Skip to main content
NCBI home page
Applied and Environmental Microbiology logoLink to Applied and Environmental Microbiology
. 1978 Dec;36(6):851–856. doi: 10.1128/aem.36.6.851-856.1978

Isolation and identification of the cytoplasmic membrane from Saccharomyces carlsbergensis by radioactive labeling.

M J Lewis, P C Patel
PMCID: PMC243157  PMID: 153726

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.

Full text

PDF
851

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. BROWN A. D. THE PERIPHERAL STRUCTURES OF GRAM-NEGATIVE BACTERIA.IV. THE CATION-SENSITIVE DISSOLUTION OF THE CELL MEMBRANE OF THE HALOPHILIC BACTERIUM, HALOBACTERIUM HALOBIUM. Biochim Biophys Acta. 1963 Nov 29;75:425–435. doi: 10.1016/0006-3002(63)90630-9. [DOI] [PubMed] [Google Scholar]
  2. Bretscher M. S. Membrane structure: some general principles. Science. 1973 Aug 17;181(4100):622–629. doi: 10.1126/science.181.4100.622. [DOI] [PubMed] [Google Scholar]
  3. Bretscher M. S. On labelling membranes. Nat New Biol. 1973 Sep 26;245(143):116–117. doi: 10.1038/newbio245116a0. [DOI] [PubMed] [Google Scholar]
  4. DODGE J. T., MITCHELL C., HANAHAN D. J. The preparation and chemical characteristics of hemoglobin-free ghosts of human erythrocytes. Arch Biochem Biophys. 1963 Jan;100:119–130. doi: 10.1016/0003-9861(63)90042-0. [DOI] [PubMed] [Google Scholar]
  5. Engelman D. M., Terry T. M., Morowitz H. J. Characterization of the plasma membrane of Mycoplasma laidlawii. I. Sodium dodecyl sulfate solubilization. Biochim Biophys Acta. 1967 Jul 3;135(3):381–390. doi: 10.1016/0005-2736(67)90028-4. [DOI] [PubMed] [Google Scholar]
  6. King L. E., Jr, Morrison M. Calcium effects on human erythrocyte membrane proteins. Biochim Biophys Acta. 1977 Nov 15;471(1):162–168. doi: 10.1016/0005-2736(77)90404-7. [DOI] [PubMed] [Google Scholar]
  7. LEWIS M. J., PHAFF H. J. RELEASE OF INTROGENOUS SUBSTANCES BY BREWER'S YEAST. 3. SHOCK EXCRETION OF AMINO ACIDS. J Bacteriol. 1964 Jun;87:1389–1396. doi: 10.1128/jb.87.6.1389-1396.1964. [DOI] [PMC free article] [PubMed] [Google Scholar]
  8. LOWRY O. H., ROSEBROUGH N. J., FARR A. L., RANDALL R. J. Protein measurement with the Folin phenol reagent. J Biol Chem. 1951 Nov;193(1):265–275. [PubMed] [Google Scholar]
  9. Longley R. P., Rose A. H., Knights B. A. Composition of the protoplast membrane from Saccharomyces cerevisiae. Biochem J. 1968 Jul;108(3):401–412. doi: 10.1042/bj1080401. [DOI] [PMC free article] [PubMed] [Google Scholar]
  10. Maddy A. H., Malcolm B. R. Protein conformations in the plasma membrane. Science. 1965 Dec 17;150(3703):1616–1618. doi: 10.1126/science.150.3703.1616. [DOI] [PubMed] [Google Scholar]
  11. Marriott M. S. Isolation and chemical characterization of plasma membranes from the yeast and mycelial forms of Candida albicans. J Gen Microbiol. 1975 Jan;86(1):115–132. doi: 10.1099/00221287-86-1-115. [DOI] [PubMed] [Google Scholar]
  12. Marriott M. S. Mannan-protein location and biosynthesis in plasma membranes from the yeast form of Candida albicans. J Gen Microbiol. 1977 Nov;103(1):51–59. doi: 10.1099/00221287-103-1-51. [DOI] [PubMed] [Google Scholar]
  13. Mendoza C. G., Villanueva J. R. Preparation and composition of the protoplast membrane of Candida utilis. Biochim Biophys Acta. 1967 May 2;135(2):189–195. doi: 10.1016/0005-2736(67)90113-7. [DOI] [PubMed] [Google Scholar]
  14. Nurminen T., Taskinen L., Suomalainen H. Distribution of membranes, especially of plasma-membrane fragments, during zonal centrifugations of homogenates from glucose-repressed Saccharomyces Cerevisiae. Biochem J. 1976 Mar 15;154(3):751–763. doi: 10.1042/bj1540751. [DOI] [PMC free article] [PubMed] [Google Scholar]
  15. Nurminen T., Taskinen L., Suomalainen H. Distribution of plasma-membrane fragments during zonal centrifugations of homogenates from aerobic Saccharomyces cerevisiae. J Gen Microbiol. 1977 Jan;98(1):301–304. doi: 10.1099/00221287-98-1-301. [DOI] [PubMed] [Google Scholar]
  16. Schibeci A., Rattray J. B., Kidby D. K. Isolation and identification of yeast plasma membrane. Biochim Biophys Acta. 1973 Jun 7;311(1):15–25. doi: 10.1016/0005-2736(73)90250-2. [DOI] [PubMed] [Google Scholar]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)

RESOURCES