Solubilization and purification of alpha-mannosidase, a marker enzyme of vacuolar membranes in Saccharomyces cerevisiae.
Yeast alpha-mannosidase, a marker enzyme of vacuolar membranes, was solubilized and purified from commercial bakers' yeast. The alpha-mannosidase was solubilized efficiently with 10 mM Na2CO3. A high pH (greater than 8.5) and a sufficient amount of a detergent such as 0.2% (w/v) Triton X-100 were required to keep the enzyme in a soluble state. This suggested that the enzyme is either a peripheral membrane protein or an ecto-type integral membrane protein. After 4,300-fold purification by conventional chromatography, the alpha-mannosidase gave a single band on nondenaturing polyacrylamide gel electrophoresis, but could be fractionated into active isoforms, which consisted of 107-, 73-, and 31-kDa polypeptides, with a Mono Q anion exchange fast protein liquid chromatography system. Apparent molecular weight of the native enzyme was determined as 560,000. It suggested that the composition of isoforms will be described as (107 kDa)n (73 kDa)6-n (31 kDa)6-n, where n is 0-6. The 107- and 73-kDa polypeptides were purified further under denaturing conditions. One-dimensional peptide map analysis and immunological analysis of these polypeptides indicated that they are closely related proteins. Immunoblotting of crude cell lysates revealed that the 107-kDa polypeptide appeared first, and then the 73-kDa polypeptide appeared along growth phase. It suggested that proteolytic conversion of the 107-kDa polypeptide occurs to form the 73- and 31-kDa polypeptides and leads to formation of isoforms of the enzyme.
Yoshihisa
T
T
Department of Biology, Faculty of Science, University of Tokyo, Japan.
Ohsumi
Y
Y
Anraku
Y
Y
D002851:Chromatography, High Pressure Liquid; D004591:Electrophoresis, Polyacrylamide Gel; D007163:Immunosorbent Techniques; D007425:Intracellular Membranes / Q000201:enzymology; D008361:Mannosidases / Q000302:isolation & purification*; D008970:Molecular Weight; D009940:Organoids / Q000201:enzymology*; D012441:Saccharomyces cerevisiae / Q000166:cytology / Q000201:enzymology*; D012995:Solubility; D014617:Vacuoles / Q000201:enzymology*; D043323:alpha-Mannosidase
D008361:Mannosidases; D043323:alpha-Mannosidase