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.
Ruoho
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Department of Pharmacology, University of Wisconsin, Madison 53706.
Woldegiorgis
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Kobayashi
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Shrago
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E
D000214:Acyl Coenzyme A / Q000138:chemical synthesis* / Q000494:pharmacology; D000345:Affinity Labels; D000818:Animals; D001278:Atractyloside / Q000031:analogs & derivatives / Q000494:pharmacology; D001386:Azides / Q000138:chemical synthesis* / Q000494:pharmacology; D002417:Cattle; D003065:Coenzyme A / Q000031:analogs & derivatives*; D066298:In Vitro Techniques; D008929:Mitochondria, Heart; D000226:Mitochondrial ADP, ATP Translocases / Q000037:antagonists & inhibitors*; D009713:Nucleotidyltransferases / Q000037:antagonists & inhibitors*; D010777:Photochemistry
D000214:Acyl Coenzyme A; D000345:Affinity Labels; D001386:Azides; C058676:N-(3-iodo-4-azidophenylpropionamido)cysteinyl-5-(2'-thiopyridyl)cysteine-coenzyme A; D001278:Atractyloside; D000226:Mitochondrial ADP, ATP Translocases; D009713:Nucleotidyltransferases; D003065:Coenzyme A; C003853:carboxyatractyloside