Abstract
The plasma membrane from Saccharomyces cerevisiae X2180-1A and a secretion-blocked mutant, secl (P. Novick and R. Schekman, Proc. Natl. Acad. Sci. U.S.A. 76:1858-1862, 1979) has been purified. Cell walls were digested by treatment with lyticase followed by concanavalin A coating of spheroplasts. alpha-Methylmannoside treatment after lysis, sonication at high salt concentration, and fractionation on a Renografin gradient resulted in two highly purified membrane fractions sedimenting at densities of 1.15 and 1.17 g/cm3. Yields determined by recovery of vanadate-sensitive ATPase activity were 11 to 18%, and those determined by recovery of the spheroplast surface label 125I were 17 to 29%. Iodinated cells have most of their label in sedimentable, nonspheroplast material. However, both membrane populations contain some 125I surface label and show ATPase activity with pH optima only at 5.5. The apparent Vmax of the plasma membrane ATPase equals 360 to 560 nmol of ATP hydrolyzed per min per mg of protein, with a Km for ATP of 0.7 mM. ATPase specific activity is not decreased in mutant plasma membrane. Analysis of 125I-labeled plasma membrane proteins by two-dimensional gel electrophoresis revealed seven major proteins on the plasma membrane surface.
Publisher
American Society for Microbiology
Subject
Molecular Biology,Microbiology
Reference47 articles.
1. Two-dimensional gel electrophoresis of membrane proteins;Ames G. F.;Biochemistry,1976
2. Colorimetric analysis of sugars;Ashwell C.;Methods Enzymol.,1957
3. Structure and biosynthesis of the mannan component of the yeast cell envelope;Ballou C.;Adv. Microb. Physiol.,1976
4. Inhibition of red cell NaK-ATPase by vanadium: kinetics of reversal by catecholamines;Bond G. H.;Fed. Proc.,1978
5. Characterization of plasma membrane adenosine triphosphatase of Neurospora crassa;Bowman B. J.;J. Biol. Chem.,1977
Cited by
24 articles.
订阅此论文施引文献
订阅此论文施引文献,注册后可以免费订阅5篇论文的施引文献,订阅后可以查看论文全部施引文献