Solubilization of an α-(1→3)-D-mannosyltransferase from pancreas which utilizes synthetic dolichyl pyrophosphate trisaccharide β-Man-(1→4)-β-GlcNAc-(1→4)GlcNAc as substrate

Abstract

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 β-Man-(1→4)-β-GlcNac-(1→4)GlcNAc stimulated the synthesis of labeled lipid-bound tetrasaccharide 50–100-fold. The labeled tetrasaccharide thus formed was identified as α-Man-(1→3)-β-Man-(1→4)-β-GlcNAc-(1→4)GlcNAc by its chromatographic properties and by its sensitivity to α-mannosidase and to endo-β- N-acetylglucosaminidase D. The solubilized α-(1→3)mannosyltransferase did not require divalent cation and was active in the presence of 10 mM EDTA.