Hypoxanthine, Guanine, Xanthine Phosphoribosyltransferase Activity inCryptosporidium parvum

Abstract

Doyle, P. S., Kanaani, J., and Wang, C. C. 1998. Hypoxanthine, guanine, xanthine phosphoribosyltransferase activity inCryptosporidium parvum. Experimental Parasitology89,9–15. All parasitic protozoa examined to date are incapable ofde novosynthesis of purine nucleotides and rely on salvage mechanisms for survival. We have identified hypoxanthine, guanine, xanthine phosphoribosyl-transferase activities in crude cell-free extracts ofCryptosporidiumsporulated oocysts utilizing radiolabeled substrates. Guanine, hypoxanthine, and xanthine were converted to their corresponding mononucleotides with specific activities of 346, 280, and 108 nmol/min/ mg protein, respectively. The conversion of the radiolabeled purines was examined in the presence of another, unlabeled, purine base. These competition assays showed that both hypoxanthine and guanine were capable of inhibiting conversion of hypoxanthine, guanine, and xanthine to the corresponding nucleotides. Xanthine had a much lower inhibitory effect on the conversion of guanine and hypoxanthine to the nucleotides, whereas adenine had no effect at all. Autoradio-graphic studies ofCryptosporidium-infected Madin—Darby canine kidney (MDCK) cells showed that radiolabeled hypoxanthine, guanine, and adenine were primarily incorporated by intracellularCryptosporidiumas well as by MDCK nuclei. No apparent incorporation of xanthine by either host cells or intracellular parasites occurred. Radiolabeled glycine and formate were incorporated only into the nuclei of MDCK cells, suggesting a lack ofde novosynthesis of purine nucleotides inCryptosporidium. Radiolabeled hypoxanthine and guanine were also incorporated by excystingCryptosporidiumsporozoites. Altogether, our results indicate the presence of HPRTase, GPRTase, and XPRTase activities. These activities may play an important role in purine salvage, and may localize to a single HGXPRTase enzyme, as in the case ofEimeria, Toxoplasma,and Plasmodium.