Acyl-CoA synthetase activity of brown adipose tissue mitochondria. Substrate specificity and its relation to the endogenous pool of long-chain fatty acids.
1. A new assay of acyl-CoA synthetase (acid:CoA ligase (AMP-forming), EC 6.2.1.3) activity was developed for application to measurements on isolated intact mitochondria. The assay was based on the formation of the product AMP as determined by high-performance liquid chromatography (HPLC), making corrections for the partial conversion of AMP to ADP by the adenylate kinase reaction. 2. The substrate specificity of the long-chain acyl-CoA synthetase was measured in brown adipose tissue mitochondria, isolated from cold-acclimated guinea-pigs. Using mitochondria largely deficient of endogenous fatty acids, the synthetase activity (V') revealed an optimum at 12 :0 and 13 : 0 with saturated fatty acids as the substrate. In contrast to other tissues, the highest V' values were observed with unsaturated fatty acids 18 : 1 (9) (cis) and 18 : 2 (9, 12) (all cis). The apparent Michaelis constant varied within a narrow range and revealed no systematic dependence on the chain-length as was the case for V'. Since the mitochondria have a high capacity of fatty acid binding, with unknown affinity, the estimated K'm values are, however, of questionable significance. 3. The pattern of chain-length specificity in the synthetase reaction of the mitochondria compares well with the pattern of endogenous long-chain fatty acids in which mainly four species contribute, i.e. 18 : 1 (42 mol%) and 18 : 2 (18 mol%), 16 : 0 (26 mol%) and 18 : 0 (14 mol%).
D000106:Acetate-CoA Ligase / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000096:biosynthesis; D000255:Adenosine Triphosphate / Q000378:metabolism; D002001:Adipose Tissue, Brown / Q000201:enzymology*; D000818:Animals; D003066:Coenzyme A Ligases / Q000378:metabolism*; D003080:Cold Temperature; D005227:Fatty Acids / Q000378:metabolism; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008928:Mitochondria / Q000201:enzymology*; D013379:Substrate Specificity
D005227:Fatty Acids; D000249:Adenosine Monophosphate; D000255:Adenosine Triphosphate; D003066:Coenzyme A Ligases; D000106:Acetate-CoA Ligase