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2-Azido-4nitrophenol (NPA) is a potent, water-soluble uncoupler of oxidative phosphorylation.It is photochemically active and capable of covalent labeling of the mitochondrial components presumably concerned with the uncoupling process.Acrylamide gel electrophoresis of NPAlabeled mitochondria digested with sodium dodecyl sulfate and mercaptoethanol showed that 40% of NPA radioactivity was associated with protein bands in the molecular weight region of 20,000 to 30,000.Equilibrium binding studies with NPA, under nonphotolytic conditions, have shown that bovine heart mitochondria contain 0.56 f 0.13 nmole of a uniformly reacting uncoupler binding site per mg of protein.The binding of NPA to these sites is competitively inhibited by other uncouplers, but not by antimycin A, mtamycin, valinomycin, or arsenate.The uncouplers tested against NPA were sodium azide, 2,4dinitrophenol, pentachlorophenol, carbonylcyanide m-chlorophenylhydrazone, and 5chloro -3 -t -butyl -2' -chloro -4' -nitrosalicylanilide.The uncoupler binding sites are located in the inner membrane, and do not appear to involve F1 (ATPase).They are unaffected with respect to NPA binding by changes in the respiratory state or the energization state of mitochondria.Data regarding dissociation constants and the uncoupler potencies of NPA, 2,4-dinitrophenol, and azide have suggested that the uncoupler binding sites of mitochondria are functionally involved in the act of uncoupling.The mechanism of oxidative phosphorylation has been studied through intensive and highly imaginative approaches focused on the electron transport system and the terminal steps of ADP phosphorylation (l-5).A third approach, which might yield more crucial information regarding the details of energy coupling, is the study of the mechanism of uncoupling and the mitochon-
Hanstein et al. (Fri,) studied this question.