ATPase activity of purified multidrug resistance-associated protein.
Human multidrug resistance protein (MRP) was expressed at high levels in stably transfected baby hamster kidney (BHK-21) cells. These cells exhibited a pattern of cross-resistance to several different drugs typical of an MRP-mediated phenotype despite the addition of 10 histidine residues at the C terminus to facilitate purification. Consistent with this functional evidence of the presence of MRP at the surface of these transfectants, strong signals were detected by immunoblotting and immunofluorescence using a specific monoclonal antibody to MRP. There was intense uniform staining of the cell surface as well as weaker staining of intracellular membranes. MRP-containing membranes were solubilized in 1% N-dodecyl-beta-D-maltoside in the presence of 0.4% sheep brain phospholipids. Two sequential affinity purification steps on Ni-NTA agarose and wheat germ agglutinin agarose provided substantial enrichment, and contaminating bands were not detected. ATPase activity of the purified protein was assayed in the presence of the phospholipids, which had been maintained throughout all purification steps. ATP was hydrolyzed in proportion to the amount of purified protein assayed, and typical Michaelis-Menten behavior was exhibited, yielding estimations of Km of approximately 3.0 mM and Vmax of 0.46 micromol mg-1 min-1. This activity was moderately stimulated by the drugs that others have shown to be transported by MRP-containing membrane vesicles. This stimulation was enhanced by reduced glutathione as is its drug transport, and oxidized glutathione, itself a substrate for transport, caused a strong stimulation. These data describe the first purification of MRP and provide the first direct evidence that the molecule possesses drug-stimulated ATPase activity.
Chang
X B
XB
S. C. Johnson Medical Research Center, Mayo Clinic Arizona, Scottsdale, Arizona 85259, USA.
Hou
Y X
YX
Riordan
J R
JR
D018528:ATP-Binding Cassette Transporters / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D003001:Cloning, Molecular; D006224:Cricetinae; D018076:DNA, Complementary / Q000737:chemistry; D018432:Drug Resistance, Multiple; D006367:HeLa Cells; D006801:Humans; D007700:Kinetics; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins / Q000378:metabolism*; D014162:Transfection
D018528:ATP-Binding Cassette Transporters; D000251:Adenosine Triphosphatases; D018076:DNA, Complementary; D027425:ATP-Binding Cassette, Sub-Family C Proteins; D009363:Neoplasm Proteins