Specific labeling of beef heart mitochondrial ADP/ATP carrier with N-(3-iodo-4-azidophenylpropionamido)cysteinyl- 5-(2'-thiopyridyl)cysteine-coenzyme A (ACT-CoA), a newly synthesized 125I-coenzyme A derivative photolabel.

An azido-125I-CoA photolabel was synthesized from N-(3-iodo-[125I]4-azidophenylpropionamido)cysteinyl- 5-(2'thiopyridyl) cysteine ([125I]ACTP) and CoASH, separated by chromatography on a silica gel TLC, and identified by autoradiography. Synthesis of [125I]ACT-CoA from [125I]ACTP and CoA was further confirmed by monitoring the release of one of the products, thiopyridone, at 343 nm and concurrent formation of [125I] ACT-CoA. Beef heart mitochondria were incubated in the presence of the 125I-CoA derivative with or without specific inhibitors and/or substrates of the ADP/ATP carrier, and immediately photolyzed for 5 s. Sodium dodecyl sulfate-gel electrophoresis and autoradiography of the separated proteins revealed exclusive photolabeling of a 30-kDa protein in the absence of inhibitors of the carrier. This specific photolabeling of the 30-kDa protein was prevented in a concentration-dependent manner by either carboxyatractylate or palmitoyl-CoA (0.1-5 microM), two known inhibitors of the ADP/ATP carrier. ADP reduced the extent of photolabeling of the 30-kDa protein, but palmitic acid, free CoASH, and dinitrophenol were ineffective, indicating the specificity of the reaction. CoA photolabels may be useful in probing ligand and/or substrate binding sites and in determining the structure-function relationship of the ADP/ATP carrier.