosine kinase <0.1* <0.1* which the optimal pH is at 7.0-7.5, total inactivation is at pH 5.4, and 50% inactivation occurs at pH 9.0. * Three independent trials. The enzyme, designated hypoxanthine-xanthine-guanine t Six independent trials. * phosphoribosyltransferase (HXGPRTase), was purified by GMP- Two independent trials. Downloaded by guest on September 29, 2021 Biochemistry: Wang and Simashkevich Proc. NatL Acad. Sci. USA 78 (1981) 6621

Mr x 10-3

67- -_ 60 *w .M 47 ---

.1

25 -_ lo 'M 4o _w q

.4- 1360 12.5- * 40

20 1 2 3 4 5 6 0sIJ FIG. 5. NaDodSOopolyacrylamide gel electrophoresis of E. te- 0 1 2 3 nella HXGPRTase. Lanes: 2, 2.5 yg of purified protein; 3, 5.0 tig of purified protein; 4, 40 ,gofpurified protein; 6, crudeE. tenella extract Hours containing 2 gg of protein; 1 and 5, protein standards (0.2 pg each) [serum albumin (Mr 67,000), catalase (Mr 60,000), hen albumin (Mr FIG. 3. Time course of heat inactivation of purine phosphoribo- 47,000), chymotrypsinogen (Mr 25,000 K), and cytochrome c (Mr syltransferase activities in E. tenella oocyst extracts. The incubation 12,500)]. was carried out at 3700 and the time points were assayed in duplicate. *, APRTase; *, HPRTase; e, GPRTase; *, XPRTase. PRPP/1 mM dithiothreitol at -1960C for weeks without ap- preciable loss ofactivity. resents the subunit ofE. tenella HXGPRTase, the enzyme must The chicken liver enzyme was partially purified by using a have been purified to near homogeneity in one step by GMP- procedure for human erythrocyte HGPRTase (31). HPRTase agarose affinity column chromatography. Inclusion of protein and GPRTase activities copurified to a similar extent, suggest- standards in the NaDodSOJpolyacrylamide gel electrophore- ing that the chicken liver has a single enzyme HGPRTase. sis (Fig. 5) allowed an estimation ofthe molecular weight ofthe Kinetic Studies ofE. teneUa HXGPRTase. For a saturating purified protein (30); results from two such runs gave a value concentration of Mg2ePRPP, initial rates were calculated from of22,400-23,200 daltons. measurements made on reactions terminated 1, 2, and 4 min The purified enzyme can be stored in TMK buffer/10 mM after initiation (Table 3). Data obtained within this time frame gave linear rates with a SEM of± 15%. Lineweaver-Burke plots of the data were then constructed by weighted least-squares _- TMK analysis to produce various kinetic constants; for hypoxanthine, -TMK + PRPP the value ofKa is lower and that of Vmi is higher for the E. te- ne11a enzyme than for chicken liver HGPRTase. 300 Allopurinol was tested on the two enzyme preparations and shown tobearelativelypotentinhibitorofE. tenella HXGPRTase. 200 0 Because ofthe structural similarity between allopurinol and the purine substrate, it was apparent that allopurinol is a compet- itive inhibitor and that initial velocity data at saturating con- C.2 centrations of Mg2ePRPP are adequately described by Eq. 1: 150 -1.5 v=A x +I x (+ I E Vma/[(A I [1]

