Facile preparation of inside-out plasma membrane vesicles from tumor cells for functional studies of pharmacologically relevant translocating ATPases

Abstract

A reasonably facile and effective procedure is described for the preparation of inside-out plasma membrane vesicles from tumor cells. The method incorporates nitrogen cavitation, optimized with respect to the applied N2 pressure, in the absence of added divalent cations followed by differential centrifugation and discontinuous, sucrose gradient centrifugation. With the three tumor cell types utilized, multidrug-resistant (MEL/VCR-6) and parental (MEL/O) murine erythroleukemia cells and methotrexate-resistant (L1210/R24) L1210 leukemia cells, yields were in the range of 8-12 mg of plasma membrane vesicles/1010 cells at a purity of 87- 94% with average inside-out sidedness among preparations varying from 65 to 93% depending upon the cell type. Inside-out plasma membrane vesicles so derived were capable of sustaining ATP-dependent transport inward of two common anti-tumor cytotoxic agents, vinblastine and methotrexate. The former was demonstrated with inside-out vesicles from only P-glycoprotein- overexpressing, multidrug-resistant MEL/VCR-6 cells, while the latter was readily demonstrated in inside-out vesicles from all three cell types. © 1995 Academic Press, Inc.