ISOLATION AND CHARACTERIZATION OF UDP-GLUCOSE DOLICHYL-PHOSPHATE GLUCOSYLTRANSFERASE FROM HUMAN LIVER

The enzyme UDP‐glucose dolichyl‐phosphate glucosyltransferase has been purified to near homogeneity from human liver microsomes. A 1100‐fold enrichment over starting microsomal membranes was achieved by selective solubilization followed by anion‐ and cation‐exchange chromatography, 5‐HgUDP‐thiopropyl‐Sepharose affinity chromatography, butylagarose chromatography and hydroxyapatite chromatography. The glucosyltransferase was shown to be separated from other dolichyl‐phosphate‐dependent glycosyltransferases catalyzing the formation of dolichyl diphospho‐N‐acetylglucosamine and dolichyl phosphomannose. Sodium dodecyl sulfate/polyacryl‐amide gradient gel electrophoresis of the purified enzyme under reducing conditions revealed a protein band of Mr 36 000. Protection of the solubilized enzyme against rapid inactivation was achieved by its competitive inhibitor uridine. The purified glucosyltransferase activity exhibited a specific requirement for the presence of phospholipids. Phosphatidylethanolamine was the most effective activator of enzyme activity. Copyright © 1990, Wiley Blackwell. All rights reserved