0 where v is the initial velocity, A is the concentration ofsubstrate, E and I is the concentration of inhibitor. Initial velocity data ob- >% 100 1.0 tained at various substrate and inhibitor concentrations were C.4- fit to this equation (32) by using a Gauss-Newton method of nonlinear regression in a commerically available statistical anal- ysis package. The data give allopurinol a K. of2.2 + 0.9 x 10-5 M versus hypoxanthine, which is =85 times higher than the K, 50 .5 ofhypoxanthine. Similar kinetic studies using xanthine or gua- nine as the substrate have been also carried out. The results (Table 3) suggest competition between allopurinol and the two other purines with similar K1 values, which could be also taken as an indication that there is but a single enzyme HXGPRTase 0 5 10 15 20 50 55 60 in E. tenella. Allopurinol, however, has only a weak inhibitory effect on chicken liver HGPRTase. The compound has an ap- Fraction No. parent K1 of9.3 ± 2.7 x 10-' M (Table 3), which is more than FIG. 4. Purification of E. tenella HXGPRTase by GMP-agarose 40 times that for the E. tenela enzyme. Conversely, 6-mercap- affinity column chromatography. A, APRTase; v, HPRTase; o, GPRTase; topunne and 6-thioguanine were more potent inhibitors of n, XPRTase; *, A2N. chicken liver HGPRTase than ofthe E. tenella enzyme, the 50% Downloaded by guest on September 29, 2021 6622 Biochemistry: Wang and Simashkevich Proc. Nad Acad. Sci. USA 78 (1981) Table 3. Kinetic constants of E. tenella HXGPRTase and chicken specific binding to GMP that cannot be dissociated by 0.1 M liver HGPRTase MgCl2/0.5 M KC1 at neutral pH. Vmaw The preferential inhibition of E. tenela HXGPRTase by al- (pmol/sec)/ Ki lopurinol is interesting because the drug is known to inhibit the Purine Crude mg of (allopurinol), growth ofE. tenela in cultured embryonic chicken ep- substrate extract Ka, M protein M x 10-5 ithelial cells at 100 ppm (34). This in vitro anticoccidial activity Hypoxan- Chicken of allopurinol may be due to its competitive inhibition of thine liver 1.4 ± 0.2 x 10-6 10.1 ± 0.4 93 ± 27 HXGPRTase, or by incorporation into the RNA of E. tenella. Hypoxan- For the latter, one would have to assume that E. tenella ad- thine E. tenella 2.5 ± 1.1 x 10-7 95.0 ± 2.8 2.2 ± 0.9 enylosuccinate synthetase and lyase can also recognize allopu- Guanine E. tenelka 3.7 ± 1.0 x 10-7 43.1 ± 2.7 1.4 ± 0.4 rinol ribonucleotide and succinoallopurinol ribonucleotide as Xanthine E. tenella 1.5 ± 0.4 x 10-5 95.4 ± 10.8 1.2 ± 0.3 their substrates, as is the case in L. donovani (35). Results are mean ± SEM in the presence of 1.0 mM PRPP/7.0 mM MgCI2. The Ka values for Mg2e PRPP for the two enzymes are esti- We wish to thank Dr. Herb Bull and Ms. Nancy Thornberry for their mated to be 0.03-0.10 mM (unpublished observation). helpful discussions and nonlinear regression analysis of our enzyme kinetic data. inhibition concentrations being <1 X 10-5 and 1.6 x 10-5 M, respectively, against chicken liver versus 4.6 X 10-5 M and 3.7 X 1. Gutteridge, W. E. & Gaborak, M. (1979) Int. J. Biochem. 10, 10`5 M versus E. tenella HPRTase activities. There is thus 415-422. a difference in the inhibitor specificity between the host and 2. Marr, J. J., Berens, R. L. & Nelson, D. J. (1978) Biochim. Bio- the parasite enzymes. phys. Acta 544, 360-371. 3. Walsh, C. J. & Sherman, I. W. (1968) J. ProtozooL 15, 763-770. DISCUSSION 4. Bone, G. J. & Steinert, M. (1956) Nature (London) 178, 308-309. 5. Konigk, E. (1977) Bull W. H. 0. 55, 249-252. This study has shown lack ofchange oflabeled glycine and for- 6. Sherman, I. W. (1979) Mwrobiot Rev. 43, 453-495. mate after prolonged incubation with extracts of E. tenella 7. Hensen, B. D., Sleeman, H. K. & Pappas, P. W. (1980)J. Par- oocysts under conditions allowing expression of the activities asitoL 66, 205-212. 