The major objective of this project is to synthesize by a newly divised method a sufficient quantity of 2-azido ?32p! palmitoyl CoA for photolabeling specific mitochondrial membrane proteins, namely the ADP/ATP carrier from beef heart mitochondria and the uncoupling protein from brown fat mitochondria. Following purification and cleavage of the photolabeled proteins, the radioactive peptide amino acids will be sequenced to mp the acyl CoA and possibly the nucleotide binding sites. The ADP/ATP carrier will be photolabeled from both the cytosolic and matrix sides of the inner mitochondrial membrane with the aim of differentiating between a single or two distinct binding sites. The amino acid profiles of the acyl CoA binding sites on the ADP/ATP carrier and uncoupling protein will be compared for the degree of homology of these two similar proteins. Long chain acyl CoA esters are recognized to be biological regulatory ligands for a number of important enzymes. These experiments will provide for the first time pertinent biochemical information on the mechanism of regulation of cellular bioenergetics by acyl CoA esters. Furthermore, it is anticipated that 2-azido palmitoyl CoA will prove very useful in screening for the co-valent binding of a number of membrane and soluble proteins which are modulated by acyl CoA esters.
