THE ISOLATION AND CHARACTERIZATION OF A PLASMA MEMBRANE AND A MYELIN FRACTION DERIVED FROM OLIGODENDROGLIA OF CALF BRAIN

Abstract

—A method is described for the fractionation of bulk isolated oligodendroglial cells from calf brain to produce both a plasma membrane and an attached myelin fraction. The cells are homogenized in a sucrose solution containing Mg2+ and K+ at a pH of 6·5. Crude membrane fractions are obtained from this homogenate by discontinuous sucrose density gradient centrifugation. After being subjected to osmotic shock, these fractions are purified by continuous sucrose density gradient centrifugation. The plasma membrane fraction, which bands at 1·0 m-sucrose, was identified by its morphology and enzyme content. Electron microscopy showed it to be a homogeneous preparation of vesicles composed, for the most part, of smooth trilaminar membranes. Enzymatic analysis revealed the presence of high specific activities of Na+, K+-ATPase, 5′-nucleotidase and 2′,3′-cyclic AMPase. Lipid analysis showed a higher galactolipid and lower phospholipid content than has been reported for neuronal and synaptic membranes. The attached myelin fraction, which bands at 0·7 m-sucrose has the typical multilamellar appearance of myelin, but differs considerably from normal myelin in having high concentrations of plasma membrane marker enzymes, and a lipid composition intermediate between normal myelin and the plasma membrane fraction. The ganglioside content and protein patterns of these fractions have also been examined.