Glycoprotein biosynthesis in Saccharomyces cerevisiae. Purification of the alpha-mannosidase which removes one specific mannose residue from Man9GlcNAc.
A soluble form of the specific alpha-mannosidase from Saccharomyces cerevisiae, which catalyzes the following reaction, was purified at least 100,000-fold by conventional chromatography procedures: (Formula: see text). The purified enzyme migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis as a single band of about 60 kDa in the absence of reducing agent, and as two bands of about 44.5 kDa and 22.5 kDa in the presence of reducing agent. The apparent molecular weight of the soluble enzyme is about 75,000 by gel filtration on Sephacryl S-200. The specific alpha-mannosidase does not require the addition of divalent cation for activity, but it is inhibited by Tris, EDTA, Mn2+, Co2+, Zn2+, and Mg2+. The inhibition caused by EDTA can be reversed completely by Ca2+ and partially by Mg2+, but not by other divalent cations. The soluble alpha-mannosidase arises from a larger hydrophobic form of the enzyme which is found in the detergent phase during partition in Triton X-114. The formation of the soluble enzyme, which is recovered in the aqueous phase during partition in Triton X-114, is time- and temperature-dependent and is prevented by pepstatin, but not by other protease inhibitors. These results indicate that the purified soluble alpha-mannosidase represents the catalytically active domain of the enzyme which has been proteolytically released from its membrane-bound form.
D002240:Carbohydrate Sequence; D002850:Chromatography, Gel; D002852:Chromatography, Ion Exchange; D006023:Glycoproteins / Q000096:biosynthesis*; D007700:Kinetics; D008361:Mannosidases / Q000302:isolation & purification* / Q000378:metabolism; D008970:Molecular Weight; D012441:Saccharomyces cerevisiae / Q000201:enzymology*; D013379:Substrate Specificity; D043323:alpha-Mannosidase
D006023:Glycoproteins; D008361:Mannosidases; D043323:alpha-Mannosidase