Isolation and initial characterization of the single polypeptide that synthesizes 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), has been 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 amolecular weight of approximately 51500 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 molecular weight of about 50000. Two-dimensional electrophoresis of UMP synthase shows that the protein exists as two isomeric forms with isoelectric points of 5.85 (major form) and 5.65 (minor form). Both forms have the same molecular weight of 51500 and contain both active centers. These results clearly show that the last two enzyme activities of de novo UMP biosynthesis occur on a single polypeptide chain of approximately 51500 daltons and that this polypeptide exists in at least two isomeric forms.

• Authors: R W McClard, M J Black, L R Livingstone, M E Jones
• Journal: Biochemistry (Biochemistry) Vol. 19 Issue 20 Pg. 4699-706 (Sep 30 1980) ISSN: 0006-2960 [Print] United States
• PMID: 6893554 (Publication Type: Journal Article, Research Support, U.S. Gov't, Non-P.H.S., Research Support, U.S. Gov't, P.H.S.)

Chemical References

• Multienzyme Complexes
• uridine 5'-monophosphate synthase
• Pentosyltransferases
• Orotate Phosphoribosyltransferase
• Carboxy-Lyases
• Orotidine-5'-Phosphate Decarboxylase

Topics

• Animals
• Carboxy-Lyases (isolation & purification)
• Carcinoma, Ehrlich Tumor (enzymology)
• Centrifugation, Density Gradient
• Chromatography, Gel
• Electrophoresis, Polyacrylamide Gel
• Isoelectric Focusing
• Male
• Mice
• Multienzyme Complexes (isolation & purification, metabolism)
• Orotate Phosphoribosyltransferase (isolation & purification, metabolism)
• Orotidine-5'-Phosphate Decarboxylase (isolation & purification, metabolism)
• Pentosyltransferases (isolation & purification)