Isolation of Cell Surface Membranes from Cultured C6 Glioblastoma Cells

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Abstract
Plasma membranes were isolated from C6 [rat] glioblastoma cells by 2 methods. In the 1st method, cells were treated with concanavalin A and lysed in hypotonic medium. After partial separation of plasma membranes from other cell material, the lectin was displaced with .alpha.-methyl-D-mannoside. In the 2nd method, untreated cells or cells iodinated in a lactoperoxidase-catalyzed reaction were homogenized in isotonic medium. Membrane fractions obtained by either homogenization procedure were further purified by rate zonal and equilibrium centrifugations into linear density gradients. Disruption of the glioblastoma cell membrane gives rise to heterogeneous assemblies of membrane fragments. Two populations of plasma membranes were isolated from untreated and from iodinated cells: a lighter membrane fraction characterized by relatively lower sedimentation velocity and buoyant density, and a heavier membrane fraction of relatively faster sedimentation velocity and higher buoyant density. Both fractions showed electrophoretic patterns similar to those of 125I-labeled cell surface proteins. Their specific (Na+ + K+)-ATPAse activity was 7- to 8-fold the homogenate activity (recovery, 13.1%). Both fractions were still contaminated by smooth endoplasmic reticulum, as judged from the activity of NADPH-dependent cytochrome C reductase (recovery, 2.4%). Plasma membrane fragments present in the 2 fractions might differ in the organization of their structures, e.g., membrane vesicle intactness and membrane orientation.