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 approximately 90% of the alpha-1,2-ManT. 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 approximately 62 kDa corresponded to the alpha-1,6-ManT and another band of approximately 66 kDa, which was eluted from the Sephacryl S-200 column slightly earlier, corresponded to the alpha-1,2-ManT.
Romero
P A
PA
McGill Cancer Centre, McGill University, Montreal, Quebec, Canada.
Sleno
B
B
Herscovics
A
A
D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005656:Fungal Proteins / Q000096:biosynthesis*; D006023:Glycoproteins / Q000096:biosynthesis*; D007700:Kinetics; D008364:Mannosyltransferases / Q000302:isolation & purification* / Q000378:metabolism*; D012441:Saccharomyces cerevisiae / Q000201:enzymology*
D005656:Fungal Proteins; D006023:Glycoproteins; D008364:Mannosyltransferases; C058478:alpha 1,6-mannosyltransferase