Salvage syntheses and their relationship to nucleic acid metabolism

Bulletin of the World Health Organization, 55 (2-3): 249-252 (1977) Salvage syntheses and their relationship to nucleic acid metabolism E. KONIGK 1 The intraerythrocytic stages ofplasmodia are capable ofsynthesizing purine nucleotides and apparently deoxycytidylate by salvage syntheses. Data obtained by studying the incorporation ofradioactive precursor molecules into intact cells and kinetic experiments on purified enzyme preparations suggest biosynthetic routes whiAh, generally, are similar to those of the host's cell metabolism. However, details on the regulation of both the uptake of nucleosides and bases into the intraerythrocytic stages ofplasmodia and of the metabolic routes involved in this incorporation are still lacking. Mammalian erythrocytes do not synthesize DNA and RNA; however, they are able to synthesize nucleotides. The synthesis of purine nucleotides is not performed de novo, but via metabolic salvage pathways. Mature erythrocytes, therefore, rely on an external source of preformed purines that originate from the liver and probably other organs (1, 2). The passage of nucleosides and bases across the plasma membranes is a process mediated by transport elements of the plasma membrane and is apparently closely linked to reactions that transform nucleo- sides and bases into impermeable nucleotides (3, 4). The intraerythrocytic stages of plasmodia possess high rates of DNA-synthesis and protein synthesis, and they require a vast supply of precursor molecules. The host erythrocytes have the remarkable capacity of providing the essential precursor molecules in amounts that will allow the successful growth and multiplication of the parasite within the cell. While the synthesis of dihydrofolate is an example of a parasite-specific sequence of reactions, the transfor- mation of energy and the synthesis of nucleotides in the malaria parasite occur via metabolic routes very similar to those of their host cells. The experiments performed by Biungener & Nielsen (5, 6) clearly indicated the existence of a salvage synthesis of purine nucleotides and the apparent lack of the incorporation of 3H-thymidine into DNA of rodent plasmodia. Incorporation studies have proved to be of value in studying the nucleotide metabolism of avian and mammalial plasmodia and conditions for the cultivation of plasmodia, 1 Bernhard Nocht Institute, Bernhard-Nocht-Strasse 74, 2000 Hamburg 4, Federal Republic of Germany. and have yielded information on the mode of action of antimalarial drugs (7-18). Pyrimidine nucleotides are obviously synthesized de novo (7, 10, 19). The separation of nucleotides, nucleosides, and bases by TL-chromatography on PEI-cellulose- coated Polygram sheets greatly facilitated the analyti- cal work essential in performing sensitive enzyme assays and detailed studies on the fate of incor- porated radioactive precursor compounds (20, 21). By combining data from incorporation experiments in P. berghei and kinetic data obtained from enzyme studies with the supernatant of plasmodial homo- genate and partly purified enzymes of P. chabaudi, the following metabolic scheme for purine salvage synthesis in the malaria parasite can be suggested (Fig. 1) (22-26): hypoxanthine, obtained either directly or by a sequence of perhaps membrane- associated reactions in which adenosine is taken Erythrocyte Pchabaudi hypoxanthnie hypoxanthine IMP lnosne t adenosine adenosine - AMP adenne adenine - AMP Fig. 1. Synthesis of adenosine 5'-monophosphate (AMP) in Plasmodium chabaudi (IMP = inosine 5'-monophosphate). 