Purification and properties of long-chain acyl-coenzyme-A synthetase from rat liver.

Long-chain acyl-coenzyme-A synthetase from the microsomes as well as from the mitochondrial fraction of rat liver has been purified to homogeneity as evidenced by dodecylsulfate/polyacrylamide gel electrophoresis, amino-terminal analysis and the elution profile at the final chromatography step. The purification procedure involves resolution of the cellular particles with Triton X-100 and chromatography on Blue-Sepharose, hydroxyapatite and phosphocellulose. The purified enzymes from both sources have a specific activity of 26–29 units/mg protein at 35°C, which is more than 100-fold higher than those of long-chain acyl-CoA synthetases of animal and bacterial origin hitherto reported. The purified enzymes exhibit a molecular weight of approximately 76000 as estimated by dodecylsulfate/polyacrylamide gel electrophoresis and catalyze the activation of saturated fatty acids with 10–18 carbon atoms and unsaturated fatty acids with 16–20 carbon atoms most efficiently. The purified enzyme from the microsomes and that from the mitochondrial fraction, which are obtained by essentially identical procedures, are indistinguishable from each other with respect to all molecular and catalytic properties examined, including molecular weight, amino acid composition, amino-terminal residue, heat stability, specific activity, pH optimum and substrate specificity regarding fatty acid, acyl acceptor and nucleoside 5′-triphosphate.