Figure 2. Reaction and ATPase activity of mutant L65C treated with MTS-verapamil.
(A) Schematic reaction of a thiol group with MTS-verapamil and attachment of verapamil to the protein via a disulphide bond. (B) His-tagged Cys-less or mutants M51C–V71C P-gps were expressed in HEK-293 cells and solubilized with n-dodecyl-β-D-maltoside. Insoluble material was removed by centrifugation and the supernatants were treated with or without 1 mM MTS-verapamil. His-tagged P-gps were then isolated by nickel-chelate chromatography. Equivalent amounts of P-gp were mixed with lipid, sonicated and assayed for ATPase activity in the absence of drug substrate. The fold stimulation is the ratio of activity of a sample treated with MTS-verapamil to that in an untreated sample. Each value is the average of three different experiments. ND, not determined because of low expression. (C) His-tagged mutant L65C expressed in HEK-293 cells was solubilized with n-dodecyl-β-D-maltoside, treated with various concentrations of MTS-verapamil and isolated by nickel-chelate chromatography. The ATPase activities of the isolated protein were determined as described above.