3603 -249 E. KONIGK up by the parasite, deaminated to inosine, and dephosphorylated to hypoxanthine, can be trans- formed to inosine 5'-monophosphate (IMP) by the catalytic action of hypoxanthine phosphoribosyl- transferase (EC 2.4.2.8). IMP can be transformed to adenosine 5'-monophosphate (AMP) via the reac- tions that are catalysed by adenylosuccinate syn- thetase (EC 6.3.4.4) and adenylosuccinate lyase (EC 4.3.2.2). AMP can also be synthesized either from adenosine by a phosphorylating reaction catalysed by adenosine kinase (EC 2.7.1.20) or from adenine by adenine phosphoribosyltransferase (EC 2.4.2.7). Such salvage syntheses have also been reported from erythrocytes, leucocytes, and throm- bocytes. A similar pathway of adenosine utilization is found in a system that regulates the coronary blood flow of the heart muscle (27). Guanosine 5'-mono- phosphate can be synthesised by the action of hypoxanthine phosphoribosyltransferase and via IMP. The latter reaction has not been confirmed yet in plasmodia. The salvage syntheses of purine nucleotides provide the precursor molecules for the biosynthesis of nucleic acids and the nucleotide coenzymes. An important precursor molecule of the latter group is guanosine 5'-triphosphate, the initial compound of the plasmodia-specific synthesis of dihydrofolate. Autoradiographic studies with 3H-thymidine (5) and the failure to measure activities of thymidine kinase (EC 2.7.1.75) suggest the absence of a salvage synthesis of thymidylate in plasmodia. These observations furnish an additional explanation for the inhibitory effect on the DNA synthesis of plasmodia by analogues of p-aminobenzoate and folate. Deoxycytidine has been reported to be the major deoxynucleoside in the circulating blood (28, 29). The uptake and very low rates of transformation of deoxycytidine in P. chabaudi-infected red cells in vitro have been observed (30). This pathway could provide deoxycytidylate, which becomes deaminated to deoxyuridylate, the substrate of the thymidylate synthetase reaction (30) (Fig. 2). Any discussion on the regulation of the salvage synthesis of purine nucleotides will remain incom- plete without considering the mechanisms of membrane transport and aspects of compartment- alization. However, kinetic data obtained from partly purified enzyme preparations suggest that the utilization of adenosine may proceed at low concentrations via the reaction catalysed by adeno- sine kinase and at higher concentrations via that catalysed by adenosine deaminase (EC 3.5.4.4) (Table 1). Adenosine kinase, which may operate in close association with the membrane, is capable of using adenosine analogues as substrates. However, DNA t t thyxmde knasethymdne dTMP 5-CH3dCMP thyr dCMPanno - CTP -* dCTP * dCMP -. dUMP (CDP) (dCDP) A GTP folate I- cdhydrofodate nidylate synthetase " NSN1methylene - FH4 H2-pterin -CH2-0-P-P+ p-anirinobenzoate dIhydropteroate 1dd syther tase ~~~~~dihydropteroate IdCR knase dCR Fig. 2. Biosynthesis of deoxynucleotides (CDP = cytidine 5'-diphosphate; CTP = cytidine 5'-triphosphate; dCDP = deoxycytidine 5'-diphosphate; dCMP = deoxycytidine 5'-monophosphate; dCR = deoxycytidine; dCTP = deoxycytidine 5'-triphosphate; dTMP = thymidine 5'-monophosphate; dUMP = deoxyuridine 5'-mono- phosphate; GTP = guanosine 5'-triphosphate). 