Solubilization of an alpha-(1 leads to 3)-D-mannosyltransferase from pancreas which utilizes synthetic dolichyl pyrophosphate trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc as substrate.
Calf pancreas microsomes were treated with 0.5-1% Triton X-100 and the resulting soluble enzyme preparation was incubated with GDP-D-[14C]mannose. The addition of synthetic Dol-PP derivative of the trisaccharide beta-Man-(1 leads to 4)-beta-GlcNAc-(1 leads to 4)GlcNAc stimulated the synthesis of labeled lipid-bound tetrasaccharide 50-100-fold. The labeled tetrasaccharide thus formed was identified as alpha-Man-(1 leads to 3)-beta-Man-(1 leads to 4)-beta-GlcNAc- (1 leads to 4)GlcNAc by its chromatographic properties and by its sensitivity to alpha-mannosidase and to endo-beta-N-acetylglucosaminidase D. The solubilized alpha-(1 leads to 3)mannosyltransferase did not require divalent cation and was active in the presence of 10 mM EDTA.
D000118:Acetylglucosaminidase; D000818:Animals; D002417:Cattle; D006602:Hexosyltransferases / Q000302:isolation & purification*; D008361:Mannosidases; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase; D008364:Mannosyltransferases / Q000302:isolation & purification*; D008861:Microsomes / Q000201:enzymology; D010179:Pancreas / Q000201:enzymology*; D011104:Polyisoprenyl Phosphate Oligosaccharides / Q000378:metabolism*; D011105:Polyisoprenyl Phosphate Sugars / Q000378:metabolism*; D012995:Solubility; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D011104:Polyisoprenyl Phosphate Oligosaccharides; D011105:Polyisoprenyl Phosphate Sugars; C038433:dolichol pyrophosphate Man(1-4)-GlcNAc(1-4)GlcNAc; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C033143:glycolipid 3-alpha-mannosyltransferase; D008361:Mannosidases; D043323:alpha-Mannosidase; D000118:Acetylglucosaminidase; D017038:Mannosyl-Glycoprotein Endo-beta-N-Acetylglucosaminidase