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Plasma membrane of cultured oligodendrocytes: I. Isolation, purification, and initial c...
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Plasma membrane of cultured oligodendrocytes: I. Isolation, purification, and initial characterization
作者:
Paul E. Polak,
Sara Szuchet,
期刊:
Glia
(WILEY Available online 1988)
卷期:
Volume 1,
issue 1
页码: 39-53
ISSN:0894-1491
年代: 1988
DOI:10.1002/glia.440010106
出版商: Wiley Subscription Services, Inc., A Wiley Company
关键词: Subcellular fractionation;Enzyme markers;2′,3′ Cyclic nucleotide phosphodiesterase;Glial cells
数据来源: WILEY
摘要:
AbstractOligodendrocytes generate myelin as extensions of the plasma membrane. Myelin has been well characterized, yet little is known concerning oligodendrocyte plasma membrane. We have developed a reproducible method for the isolation of an oligodendrocyte plasma membrane‐rich fraction (F2.2). F2.2 has a 25‐fold enrichment in K+‐dependent p‐nitrophenyl phosphatase, a plasma membrane marker. Impurities are composed of Golgi elements (8–12%), microsomes (4–6%), and lysosomal membranes (1–5%). Our starting material was oligodendrocytes kept in culture in a nonattached state for 3 to 5 d. After disrupting the cells and removing nuclei (P1), the supernatant (SP1) was fractionated on a self‐generating 20% Percoll gradient into three bands: F1, F2, and F3. F1 had only 3% of the applied protein and was not characterized. F2, with 11% of the protein, was fivefold enriched in plasma membrane. F3 had 27% of initial protein; it consisted of a crude mitochondrial and lysosomal fraction. F2 was further purified by first washing it hypotonically, treating it with Mg2+, and then fractionating it on a Ficoll step gradient that yielded F2.2 at the interphase. Morphologically F2.2 comprises (1) membranous sheets, often with more than one membrane in close apposition; (2) membrane vesicles of various sizes and shapes frequently filled with amorphous material; (3) Golgi elements; and (4) unrecognizable profiles. The sodium dodecyl sulfate‐polyacrylamide gel electrophoresis protein profile of F2.2 reveals CNPase as a major component in agreement with the high CNPase specific activity (3,860 μmol/mgP/h) found in F2.2 Other significant polypeptides have Mr= 170,000, 135,000, 108,000, 80,000, 53,000, 38,500,
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