ISSN:
1432-1424
Source:
Springer Online Journal Archives 1860-2000
Topics:
Biology
,
Chemistry and Pharmacology
Notes:
Summary Plasma membranes have been isolated on a large scale from cultured human skin fibroblasts by methods which avoid treatment with either proteases or heavy metals. Cells (0.5–1.0×109) were harvested from roller, bottles and disrupted by manual agitation in hypotonic medium. A plasma membrane-enriched fraction (10–15 mg protein) was then obtained by conventional differential and density gradient centrifugation. Fractionation was monitored by phase-contrast and electron microscopy, measurement of activities of marker enzymes and recovery of125I wheat germ agglutinin which was bound to the cell surface prior to disruption. The membranes obtained were mainly vesicular (0.1–2 μm diameter) and banded at a sucrose density of 1.12. 5′-Nucleotidase was purified approximately 20-fold with respect to the cell homogenate and nearly 50% of this enzyme was recovered in the plasma membrane-enriched fraction. Activities of Mg2+-ATPase, (Na++K+)Mg2+-ATPase and prostaglandin E, and fluoride-stimulated adenylate cyclases were also maximal in this fraction. Recovery and specific activity of125I wheat germ agglutinin bound to the cell surface was greatest in these membranes. The only contaminating structures identifiable in electron micrographs resembled the flat cisternal plates of the Golgi apparatus. The detection of sialyl and galactosyl transferase activities was consistent with the presence of these structures. Some further subfractionation of the various markers in the preparation was possible. A similar plasma membrane-enriched fraction also was obtained by a second method in which cells were harvested by scraping from the roller bottles and disrupted by homogenization in isotonic media.
Type of Medium:
Electronic Resource
URL:
http://dx.doi.org/10.1007/BF01868151
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