Lysine 268 adjacent to transmembrane helix 5 of hamster P‐glycoprotein is the major photobinding site of iodomycin in CHO B30 cells

P‐glycoprotein (Pgp) detoxifies cells by exporting hundreds of chemically dissimilar hydrophobic and amphipathic compounds and is implicated in multidrug resistance (MDR) in the treatment of cancers. Photoaffinity labeling of plasma membrane vesicles of MDR CHO B30 cells with the anthracycline [(125)I]‐iodomycin, subsequent sequential cleavage with BNPS‐skatol and endoproteinase Lys‐C, and the Edman sequencing of the purified photoaffinity‐labeled peptide identified the lysine residue at position 268 in the hamster Pgp primary sequence as the major photobinding site of iodomycin in CHO B30 cells. Lysine 268 is located adjacent to the cytosolic terminus of transmembrane 5. According to thermodynamic and kinetic analyses, this location should present the equilibrium binding site of ATP‐free Pgp for daunomycin and iodomycin in B30 cells.