Photoaffinity labeling of uncoupler binding sites on mitochondrial membrane.
3H 2-azido-4-nitrophenol, a photoactive uncoupler, has been synthesized, and its uncoupling action on oxidative phosphorylation and its binding to the mitochondrial membrane have been studied. The uncoupler bound covalently to the mitochondrial membrane on photoirradiation was 3-4 times that bound reversibly in the absence of light. When irradiation was carried out in the presence of serum albumin, covalent binding was significantly depressed. The pattern of loss of ATP-Pi exchange activity with increasing amounts of the uncoupler suggests that serum albumin prevents the binding of the uncoupler to the functional sites as well. Polyacrylamide gel electrophoresis of photoaffinity labeled submitochondrial particles in the presence of sodium dodecyl sulfate revealed that a 9000 dalton peptide bound high levels of uncoupler. Other proteins in the molecular weight range of 20,000-40,000 and 55,000 were also labeled. Photolysis in the presence of serum albumin or ATP decreased the covalent binding of the uncoupler to all the proteins, but particularly to the 20,000 dalton component. Soluble ATPase and the mitochondrial proteolipid purified from labeled mitochondria showed the presence of label.
D000255:Adenosine Triphosphate / Q000494:pharmacology; D000818:Animals; D001665:Binding Sites; D002417:Cattle; D066298:In Vitro Techniques; D008027:Light; D008566:Membranes / Q000187:drug effects / Q000378:metabolism; D008928:Mitochondria / Q000187:drug effects / Q000378:metabolism*; D009596:Nitrophenols / Q000494:pharmacology*; D010085:Oxidative Phosphorylation / Q000187:drug effects*; D051381:Rats; D012709:Serum Albumin / Q000494:pharmacology; D014475:Uncoupling Agents / Q000494:pharmacology*
D009596:Nitrophenols; D012709:Serum Albumin; D014475:Uncoupling Agents; D000255:Adenosine Triphosphate