Uridine phosphorylase from Novikoff rat hepatoma cells: purification, kinetic properties, and its role in uracil anabolism.
Uridine phosphorylase activity was detected in sonic extracts of six different mammalian cell lines and, in conjunction with uridine kinase, provides a route for the conversion of uracil to UMP via uridine. Uracil phosphoribosyl transferase activity was not detected in any of eight different mammalian cell lines. Uridine phosphorylase was purified 5,330-fold from Novikoff rat hepatoma cells by ammonium sulfate precipitation, DEAE-Sephadex chromatography, hydroxyapatite chromatography, and Sephadex G-200 fractionation. The molecular weight of the enzyme by gel filtration was approximately 45,000. The kinetics of the purified enzyme were analyzed with respect to all four substrates at saturating cosubstrate concentration, yielding the parameters KmUra = 360 microM, KmRib-1-P = 88 microM, KmUrd = 16 micron, and KmPi = 130 microM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 microM. Novikoff cells treated with 0.5 mM inosine exhibited an increase in uracil uptake rate which was proportional to an observed increase in intracellular ribose-1-phosphate. Nevertheless, in cells whose de novo synthesis of pyrimidines was blocked by pyrazofurin or N-(phosphonacetyl)-L-aspartate ("PALA"), the uptake of uracil was insufficient to support proliferation, even when enhanced by inosine. These observations are consistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose-1-phosphate plays a rate-limiting role in the uptake of uracil mediated by uridine phosphorylase.
McIvor
R S
RS
Wohlhueter
R M
RM
Plagemann
P P
PP
D000818:Animals; D006528:Carcinoma, Hepatocellular / Q000201:enzymology* / Q000473:pathology; D002455:Cell Division / Q000187:drug effects; D002460:Cell Line; D006224:Cricetinae; D006801:Humans; D007288:Inosine / Q000494:pharmacology; D007700:Kinetics; D008113:Liver Neoplasms; D051379:Mice; D010430:Pentosyltransferases / Q000378:metabolism*; D051381:Rats; D011919:Rats, Inbred Strains; D012267:Ribosemonophosphates / Q000378:metabolism; D014498:Uracil / Q000378:metabolism* / Q000494:pharmacology; D014543:Uridine Phosphorylase / Q000302:isolation & purification / Q000378:metabolism*
D012267:Ribosemonophosphates; C031154:ribose 1-phosphate; D014498:Uracil; D007288:Inosine; D010430:Pentosyltransferases; D014543:Uridine Phosphorylase