Purine Metabolism in the Protozoan Parasite Eimeria Tenella (Hypoxanthine-Xanthine-Guanine Phosphoribosyltransferase/GMP-Agarose Affinity Column/Alopurinol) C
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Proc. NatL Acad. Sci. USA Vol. 78, No. 11, pp. 6618-6622, November 1981 Biochemistry Purine metabolism in the protozoan parasite Eimeria tenella (hypoxanthine-xanthine-guanine 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 glycine or formate into purine nucleotides; this suggests lack of grown in cell culture (13, 14). Purine metabolism 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 enzymes: hypoxanthine, guanine, xanthine, and adenine cumulate in avian tissues phosphoribosyltransferases and adenosine 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 enzyme 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 Allopurinol, 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 liver HGPRTase. Allopurinol conversion to allopurinol ribonucleotide 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 nucleic acid 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-guanosine-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 nucleoside 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). nucleotide 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 ribonucleoside 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]inosine (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 ribonucleotides 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