FIGURE 2.

Cross-linking of the NBDs with M4M highly stimulates ATPase activity. A, membranes prepared from HEK 293 cells expressing the NBD1/NBD2 mutant P517C/I1050C were treated without (Control) or with cross-linkers M4M or M17M. The reactions were stopped by addition of SDS sample buffer containing no thiol-reducing agent and samples subjected to immunoblot analysis. The positions of the cross-linked (X-link) and mature (170 kDa) P-gps are indicated. B, membranes expressing mutant P517C/I1050C or Cys-less P-gp were treated with or without (None) cross-linker and histidine-tagged P-gp isolated by nickel-chelate chromatography. The isolated P-gps were mixed with lipid and ATPase activities were measured in the absence (−) or presence (+) of 0.3 mm verapamil. In some cases the samples were treated with 10 mm dithiothreitol (+DTT) before assay. Each value is the mean ± S.D. (n = 3).