Long-chain acyl-CoA synthetase and "outer" carnitine long-chain acyltransferase activities of intact brown adipose tissue mitochondria.
1. The activities of long-chain acyl-CoA synthetase (acid: CoA ligase (AMP-forming), EC 6.2.1.3) and the "outer" carnitine long-chain acyltransferase (palmitoyl-CoA: L-carnitine O-palmitoyltransferase, EC 2.3.1.21) have been estimated in intact brown adipose tissue mitochondria. The assay of both enzymes is based on a coupled reaction in which the intramitochondrial (matrix) CoASH is the final acyl acceptor and the oxidation-reduction state of the flavoproteins in the acyl-CoA dehydrogens pathway is used to determine the intramitochondrial level of acyl-CoA. 2. Using endogenous fatty acids as the substrate, the progress curve of acyl-CoA synthetase activity was in most mitochondrial preparations linear within the first 30 s. When initial rates were measured, the Km value for CoASH (2.4 micron) was lower than previously determined for the acyl-CoA synthetase in brown adipose tissue mitochondria as well as in mitochondria of other tissues. The pH activity curve indicates that the unprotonated form of the fatty acids represents the substrate of acyl-CoA synthetase, i.e. similar to the effect of pH on the binding of fatty acids to bovine serum albumin. 3. Experimental evidence is presented that at temperatures higher than the transition temperature of the acyl-CoA synthetase (i.e. Tt = 19 degrees C), this enzymic reaction is rate-limiting in the sequence of coupled reactions leading to beta-oxidation in the mitochondrial matrix. 4. The initial rate of the long-chain acyl-COA synthetase reaction was estimated to v = 119 +/- 16 nmol . min-1 . mg-1 protein (mean +/- S.D., n = 5) at an optimal concentration of palmitate which exceeds that of rat heart mitochondria by a factor of 10.
D000217:Acyltransferases / Q000378:metabolism*; D002001:Adipose Tissue, Brown / Q000201:enzymology*; D000818:Animals; D002334:Carnitine O-Palmitoyltransferase / Q000378:metabolism*; D003066:Coenzyme A Ligases / Q000378:metabolism*; D006168:Guinea Pigs; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D051381:Rats; D013696:Temperature
D000217:Acyltransferases; D002334:Carnitine O-Palmitoyltransferase; D003066:Coenzyme A Ligases