250 SALVAGE SYNTHESIS OF PURINE NUCLEOTIDES IN PLASMODIA Table 1. Michaelis-Menten constants of adenosine kinase and adenosine deaminase from human erythro- cytes (31) and P. chabaudi Human P.chabaudi erythrocytes P.hbad adenosine kinase 1.9 x 10-6 mol/litre 6.5 x 10-6 mol/litre adenosine deaminase 4.0 x 10-5 mol/litre 1.3 x 10-4 mol/litre the further transformation of the nucleotide ana- logues may vary because of differences in the specificity of the adenylate kinase reaction (3). Cordycepin and tubercidin, both analogues of adenosine and substrates of adenosine kinase, have been assayed in laboratory experiments for their antimalarial and schistosomicidal effectiveness (15, 31). Plasmodia, like human erythrocytes, possess two distinct enzymes for a phosphoribosyltransferase reaction using hypoxanthine (guanine) and adenine as a source of purines (25). The Michaelis-Menten constant for hypoxanthine of the hypoxanthine phosphoribosyltransferase reaction was 2.5 x 10-6 mol/litre, for adenine of the adenine phosphoribosyl- Table 2. Michaelis-Menten constants and inhibitor constants of partly purified hypoxanthine phospho- ribosyltransferase (HGPRTase) and adenine phorpho- ribosyltransferase (APRTase) Km Ki Enzyme Substrate or inhibitor (x 10-6 (x 10-6 mol/litre) mol/litre) HGPRTase hypoxanthine 2.5 guanine 2.0 6-mercaptopurine 7.0 2-amino-6-mercaptopurine 4.0 6-thioguanine 5.0 APRTase adenine 5.0 transferase reaction it was 5.0 x 10-6 mol/litre. Only the former reaction was competitively inhibited by the purine analogues 6-mercaptopurine, 2- amino-6-mercaptopurine, and 6-thioguanine (Table 2). Isoenzymes of hypoxanthine phos- phoribosyltransferase that have been reported from various systems have not been found in P. chabaudi. The molecular weights of 71 000 for hypoxanthine phosphoribosyltransferase and 34000 for adenine phosphoribosyltransferase correspond with those obtained from human erythrocytes (68 000 and 34 000, respectively (25, 32, 33)). The isoelectric point of plasmodial hypoxanthine phosphoribosyl- transferase of 5.25 differed from those reported from human erythrocytes (25, 32, 33). The kinetic data provided suggest that hypoxanthine phos- phoribosyltransferase functions as a key enzyme in the reutilization of purines. Allopurinol added to the drinking-water of mice stimulated the parasitization of mature erythrocytes in the P. berghei-infected mouse and the multiplication of P. vinckei and P. chabaudi in mice and rats. This indicates also that purines from exoerythrocytic sources are limiting factors of the multiplication of intra- erythrocytic plasmodia (34). The concentration of phosphoribosylpyrophosphate (PRPP) and of ribose 5'-phosphate may be of regulating importance (35). In Lesh-Nyhan red cells with decreased activity of hypoxanthine phosphoribosyltransferase, the con- centration of PRPP is elevated by up to ten times. This elevation of PRPP may increase the activity of adenine phosphoribosyltransferase by stabilizing the enzyme (36). Plasmodia, obviously, are capable of providing the nucleotides that they need for the synthesis of their nucleic acids, mainly by simulating reactions that also exist in their host cells. However, minor differences may exist in the sequence and regulation of some reactions-e.g., the synthesis of AMP from IMP has not been reported from human erythrocytes because of an apparent lack of adenylo- succinate synthetase and lyase-enzymes that have been observed in rabbit reticulocytes and in prepara- tions of P. chabaudi. R1tSUMIt LES SYNTHtSES PAR RECUPERATION ET LEURS RELATIONS AVEC LE METABOLISME DES ACIDES NUCLEIQUES Les stades intra-erythrocytaires des plasmodies sont capables de synthetiser par recuperation les nucleotides puriques et