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Plasma membrane of cultured oligodendrocytes: I. Isolation, purification, and initial characterization
Abstract:
Oligodendrocytes 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 mumol/mgP/h) found in F2.2. Other significant polypeptides have Mr = 170,000, 135,000, 108,000, 80,000, 53,000, 38,500, 32,000, and 22,200.
Insights
Researchers developed a new method to isolate oligodendrocyte plasma membranes, a crucial component of myelin. This technique yields a purified fraction enriched in plasma membrane markers, aiding the study of these vital cells.
Area of Science:
- Neuroscience
- Cell Biology
- Biochemistry
Background:
- Oligodendrocytes are glial cells responsible for producing myelin sheaths around axons in the central nervous system.
- While myelin composition is well-studied, the oligodendrocyte plasma membrane itself remains poorly characterized.
- Understanding the oligodendrocyte plasma membrane is crucial for insights into myelin formation and CNS health.
Purpose of the Study:
- To develop a reproducible method for isolating a plasma membrane-rich fraction from oligodendrocytes.
- To characterize the biochemical and morphological properties of the isolated oligodendrocyte plasma membrane fraction.
Main Methods:
- Oligodendrocytes were cultured in a non-attached state for 3-5 days.
- Cells were disrupted, and nuclei were removed to obtain a supernatant (SP1).
- SP1 was fractionated using a self-generating 20% Percoll gradient, yielding fractions F1, F2, and F3.
- Fraction F2 was further purified through hypotonic washing, Mg2+ treatment, and a Ficoll step gradient to obtain the final F2.2 fraction.
Main Results:
- A reproducible method yielded an oligodendrocyte plasma membrane-rich fraction (F2.2).
- F2.2 showed a 25-fold enrichment in K+-dependent p-nitrophenyl phosphatase, a key plasma membrane marker.
- SDS-PAGE revealed CNPase as a major component, with other significant polypeptides identified.
- Morphological analysis of F2.2 showed membranous sheets, vesicles, and Golgi elements, with minimal contamination from microsomes and lysosomal membranes.
Conclusions:
- The developed method successfully isolates a highly enriched oligodendrocyte plasma membrane fraction (F2.2).
- This purified fraction serves as a valuable resource for studying oligodendrocyte plasma membrane composition and function.
- The characterization of F2.2 provides a foundation for future investigations into oligodendrocyte biology and CNS disorders.