Hypoxanthine, guanine, xanthine phosphoribosyltransferase activity in Cryptosporidium parvum.
All parasitic protozoa examined to date are incapable of de novo synthesis of purine nucleotides and rely on salvage mechanisms for survival. We have identified hypoxanthine, guanine, xanthine phosphoribosyl-transferase activities in crude cell-free extracts of Cryptosporidium sporulated 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. Autoradiographic studies of Cryptosporidium-infected Madin-Darby canine kidney (MDCK) cells showed that radiolabeled hypoxanthine, guanine, and adenine were primarily incorporated by intracellular Cryptosporidium as 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 of de novo synthesis of purine nucleotides in Cryptosporidium. Radiolabeled hypoxanthine and guanine were also incorporated by excysting Cryptosporidium sporozoites. 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 of Eimeria, Toxoplasma, and Plasmodium.
Doyle
P S
PS
Department of Pharmaceutical Chemistry, University of California, San Francisco, 94143, USA.
Kanaani
J
J
Wang
C C
CC
D000225:Adenine / Q000378:metabolism; D000818:Animals; D001345:Autoradiography; D001667:Binding, Competitive; D002460:Cell Line; D016785:Cryptosporidium parvum / Q000201:enzymology*; D004285:Dogs; D005561:Formates / Q000378:metabolism; D005998:Glycine / Q000378:metabolism; D006147:Guanine / Q000378:metabolism; D019271:Hypoxanthine / Q000378:metabolism; D007041:Hypoxanthine Phosphoribosyltransferase / Q000378:metabolism*; D010430:Pentosyltransferases / Q000378:metabolism*; D013170:Spores / Q000201:enzymology; D019820:Xanthine / Q000378:metabolism
D005561:Formates; C030544:formic acid; D019820:Xanthine; D019271:Hypoxanthine; D006147:Guanine; D010430:Pentosyltransferases; C024827:xanthine phosphoribosyltransferase; D007041:Hypoxanthine Phosphoribosyltransferase; D000225:Adenine; D005998:Glycine