Partial purification and properties of a glucosyltransferase that synthesizes Glc1Man9(GlcNAc)2-pyrophosphoryldolichol.
The glucosyltransferase that transfers the first glucose residue from dolichyl-P-glucose to Man9-(GlcNAc)2-PP-dolichol has been solubilized from porcine aorta and purified 720-fold. The purification strategy involved ammonium sulfate precipitation followed by ion-exchange, gel filtration, and hydroxylapatite column chromatographies. Analysis of the products produced by enzyme fractions at different stages of purification indicate that three different glucosyltransferases are involved in the conversion of Man9(GlcNAc)2-PP-dolichol to Glc3Man9(GlcNAc)2PP-dolichol. the first glucosyltransferase appears to be specific for dolichyl-P-glucose as the donor substrate. Man9(GlcNAc)2-PP-dolichol, Man7(GlcNAc)2-PP-dolichol, and Man5(GlcNAc)2-PP-dolichol (with two different oligosaccharide structures) were tested for their ability to accept glucose from dolichyl-P-glucose. Studies on the comparative rates of transfer of glucose to these different acceptor substrates demonstrated that Man9(GlcNAc)2-PP-dolichol accepts glucose at a higher initial rate and to a greater extent than does Man7(GlcNAc)2-PP-dolichol and the biosynthetic Man5(GlcNAc)2-PP-dolichol. The other Man5(GlcNAc)2-PP-dolichol (i.e. Man alpha 1,6[Man alpha 1,3]-Man alpha 1,6[Man alpha 1,3]Man beta 1,4GlcNAc beta 1, GlcNAc) was not an acceptor, indicating that the Man alpha 1,2-Man alpha 1,2Man alpha 1,3Man arm is necessary. Man9(Glc-NAc)2 and Man9(GlcNAc)2-protein were not acceptors, indicating that both the lipid and the oligosaccharide portion of Man9(GlcNAc)2-PP-dolichol are required for enzyme activity. The partially purified enzyme has a pH optimum of 6.5 and exhibits a requirement for divalent metal ions.
D'Souza-Schorey
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Department of Biochemistry and Molecular Biology, University of Arkansas for Medical Sciences, Little Rock 72205.
Elbein
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AD
D000818:Animals; D001011:Aorta / Q000201:enzymology; D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid; D005964:Glucosyltransferases / Q000302:isolation & purification* / Q000378:metabolism; D008861:Microsomes / Q000201:enzymology; D008969:Molecular Sequence Data; D011103:Polyisoprenyl Phosphate Monosaccharides / Q000378:metabolism; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
C066723:Glc(3)Man(9)(GlcNAc)(2)-diphosphate-dolichol; D011103:Polyisoprenyl Phosphate Monosaccharides; D011105:Polyisoprenyl Phosphate Sugars; C002968:dolichol-D-glucosylmonophosphate; D005964:Glucosyltransferases