FACILE PREPARATION OF INSIDE-OUT PLASMA-MEMBRANE VESICLES FROM TUMOR-CELLS FOR FUNCTIONAL-STUDIES OF PHARMACOLOGICALLY RELEVANT TRANSLOCATING ATPASES

A reasonably facile and effective procedure is described for the prepa ration of inside-out plasma membrane vesicles from tumor cells. The me thod incorporates nitrogen cavitation, optimized with respect to the a pplied N-2 pressure, in the absence of added divalent cations followed by differential centrifugation and discontinuous, sucrose gradient ce ntrifugation. With the three tumor cell types utilized, multidrug-resi stant (MEL/VCR-6) and parental (MEL/O) murine erythroleukemia cells an d methotrexate-resistant (L1210/R24) L1210 leukemia cells, yields were in the range of 8-12 mg of plasma membrane vesicles/10(10) 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 plasm a membrane vesicles so derived were capable of sustaining ATP-dependen t transport inward of two common antitumor cytotoxic agents, vinblasti ne and methotrexate. The former was demonstrated with inside-out vesic les from only P-glycoprotein-overexpressing, multidrug-resistant MEL/V CR-6 cells, while the latter was readily demonstrated in inside-out ve sicles from all three cell types. (C) 1995 Academic Press, Inc.