Characterization of a feedback-resistant phosphoribosylpyrophosphate synthetase from cultured, mutagenized hepatoma cells that overproduce purines.
A clone of cells in which the regulation of purine metabolism is genetically altered was selected and isolated from chemically mutagenized HTC cells (a line of rat hepatoma cells in continuous culture). The clone, designated MAU V, was selected for increased ability to salvage exogenous purines by isolating it in medium containing methylmercaptopurine ribonucleoside, adenine, and uridine, in which medium wild-type cells cannot divide. We have characterized these cells as having an increased rate of de novo purine biosynthesis, apparently as the result of an altered phosphoribosylpyrophosphate (PRPP) synthetase. The altered enzyme has normal catalytic properties but an altered sensitivity to feedback inhibition by purine and pyrimidine nucleotides. The types of inhibitions (competitive and uncompetitive) exerted by AMP, ADP, and TDP on the wild-type enzyme have been maintained in the altered enzyme, but values for K(i) have been increased by factors of 10, 17.5, and 5, respectively. The specific catalytic activities of AMP: pyrophosphate phosphoribosyltransferase and IMP:pyrophosphate phosphoribosyltransferase are normal. The mutant cell may serve as a model for a specific human disease, one type of dominantly inherited overproduction hyperuricemia.
D000244:Adenosine Diphosphate / Q000494:pharmacology; D000249:Adenosine Monophosphate / Q000494:pharmacology; D000818:Animals; D002250:Carbon Radioisotopes; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D002999:Clone Cells; D005246:Feedback; D007700:Kinetics; D008113:Liver Neoplasms / Q000201:enzymology*; D009154:Mutation; D009374:Neoplasms, Experimental / Q000201:enzymology; D010428:Pentosephosphates; D010756:Phosphoric Acids; D010770:Phosphotransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D011685:Purine Nucleotides / Q000494:pharmacology; D011687:Purines / Q000096:biosynthesis*; D011742:Pyrimidine Nucleotides / Q000494:pharmacology; D051381:Rats; D013942:Thymine Nucleotides / Q000494:pharmacology
D002250:Carbon Radioisotopes; D010428:Pentosephosphates; D010756:Phosphoric Acids; D011685:Purine Nucleotides; D011687:Purines; D011742:Pyrimidine Nucleotides; D013942:Thymine Nucleotides; D000249:Adenosine Monophosphate; D000244:Adenosine Diphosphate; D010770:Phosphotransferases