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

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. Copyright © 1989, Wiley Blackwell. All rights reserved