Isolation and initial characterization of the single polypeptide that ssynthesizes uridine 5'-monophosphate from orotate in Ehrlich ascites carcinoma. Purification by tandem affinity chromatography of uridine-5'-monophosphate synthase

Abstract

UMP synthase or multienzyme pyr-5,6 (orotate phosphoribosyltransferase:orotidine monophosphate decarboxylase) was purified from Ehrlich ascites carcinoma to apparent homogeneity. The purification was achieved by the use of 5-[2-[N-(2-aminoethyl)carbamyl]ethyl]-6-azauridine 5''-monophosphate-agarose and phosphocellulose affinity columns linked in tandem by a flow dialysis system. The purified protein has a MW of .apprx. 51,500 as judged by polyacrylamide gel electrophoresis in sodium dodecyl sulfate. Both enzyme activities cosediment with an S20,w value of 3.7 S, which corresponds to a MW of about 50,000. Two-dimensional electrophoresis of UMP synthase shows that the protein exists as 2 isomeric forms with isoelectric points of 5.85 (major form) and 5.65 (minor form). Both forms have the same MW of 51,500 and contain both active centers. The last 2 enzyme activities of de novo UMP biosynthesis occur on a single polypeptide chain of .apprx. 51,500 daltons and this polypeptide exists in at least 2 isomeric forms.