The intracellular localization of long-chain acyl-CoA synthetase in brown adipose tissue.

1. The acyl-CoA synthetase activity in brown adipose tissue of cold-exposed guinea pig has been studied by measuring the rate of palmitoylcarnitine formation in the presence of excess carnitine palmitoyltransferase. 2. The rate of palmitoylcarnitine formation in the mitochondria was found to be 161 plus or minus 64 nmol.mg-minus-1. min-minus-1 (n=9). 3. In the absence of added palmitate and bovine serum albumin a total of 35 plus or minus 1 nmol endogenous fatty acids.mg-minus-1 were activated with three different mitochondrial preparations. 4. Three different experimental approaches have been used to study the subcellular localization of the enzyme: (a) conventional differential centrifugation (De Duve, C., Pressman, B.C., Gianetto, R., Wattiaux, R. and Appelmans, F. (1955) Biochem. J. 60, 604-617) (B) the determination of the sediterm of different marker enzymes (Slinde, E. and Flatmark. T. (1973) Anal. Biochem. 56, 324-340) and (c) the determination of the stoichiometry between the activities of these enzymes sedimented at higher centrifugal effects. 5. Throughout all fractionation procedures, the long-chain acyl-CoA synthetase follows strictly the amine oxidase generally considered to be exclusively located on the mitochondrial outer membrane.