Purification of GDP mannose:dolichyl-phosphate O-beta-D-mannosyltransferase from Saccharomyces cerevisiae.
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.
Haselbeck
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Institut für Botanik, Universität Regensburg, Federal Republic of Germany.
D002462:Cell Membrane / Q000201:enzymology; D002845:Chromatography / Q000379:methods; D002848:Chromatography, DEAE-Cellulose; D004591:Electrophoresis, Polyacrylamide Gel; D006602:Hexosyltransferases / Q000302:isolation & purification*; D007106:Immune Sera; D015151:Immunoblotting; D008364:Mannosyltransferases / Q000037:antagonists & inhibitors / Q000276:immunology / Q000302:isolation & purification*; D017830:Octoxynol; D011092:Polyethylene Glycols; D011489:Protein Denaturation; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D012995:Solubility
D007106:Immune Sera; D011092:Polyethylene Glycols; D017830:Octoxynol; D006602:Hexosyltransferases; D008364:Mannosyltransferases; C022755:dolichyl-phosphate beta-D-mannosyltransferase