apparemment le desoxycytidylate. Les donnees fournies par l'etude de l'incorporation de mol6cules precurseurs radio-actives dans des cellules intactes, et par les experiences de cinetique sur des 251 252 E. KONIGK preparations enzymatiques purifiees, semblent indiquer qu'il existe des voies biosynthetiques qui, en general, sont similaires A celles du metabolisme de la cellule h6te. Toutefois, on manque encore de details sur la regulation de l'incorporation des nucleosides et des bases dans les stades intra-erythrocytaires des plasmodies et sur celle des voies metaboliques impliquees. REFERENCES 1. PRITCHARD, J. B. ET AL. American journal ofphysio- logy, 219: 1263-1267 (1970). 2. MAGUIRE, M. H. ET AL. Biochimica et biophysica acta, 262: 108-115 (1972). 3. PARKS, R. E. JR & BROWN, P. R. Biochemistry, 12: 3294-3302 (1973). 4. PICKARD, M. A. & PATERSON, A. R. P. Biochimica et biophysica acta, 455: 817-823 (1976). 5. BUNGENER, W. & NIELSEN, G. Zeitschrift fiir Tropen- medizin und Parasitologie, 18: 456-462 (1967). 6. BUNGENER, W. & NIELSEN, G. Zeitschrift fur Tropen- medizin und Parasitologie, 19: 185-197 (1968). 7. WALSH, C. J. & SHERMAN, I. W. Journal of proto- zoology, 15: 763-770 (1968). 8. TRACY, S. M. & SHERMAN, I. W. Journal of proto- zoology, 19: 541-549 (1972). 9. BOODEN, T. & HULL, R. W. Experimental parasito- logy, 34: 220-228 (1973). 10. GUTTERIDGE, W. E. & TRIGG, P. I. Journal of protozoology, 17: 89-96 (1970). 11. VAN DYKE, K. ET AL. American journal of tropical medicine and hygiene, 19: 202-208 (1970). 12. LANTZ, C. H. ET AL. Experimental parasitology, 29: 402-416 (1971). 13. NEAME, K. D. ET AL. Parasitology, 69: 329-335 (1974). 14. VAN DYKE, K. & SZUSTKIEWICZ, C. Military medicine, 134: 1000-1006 (1969). 15. TRIGG, P. I. ET AL. Transactions of the Royal Society of Tropical Medicine and Hygiene, 65: 514-520 (1971). 16. GUTTERIDGE, W. E. ET AL. Parasitology, 64: 37-45 (1972). 17. THEAKSTON, R. D. G. ET AL. Annals of tropical medicine and parasitology, 66: 295-302 (1972). 18. MCCORMICK, G. J. ET AL. Antimicrobial agents and chemotherapy, 6: 16-21 (1974). 19. POLET, H. & BARR, C. F. Journal ofpharmacology and experimental therapeutics, 164: 380-386 (1968). 20. RANDERATH, K. & RANDERATH, E. Journal of chromatography, 16: 111-125 (1964). 21. K6NIGK, E. Journal of chromatography, 37: 128-131 (1968). 22. MANANDHAR, M. S. P. & VAN DYKE, K. Experi- mental parasitology, 37: 138-145 (1975). 23. VAN DYKE, K. Tropenmedizin und Parasitologie, 26: 232-238 (1975). 24. LUKOW, I. ET AL. Zeitschrift far Tropenmedizin und Parasitologie, 24: 500-504 (1973). 25. WALTER, R. D. & KONIGK, E. Tropenmedizin und Parasitologie, 25: 227-235 (1974). 26. SCHMIDT, G. ET AL. Tropenmedizin und Parasitologie, 25: 301-308 (1974). 27. BERNE, R. M. American journal of physiology, 204: 317-322 (1963). 28. SCHNEIDER, W. C. Journal of biological chemistry, 216: 287-300 (1955). 29. Guiu, C. D. ET AL. Proceedings of the Society for Experimental Biology and Medicine, 129: 31-34 (1968). 30. K6NIGK, E. In: van den Bossche, H., ed. Bio- chemistry ofparasites and host-parasite relationships. Amsterdam, North Holland, 1976, pp. 51-58. 31. JAFFE, J. J. ET AL. Nature, 230: 408-409 (1971). 32. ARNOLD, W. J. & KELLEY, W. N. Journal ofbiological chemistry, 246: 7398-7404 (1971). 33. THOMAS, C. B. ET AL. Journal of biological chemistry, 248: 2529-2535 (1973). 34. BUNGENER, W. Tropenmedizin und Parasitologie, 25: 309-312 (1974). 35. BECKER, M. A. Journal of clinical investigation, 57: 308-318 (1976). 36. GREEN, M. L. ET AL. Science, 167: 887-889 (1970).