Isolation of Purified Plasma Membranes from Cultured Cells and Hepatomas by Two-Phase Partition and Preparative Free-Flow Electrophoresis

Abstract

This report describes and documents the isolation of plasma membranes from hepatomas and tissue culture cells by aqueous two-phase partition. The method used previously for normal liver was effective, rapid, and reproducible. Preparations from both cells and hepatomas were more than 90% plasma membrane-derived based on electron microscope morphometry and assays of marker enzyme activities. Relative enrichments over starting homogenates were near theoretical as determined by electron microscope morphometry of starting cells and tissues. Recoveries were about 10% or greater. Briefly, the membranes to be separated were mixed with a combination of two different polymers that themselves separated into two phases. For tissue culture cells and hepatomas, a mixture of 6.6% (w/w) dextran and 6.6% (w/w) polyethylene glycol containing 0.25 m sucrose and 5 mm potassium phosphate (pH 7.2) was used. A potassium-stimulated, ouabain-inhibited, p-nitrophenylphosphatase was employed as a plasma membrane marker to monitor yield and recovery. In combination with free-flow electrophoresis, the preparations of plasma membranes from cultured cells were resolved further into fractions enriched in vesicles of right side-out or inside-out orientations. Unlike centrifugation methods, the same type of two-phase separations provided useful plasma membrane fractions when applied to different types of cultured cells as well as to solid tumors and normal tissues. The lower phase membranes, after two-phase partition, provided a plasma membrane-depleted source of membranes other than plasma membrane for use as reference fractions. The procedures should find wide application to problems of cancer research where facile and decisive separations of surface and internal membranes may be required.