PHOTOAFFINITY-LABELING OF MITOCHONDRIAL PROTEINS WITH 2-AZIDO [P-32] PALMITOYL COA

A long-chain fatty acyl CoA photolabel, 2-azido [P-32]palmitoyl CoA, w as synthesized and its covalent interaction with mitochondrial membran e proteins examined, On binding of 2-azido [P-32]palmitoyl CoA to beef heart mitochondria, two polypeptides were primarily labeled, the 30 k Da ADP/ATP carrier and a 41 kDa protein of unknown identity, Carboxyat ractyloside and palmitoyl CoA completely protected against labeling of the 30 kDa protein indicating that it was the ADP/ATP carrier. With i nverted submitochondrial particles, only the 30 kDa polypeptide was la beled by 2-azido [P-32]palmitoyl CoA. The labeling was inhibited by bo ngkrekic acid and palmitoyl CoA but not carboxyatractyloside, providin g evidence that the ADP/ATP carrier was covalently bound from the matr ix side of the membrane, In brown adipose tissue mitochondria, 2-azido [P-32]palmitoyl CoA photolabeled the ADP/ATP carrier and the 32 kDa u ncoupling protein with some minor labeling of 36 and 68 kDa polypeptid es, The results indicated that this physiological photolabeling reagen t with the azido group on the CoA portion of the molecule interacts li ke 2-azido ADP with nucleotide binding sites of a number of important enzymes in cell metabolism, Moreover, the evidence strongly supports t he hypothesis that long chain fatty acyl CoA esters are natural ligand s for key nucleotide binding proteins.