The specific aims of this proposal are to synthesize a photoaffinity labeled radioactive acyl CoA to study the mechanism of interaction of long chain acyl CoA esters with the adenine nucleotide translocase (ANT) in the inner mitochondrial membrane after covalent binding of the ligand to specific receptor sites on the carrier. Since acyl CoAs are known effectors of a number of enzymes in cell metabolism, the availability of photoaffinity labeled palmitoyl CoA would also be a very useful probe for investigations of a number of these enzymes. The compound, 8-Azido (2-3H) palmitoyl CoA will be prepared from the critical intermediates including 8N3-3',5'ADP(2-3H) and S-palmitoyl 4' phosphopantetheine. Experiments will be conducted to establish: Inhibition of ANT by the photoaffinity labeled acyl CoA; the binding characteristics, approximate binding and inhibition constants; and the specificity of the binding of the photolabel to the carrier prior to photoirradiation. The photoaffinity labeled radioactive ANT will then be purified by solubilization in Triton X-100 followed by hydroxylapatite chromatography, and the homogeneous 30,000 dalton subunit acyl CoA-protein complex identified by SDS gel electrophoresis. The critical experiments will be to obtain identical gel patterns starting with either isolated mitochondria or inverted submitochondrial particles. Attempts will then be made to identify the sequence of the peptide fragment which binds the photoaffinity labeled palmitoyl CoA. The results obtained from these investigations would provide further evidence to support the specificity of interactions of acyl CoAs with the ADP/ATP carrier, and indicate that they may be the physiological ligands for the carrier and a number of other enzymes.
| Woldegiorgis, G; Lawrence, J; Ruoho, A et al. (1995) Photoaffinity labeling of mitochondrial proteins with 2-azido [32P]palmitoyl CoA. FEBS Lett 364:143-6 |
| Ruoho, A E; Woldegiorgis, G; Kobayashi, C et al. (1989) 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. J Biol Chem 264:4168-72 |
| Woldegiorgis, G; Duff, T; Contreras, L et al. (1989) Photoaffinity labeling of hamster brown adipose tissue mitochondria by an [125I] coenzyme A derivative: differential interaction with the uncoupling protein and ADP/ATP carrier. Biochem Biophys Res Commun 161:502-7 |
