ADENOSINE TRIPHOSPHATE-DEPENDENT TRANSPORT OF DOXORUBICIN, DAUNOMYCIN, AND VINBLASTINE IN HUMAN TISSUES BY A MECHANISM DISTINCT FROM THE P-GLYCOPROTEIN

Previous studies have demonstrated that a human glutathione conjugate transporter, designated as dinitrophenyl-S-glutathione ATPase(DNP-SG A TPase), catalyzed ATP hydrolysis in the presence of several amphiphili c compounds other than glutathione conjugates (Singhal, S. S., R. Shar ma, S. Gupta, H. Ahmad, P. Zimniak, A. Radominska, R. Lester, and Y. C . Awasthi. 1991. FEBS[Fed. Eur. Biochem. Soc] Lett. 281:255-257). We n ow demonstrate that DNP-SG ATPase purified from human lung and erythro cyte membranes catalyzed the hydrolysis of ATP in the presence of doxo rubicin and its metabolites. Doxorubicin-stimulated ATP hydrolysis by DNP-SG ATPase was saturable with respect to doxorubicin (K-m 1.2 and 2 .8 mu M for the lung and erythrocyte enzymes, respectively). Antibodie s against DNP-SG ATPase immunoprecipitated the ATP hydrolyzing activit y stimulated by doxorubicin, its metabolites, and glutathione conjugat es. Inside out vesicles prepared from erythrocyte membranes took up do xorubicin, daunomycin, and vinblastine in an ATP-dependent manner. The uptake was linear with respect to time and vesicle protein, was depen dent on ATP and magnesium, was inhibited by heavy metal salts or by he ating the vesicles, and was sensitive to both osmolarity and orientati on of the vesicles. The transport had an activation energy of 13 kcal/ mol, was saturable with respect to both doxorubicin and ATP (K-m value s of 1.8 mu M and 1.9 mM, respectively), and was competitively inhibit ed by glutathione conjugates as well as by a number of amphiphiles suc h as daunomycin or vinblastine. Transport was diminished upon coating the vesicles with antibodies against DNP-SG ATPase. Incorporation of i ncreasing amounts of purified DNP-SG ATPase into the vesicles resulted in a linear increase in transport of doxorubicin. These studies demon strated for the first time that a membrane protein that catalyzed the transport of anionic amphiphilic molecules such as glutathione conjuga tes could also mediate the transport of weakly cationic antitumor anti biotic, doxorubicin. Notably, the K-m of transport was in the range of doxorubicin concentration achievable in human serum after intravenous dosing of doxorubicin.