Purification of GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase from Saccharomyces cerevisiae

Eur J Biochem. 1989 May 15;181(3):663-8. doi: 10.1111/j.1432-1033.1989.tb14774.x.

Abstract

The enzyme GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase (GDP-Man:DolP mannosyltransferase) catalyzing the reaction: GDP-man + DolP in equilibrium 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.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Cell Membrane / enzymology
  • Chromatography / methods
  • Chromatography, DEAE-Cellulose
  • Electrophoresis, Polyacrylamide Gel
  • Hexosyltransferases / isolation & purification*
  • Immune Sera
  • Immunoblotting
  • Mannosyltransferases / antagonists & inhibitors
  • Mannosyltransferases / immunology
  • Mannosyltransferases / isolation & purification*
  • Octoxynol
  • Polyethylene Glycols
  • Protein Denaturation
  • Saccharomyces cerevisiae / enzymology*
  • Solubility

Substances

  • Immune Sera
  • Polyethylene Glycols
  • Octoxynol
  • Hexosyltransferases
  • Mannosyltransferases
  • dolichyl-phosphate beta-D-mannosyltransferase