Characterization of the Fatty Acid Elongation System in Soluble Extracts and Membrane Preparations of Rat Liver Mitochondria

Submitochondrial particles prepared from rat liver mitochondria or Triton extracts of these particles incorporate 1-14C-acetyl coenzyme A into fatty acids in the presence of ATP and reduced pyridine nucleotides. Added acyl-CoA primers can substitute for ATP. NADPH is 25% as active as NADH, and its activity is not explicable on the basis of its conversion to NADH. It appears that NADPH may also be a direct reductant in the elongation reaction. It was shown that ATP supports acetyl-CoA incorporation by activating endogenous fatty acids via an acyl-CoA synthetasecatalyzed reaction. Gas-liquid chromatographic separation of the products and analysis of the amount of radioactivity present in the carboxyl carbon of the synthesized fatty acids were consistent with the operation of a chain elongation mechanism. The fatty acid products of the reaction were identified as CoA esters, 2 to 4 carbon atoms longer than the primer fatty acyl-CoA. The elongation reaction was found to occur in both the inner and outer mitochondrial membranes as well as in the soluble fraction. The inner membrane and the soluble fraction were highly active with decanoyl-CoA and octanoyl-CoA but exhibited almost no activity with long chain acyl-CoAs. On the other hand, the specific activity of the outer membrane preparation with palmitoyl-CoA was three times higher than that found in the inner membrane. The elongation reaction was inhibited by oxidized nucleotides, CoA—SH, and acyl-CoAs. The incorporation of 2-14C-malonyl-CoA was considerably lower than that of acetyl-CoA and could be accounted for by the presence of a highly active malonyl-CoA decarboxylase in the preparations. Both the acyl-CoA deacylase and the malonyl-CoA decarboxylase appeared to be localized in the matrix fraction of rat liver mitochondria.