GLYCOPROTEIN-BIOSYNTHESIS IN SACCHAROMYCES-CEREVISIAE - PARTIAL-PURIFICATION OF THE ALPHA-1,6-MANNOSYLTRANSFERASE THAT INITIATES OUTER CHAIN SYNTHESIS

The alpha-1,6-mannosyltransferase (alpha-1,6-ManT) that initiates outer chain synthesis in Saccharomyces cerevisiae was partially purified along with an alpha-1,2-mannosyltransferase (alpha-1,2-ManT) that acts on alpha-methylmannoside. The enzymes were solubilized by extracting a 145 000 g pellet of S. cerevisiae mnn1 mutant with 1% Triton X-100. The extract was then passed through a concanavalin A-Sepharose column and the bound material was eluted with alpha-methylmannoside. After exhaustive dialysis, the fractions containing both mannosyltransferase activities were chromatographed on DEAE-Trisacryl which removed similar to 90% of the alpha-1,2ManT. The fractions containing alpha-1,6-ManT and residual alpha-1,2-ManT were further purified by sequential chromatography on Sephacryl S-200 and CM-Trisacryl. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE) of individual fractions eluted from Sephacryl S-200 and from CM-Trisacryl, followed by silver staining of the gels, showed two major bands whose intensity corresponded to the enzyme activities. A protein band of similar to 62 kDa corresponded to the alpha-1,6-ManT and another band of similar to 66 kDa, which was eluted from the Sephacryl S-200 column slightly earlier, corresponded to the alpha-1,2-ManT.