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/polyacrylamide 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.
Matern
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H
Department of Internal Medicine III, Aachen University of Technology, Federal Republic of Germany.
Bolz
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R
Matern
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S
D002846:Chromatography, Affinity; D017886:Durapatite; D004591:Electrophoresis, Polyacrylamide Gel; D004789:Enzyme Activation / Q000187:drug effects; D004795:Enzyme Stability; D005964:Glucosyltransferases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D006801:Humans; D006882:Hydroxyapatites; D008862:Microsomes, Liver / Q000201:enzymology*; D010743:Phospholipids / Q000494:pharmacology; D012685:Sepharose / Q000031:analogs & derivatives; D012995:Solubility
D006882:Hydroxyapatites; D010743:Phospholipids; C052754:butylagarose; D012685:Sepharose; D017886:Durapatite; D005964:Glucosyltransferases; C033917:UDPglucose dolicholphosphate glucosyltransferase