The purification and preliminary characterization of UMP synthase from human placenta.
Orotate phosphoribosyltransferase (EC 2.4.2.10) and orotidine 5'-monophosphate decarboxylase (EC 4.1.1.23) are the final two of six enzymatic steps required in the de novo biosynthesis of uridine 5'-monophosphate (UMP). Earlier work of this laboratory showed that, in the mouse Ehrlich ascites carcinoma, both of these enzymatic activities were contained on the single multifunctional polypeptide chain, UMP synthase. We report here that the placenta provided an available human source for UMP synthase with 40-fold higher orotate phosphoribosyltransferase and orotidine 5'-monophosphate decarboxylase specific activities than erythrocytes, a human source previously used by others. By using the placenta as a source of UMP synthase and by developing a novel purification procedure different from that used in the purification of UMP synthase from the Ehrlich ascites carcinoma (the only other homogeneous preparation of a mammalian UMP synthase), we achieved the purification of human UMP synthase to apparent homogeneity. This represents the first publication to homogeneity of UMP synthase from a human source as well as from a source other than malignant cell lines. Contrary to earlier reports human placental UMP synthase was found to be a multifunctional protein containing both enzymatic activities on a single polypeptide of 51,000 molecular weight. Preliminary characterization of the human placental UMP synthase showed it to be similar to UMP synthase from the Ehrlich ascites carcinoma in subunit molecular weight, native molecular weight, isozyme pattern (although not absolute pI values), pH optima of enzymatic activities, and kinetic constants for orotidine 5'-monophosphate (Km) and 6-azauridine 5'-monophosphate (Ki) at the decarboxylase site.
Livingstone
L R
LR
Department of Biochemistry and Nutrition, School of Medicine, University of North Carolina at Chapel Hill 27514.
Jones
M E
ME
D002262:Carboxy-Lyases / Q000302:isolation & purification*; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007525:Isoelectric Focusing; D009097:Multienzyme Complexes / Q000302:isolation & purification* / Q000378:metabolism; D009962:Orotate Phosphoribosyltransferase / Q000302:isolation & purification* / Q000378:metabolism; D009964:Orotidine-5'-Phosphate Decarboxylase / Q000302:isolation & purification* / Q000378:metabolism; D010430:Pentosyltransferases / Q000302:isolation & purification*; D010920:Placenta / Q000201:enzymology*
D009097:Multienzyme Complexes; C027157:uridine 5'-monophosphate synthase; D010430:Pentosyltransferases; D009962:Orotate Phosphoribosyltransferase; D002262:Carboxy-Lyases; D009964:Orotidine-5'-Phosphate Decarboxylase