1887

Abstract

SUMMARY

A method was developed for the fractionation of homogenates in order to obtain purified cytoplasmic membranes. The fractions obtained were characterized by studying their ultrastructure and enzymic properties. Since ATPase is an enzyme present in the cytoplasmic membrane, we studied the levels of this activity in our fractions as an indication of the presence of cytoplasmic membrane fragments. Since this activity is also present in mitochondria, we determined the levels of fumarase, a mitochondrial enzyme, and took the ratio of the two activities as an index of the purity of the membrane preparations.

The sediment obtained by centrifuging the cell-free extract at 40000 was fractionated in a discontinuous sucrose gradient. This led to three types of fractions. The most dense had a high ATPase/fumarase ratio and its ultrastructure showed that it contained membrane fragments having a triple-layered structure.

We concluded that this fraction was rich in cytoplasmic membrane fragments; it was clearly distinguishable from the intermediate and less dense fractions. These latter fractions had a much lower ATPase/fumarase ratio and, judging by their ultrastructure, they were respectively a mitochondrial fraction and a fraction consisting of vesicles probably related to the endoplasmic reticulum. Analysis of the cytoplasmic membrane-rich fraction showed that it consisted of protein, lipid and 30 % carbohydrate

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/content/journal/micro/10.1099/00221287-81-1-247
1974-03-01
2022-12-01
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References

  1. Boulton A. A. 1965; Some observations on the chemistry and morphology of the membranes released from yeast protoplasts by osmotic shock. Experimental Cell Research 37:343–359
    [Google Scholar]
  2. Fisre C. H., Subbarow Y. 1925; The colorimetric determination of phosphorus. Journal of Biological Chemistry 66:375–400
    [Google Scholar]
  3. Folch J., Lees M., Sloane-Stanley G. H. 1957; A simple method for the isolation and purification of total lipids from animal tissues. Journal of Biological Chemistry 226:497–509
    [Google Scholar]
  4. Garcëa-Acha I., Villanueva J. R. 1963; The use of Streptomyces enzyme in preparation OF protoplasts from mold spores. Canadian Journal of Microbiology 9:139–140
    [Google Scholar]
  5. Garcëa-Acha I., Aguirre M. J. R., Lopez-Belmonte F., Uruburu F., Villanueva J. R. 1966; Isolation of cytoplasmic membranes from Fusarium culmorum protoplasts. Transactions of the British Mycological Society 49:603–609
    [Google Scholar]
  6. Garcëa Mendoza C., Villanueva J. R. 1967; Preparation and composition of the protoplast membrane of Candida utilis. Biochimica et biophysica acta 135:189–195
    [Google Scholar]
  7. Hill R. L., Bradshaw R. A. 1969; Fumarase. In Methods in Enzymology 13 pp. 91–99 Lowenstein J. M. Edited by New York and London: Academic Press;
    [Google Scholar]
  8. Layne E. 1957; Spectrophotometric and turbidimetric methods for measuring proteins. In Methods in Enzymology 3 pp. 447–454 Colowick S. P., Kaplan N. O. Edited by New York and London: Academic Press;
    [Google Scholar]
  9. Longley R. P., Rose A. H., Knights B. A. 1968; Composition of the protoplast membrane from Saccharomyces cerevisiae. Biochemical Journal 108:401–412
    [Google Scholar]
  10. Lowry O. H., Rosebrough N. J., Farr A. L., Randall R. J. 1951; Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193:265–275
    [Google Scholar]
  11. Matile P. 1970; Properties of the purified yeast plasmalemma. In Membranes: Structure and Function, FEBS Symposium 20 p. 39 Villanueva J. R., Ponz F. Edited by New York and London: Academic Press;
    [Google Scholar]
  12. Matile P., Moor H., Mühlethaler K. 1957; Isolation and properties of the plasmalemma in yeast. Archiv für Mikrobiologie 58:201–211
    [Google Scholar]
  13. Schwencke J., Krsulovic J., Rojas M., Farias G. 1972; The enzymatic composition and electron microscopy of a purified vesicles preparation from yeast plasmalemma. Abstracts of the Third International Symposium on Yeast Protoplasts p. 90 Spain: Salamanca;
    [Google Scholar]
  14. Trevelyan W. E., Harrison J. S. 1952; Studies on yeast metabolism. I. Fractionation and microdetermination of cell carbohydrates. Biochemical Journal 50:298–303
    [Google Scholar]
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