title: Isolation and identification of the cytoplasmic membrane from Saccharomyces carlsbergensis by radioactive labeling.
abstract: The cytoplasmic membrane of Saccharomyces carlsbergensis was isolated by enzymatic digestion of the yeast cell wall, followed by lysis of the protoplasts and fractionation by ultracentrifugation in a discontinuous sucrose density gradient. Location of the cytoplasmic membrane fraction on the sucrose gradient was made by labeling intact protoplasts with [G-3H]dansyl chloride, and was settled at the 50% (wt/vol) sucrose gradient (d = 1.186 g/cm3). Approximately 80% of the radioactivity was found in the membrane fraction prepared in the presence of Mg2+ ions. However, when protease inhibitors were used in the preparation step, the membrane fraction contained over 90% of the total radioactivity. The presence of Mg2+ ions during membrane isolation and purification enhanced the aggregation of membrane components but, at higher concentrations, as well as in the prolonged presence of Mg2+ ions in the membrane suspension, it caused the breakdown of membrane components. The membrane preparation contained Mg2+-adenosine triphosphatase, which was insensitive to oligomycin and ouabain. The distribution of Mg2+-adenosine triphosphatase in different fractions during sucrose gradient is reported.
mesh_terms: D000251:Adenosine Triphosphatases / Q000032:analysis; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D002499:Centrifugation, Density Gradient; D007553:Isotope Labeling; D008274:Magnesium / Q000494:pharmacology; D008565:Membrane Proteins / Q000378:metabolism; D012440:Saccharomyces / Q000648:ultrastructure*
chemical_list: D008565:Membrane Proteins; D000251:Adenosine Triphosphatases; D008274:Magnesium