Purification of GDP mannose: dolichyl‐phosphate O‐β‐D‐mannosyltransferase from Saccharomyces cerevisiae

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Abstract
The enzyme GDP mannose: dolichyl-phosphate O-β-d-mannosyltransferase (GDP-Man: DolP mannosyltransferase) catalyzing the reaction: GDP-man + DolP⇌ DolP-Man + GDP has been purified from Saccharomyces cerevisiae to homogeneity. The purification was achieved using a combination of column chromatographic methods with preparative gel electrophoresis. The enzyme has an apparent molecular mass of 30 kDa on SDS/polyacrylamide gels. Enzymatic activity could be correlated directly with this band. Antibodies against the transferase were raised in rabbits. The immune serum obtained removed enzymatic activity from a detergent extract of yeast membranes and reacted specifically with the 30-kDa band on immunoblots. Experiments addressing the orientation of this enzyme in the endoplasmic reticulum membrane are presented by using selective trypsin and N-ethylmaleimide treatment.