Uridine phosphorylase from Novikoff rat hepatoma cells: Purification, kinetic properties, and its role in uracil anabolism

✍ Scribed by R. Scott McIvor; Robert M. Wohlhueter; Peter P. G. Plagemann

• Publisher: John Wiley and Sons
• Year: 1985
• Tongue: English
• Weight: 813 KB
• Volume: 122
• Category: Article
• ISSN: 0021-9541
• DOI: 10.1002/jcp.1041220309

✦ Synopsis

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 t h e 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 pM, KmR,b.l-P = 88 pM, KmUrd = 16 pm, and KmPi = 130 pM. However, in intact cells the phosphorolysis of uridine proceeded with an apparent Km of 231 pM. 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-I-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 con- sistent with the kinetic characteristics of the enzyme and provide evidence that the intracellular level of ribose-I-phosphate plays a rate-limiting role in the uptake of uracil mediated by uridine phosphorylase.

Although mammalian cells rapidly incorporate many different nucleosides and nucleic acid bases, uracil uptake is generally slow (Plagemann and Wohlhueter, 1980;Gotto et al., 1969;Itzhaki, 1972). It has been proposed, in fact, that any substantial uracil uptake exhibited by cultured cells is a n indication of mycoplasma contamination (Perez et al., 1972;Schneider et al., 1974;Kenny, 1975). Mammalian cells possess a rapid transport system for uracil (Wohlhueter et al., 19801, so it would appear that uracil incorporation is limited by the cell's ability to metabolize it to the nucleotide level.

Unlike many microorganisms (O'Donovan and Neuhard, 1970), mammalian cells do not possess a specific uracil phosphoribosyltransferase (UPRTase) activity, which would allow a cell to convert uracil to UMP in one enzymatic step (Reyes and Guganig, 1975;Wohlhueter et al., 1979; this paper). At physiological pH, uracil, in contrast to 5-fluorouracil (pK, = 8.01, is also a very poor substrate for orotate phosphoribosyltransferase, because the enzyme is specific for negatively charged substrates and the pK, for uracil is 9.5 (Wempen and Fox, 1964). Mammalian cells incorporate uridine rapidly via uridine kinase (Plagemann and Wohlhueter, 1980,19831, and many cell lines possess uridine phosphorylase activity (Wohlhueter et al., 1979;Plagemann et al., 1981; this paper). Since uridine is much more rapidly incorporated than uracil, the conversion of uracil to UMP via uridine would appear to be limited by uridine phosphorylase activity (EC 2.4.2.3): Uracil + Ribose-1-phosphatesuridine + Phosphate This paper presents some of the kinetic characteristics of this enzyme purified from Novikoff rat hepatoma cells and addresses the role of the cosubstrate, ribose-l-phosphate, in regulating uracil uptake.