Expression of the human multidrug resistance cDNA in insect cells generates a high activity drug-stimulated membrane ATPase.
Drug-resistant tumor cells actively extrude a variety of chemotherapeutic agents by the action of the multi-drug resistance (MDR1) gene product, the plasma membrane P-glycoprotein. In this report we show that the expression of the human MDR1 gene in cultured Sf9 insect cells via a baculovirus vector generates a high activity vanadate-sensitive membrane ATPase. This ATPase is markedly stimulated by drugs known to interact with the P-glycoprotein, such as vinblastine and verapamil, and the ability of the various drugs to stimulate the ATPase corresponds to their previously observed affinity for this transporter. The drug-stimulated ATPase is not present in uninfected or mock-infected Sf9 cells, and its appearance correlates with the appearance of the MDR1 gene product detected with a monoclonal anti-MDR protein antibody and by labeling with 8-azido-ATP. The drug-induced ATPase requires magnesium ions, does not utilize ADP or AMP as substrates, exhibits a half-maximal activation at about 0.5 mM MgATP, and its maximal activity (about 3-5 mumol/mg MDR protein/min) approaches that of the well characterized ion transport ATPases. These results provide the first direct demonstration of a high capacity drug-stimulated ATPase activity of the human multidrug resistance protein and offer a new and simple assay for the investigation of functional interactions of various drugs with this clinically important enzyme.
Sarkadi
B
B
National Institute of Hematology and Blood Transfusion, Budapest, Hungary.
Price
E M
EM
Boucher
R C
RC
Germann
U A
UA
Scarborough
G A
GA
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000255:Adenosine Triphosphate / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D000818:Animals; D001345:Autoradiography; D001386:Azides / Q000737:chemistry; D016367:Baculoviridae / Q000235:genetics; D015153:Blotting, Western; D002460:Cell Line; D004247:DNA / Q000235:genetics*; D004789:Enzyme Activation; D005814:Genes, Viral; D005822:Genetic Vectors; D006801:Humans; D006868:Hydrolysis; D007313:Insecta / Q000166:cytology; D008562:Membrane Glycoproteins / Q000235:genetics*; D008565:Membrane Proteins / Q000378:metabolism*; D008969:Molecular Sequence Data; D013379:Substrate Specificity; D001616:beta-Galactosidase / Q000378:metabolism
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D001386:Azides; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; C017040:8-azidoadenosine 5'-triphosphate; D000255:Adenosine Triphosphate; D004247:DNA; D001616:beta-Galactosidase; D000251:Adenosine Triphosphatases