Studies on a pyrimidine phosphoribosyltransferase from murine leukemia P1534J. Partial purification, substrate specificity, and evidence for its existence as a bifunctional complex with orotidine 5-phosphate decarboxylase.
A pyrimidine phosphoribosyltransferase, previously shown to utilize 5-fluorouracil and possibly also uracil and orotate (Reyes, P. (1969) Biochemistry 8, 2057-2062), has been purified about 100-fold from murine leukemia P1534J. Roughly 20% of the original activity was recovered to yield an enzyme preparation with a specific activity of 7.4 mumol of 5-fluorouracil utilized/hour/mg of protein. Disc gel electrophoresis of this preparation revealed the presence of a major band of protein accompanied by several trace contaminants. Emphasis was placed on a study of the substrate specificity of this enzyme. 5-Fluorouracil, uracil, and orotate phosphoribosyltransferase activities purified in parallel during fractionation with ammonium sulfate and protamine sulfate and eluted together from columns of Sephadex tG-150 and DEAE-cellulose. The three phosphoribosyltransferase activities eluted from the Sephadex columns with an apparent molecular weight of 55,000 to 60,000. In spite of this coordinate fractionation, preferential losses of orotate activity were experienced during DEAE-cellulose chromatography. Orotate activity continued to behave in a unique manner under other conditions, such as during proteolytic digestion. In the latter case, however, all three activities responded in parallel when digestion took place in the presence of 5mM UMP. The following results provided additional evidence to support the view that all three phosphoribosyltransferase activities may be catalyzed by the same enzyme: (a) the apparent Km for 5-phosphoribosyl 1-pyrophosphate (PP-ribose-P) did not change significantly when enzyme activity was measured with either 5-fluorouracil, uracil, or orotate; (b) 5-fluorouracil and uracil were found to be mutually competitive inhibitors; the effect of 5-fluorouracil on orotate activity was likewise competitive in nature; (c) in the absence of UMP, orotate was a noncompetitive inhibitor of 5-fluorouracil and uracil activities, but in the presence of 5mM UMP it became a competitive inhibitor of both of these activities; (d) 5-fluorouracil and orotate activities co-sedimented in 5 to 20% sucrose gradients (uracil activity was not examined); and (e) a wide variety of normal mouse tissues displayed virtually the same 5-fluorouracil to uracil to orotate activity ratio as found in P1534J enzyme preparations. The apparent Km and Ki values reported in this study indicate that the preferred pyrimidine substrate is orotate. It seems likely, therefore, that this enzyme functions in vivo as an orotate phosphoribosyltransferase. Orotate phosphoribosyltransferase and orotidine 5'-monophosphate (OMP) decarboxylase activities (a) eluted together during gel filtration on Sephadex G-150, (b) co-sedimented in 5 to 20% sucrose gradients, (c) remained associated during fractionation with ammonium sulfate and protamine sulfate, and (d) separated into a phosphoribosyltransferase and decarboxylase component when enzyme preparations previously subjected to limited proteolysis by elastase were sedimented in sucrose gradients...
D000818:Animals; D002250:Carbon Radioisotopes; D002262:Carboxy-Lyases / Q000378:metabolism; D002264:Carboxylic Acids; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002848:Chromatography, DEAE-Cellulose; D002850:Chromatography, Gel; D004591:Electrophoresis, Polyacrylamide Gel; D005472:Fluorouracil / Q000378:metabolism; D006358:Hot Temperature; D007700:Kinetics; D007942:Leukemia, Experimental / Q000201:enzymology*; D051379:Mice; D009963:Orotic Acid / Q000378:metabolism / Q000494:pharmacology; D010430:Pentosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D010447:Peptide Hydrolases / Q000494:pharmacology; D010754:Phosphoribosyl Pyrophosphate / Q000378:metabolism; D011743:Pyrimidines / Q000378:metabolism; D014498:Uracil; D014500:Uracil Nucleotides / Q000494:pharmacology; D014543:Uridine Phosphorylase / Q000378:metabolism
D002250:Carbon Radioisotopes; D002264:Carboxylic Acids; D011743:Pyrimidines; D014500:Uracil Nucleotides; D014498:Uracil; D009963:Orotic Acid; D010754:Phosphoribosyl Pyrophosphate; D010430:Pentosyltransferases; C022500:pyrimidine phosphoribosyltransferase; D014543:Uridine Phosphorylase; D010447:Peptide Hydrolases; D002262:Carboxy-Lyases; D005472:Fluorouracil