8. Kidder, G. W. & Nolan, L. L. (1979) Proc. Nat! Acad. Sci. USA of de novo purine synthesis in chicken liver extracts. Because 76, 3670-3672. glycine should be incorporated into purine nucleotides at the 9. Schmidt, G., Walter, R. D. & Konigk, E. (1975) Tropenmed. Par- early stage of glycinamide ribonucleotide formation, whereas asitol 26, 19-26. formate ought to be incorporated mostly into AICAR to make 10. Perrotto, J., Keister, D. B. & Gelderman, A. H. (1971) J. Pro- formyl AICAR toward the end ofde novo purine synthesis, the tozool 18, 470-473. lack of incorporation of both glycine and formate into purine 11. Pfefferkorn, E. R. & Pfefferkorn, L. C. (1977) Exp. ParasitoL 41, 95-104. nucleotides suggests absence of the entire de novo pathway in 12. Pfefferkorn, E. R. & Pfefferkorn, L. C. (1978) J. ParasitoL 64, the extract ofE. tenella oocysts. This finding provides a strong, 486-492. although not conclusive, indication that E. tenella is incapable 13. Wang, C. C., Simashkevich, P. M. & Stotish, R. L. (1979) of de novo purine synthesis. Biochem. Pharmacot 28, 2241-2248. The presence of many purine salvage enzymes in E. tenella 14. Wang, C. C. & Simashkevich, P. M. (1980) Mot Biochem. Par- extracts at levels several times those in chicken liver extracts asitoL 1, 335-345. 15. Edson, N. L., Krebs, H. A. & Model, A. (1936) Biochem. J. 30, tends to support the argument that, because the parasite cannot 1380-1385. make purines by itself, it has to have the ability to take up ex- 16. Marr, J. J. & Berens, R. L. (1977) J. Infect. Dis. 136, 724-732. ogenous purines. HXGPRTase appears to be a crucial purine- 17. Marr, J. J., Berens, R. L. & Nelson, D. J. (1978) Science 201, salvaging enzyme for E. tenela, not only because of the abun- 1018-1020. dant presence ofhypoxanthine in its natural environment (15), 18. Berens, R. L., Marr, J. J. & Brun, R. (1980) Mol Biochem. Par- but also because salvage ofhypoxanthine can provide both AMP asitoL 1, 69-73. 19. Tuttle, J. V. & Krenitsky, T. A. (1980) J. BioL Chem. 255, and GMP for the coccidia (11). The only other two purine-sal- 909-916. vaging enzymes known to exist in E. tenella, APRTase and aden- 20. Wang, C. C. (1976) Biochem. PharmacoL 25, 343-349. osine kinase, can provide only AMP for the parasite. Due to the 21. Rowe, P. B. (1968) AnaL Biochem. 22, 166-168. absence of AMP deaminase, there is no apparent way of con- 22. Schmidt, R., Foret, M. & Reichert, U. (1976) Clin. Chem. 22, verting AMP to GMP in E. tenella. The salvage ofadenine and 67-69. adenosine alone thus may not fulfill the growth requirement by 23. Nelson, D. J., LaFon, S. W., Tuttle, J. V., Miller, W. H., Miller, R. L., Krenitsky, T. A., Elion, G. B., Berens, R. L. & E. tenella; it may not even be essential. Marr, J. J. (1979)J. BioL Chem. 254, 11544-11549. No HXGPRTase has ever been identified as a single enzyme 24. Hoagland, V. D., Jr. & Fisher, J. R. (1967) J. BioL Chem. 242, in any other living organism. Crithidiafasciculata, also sensi- 4341-4351. tive to allopurinol in vitro, has HPRTase, GPRTase, and 25. Ashby, B. & Frieden, C. (1977) J. BioL Chem. 252, 1869-1872. APRTase in three separate enzymes (33). L. donovani promas- 26. Bradford, M. M. (1976) AnaL Biochem. 72, 248-254. tigotes contain three individual enzymes-HGPRTase, XPRTase, 27. Rowe, P. B., McCairns, E., Madsen, G., Sauer, D. & Elliott, H. (1978)1. BioL Chem. 253, 7711-7721. and APRTase (19). E. tenella HXGPRTase and L. donovani 28. Laemmli, U. K. (1970) Nature (London) 227, 680-685. HGPRTase share some common property in having relatively 29. Udenfriend, S., Stein, S., Bohlen, P., Dairman, W., Leim- high pl values (-7.0) (unpublished observation). The purifi- gruber, W. & Weigele, M. (1972) Science 178, 871-872. cation ofE. tenella HXGPRTase to nearhomogeneity also shows 30. Weber, K. & Osborn, N. (1969)J. Bio! Chem. 244, 4406-4412. that (i) the enzyme constitutes 0. 14% ofthe total soluble protein 31. Olsen, A. S. & Milman, G. (1977) Biochemistry 16, 2501-2505. in the cytosol ofE. tenella unsporulated oocysts; (ii) the enzyme 32. Cleland, W. W. (1967) Adv. EnzymoL 29, 1-32. 33. Kidder, G. W., Nolan, L. L. & Dewey, V. C. (1979) J. Parasitol. subunit is smaller than that ofhuman erythrocyte HGPRTase, 65, 520-525. which has a molecular weight of 26,000 (31); (iii) the purified 34. Ryley, J. F. & Wilson, R. G. (1972) Z. Parasitenkd. 40, 31-34. enzyme has an HPRTase specific activity that is one-fourth that 35. Spector, T., Jones, T. E. & Elion, G. B. (1979) J. BioL Chem. ofpurified human enzyme (31); and (iv) the enzyme has strong 254, 8422-8426. Downloaded by guest on September 29, 2021