Effect of palmitoyl-CoA and β-oxidation of fatty acids on the kinetics of mitochondrial citrate transporter

The kinetics of the hepatic mitochondrial citrate transporter were studied using 1,2,3-benzene tricarboxylate and the inhibitor-stop technique at 8 °C. The apparent Km for this transporter was 250 μM and the maximum velocity was 2 nmol of citrate transported per minute per milligram of mitochondrial protein. This apparent Km was increased when hepatic mitochondria were preincubated with both L-palmitoylcarnitine and CoA-SH but not with either alone. This rise in apparent Km was accompanied by a rise in the acid insoluble CoA-SH content. Removal of mitochondrial acid insoluble CoA by 'defatted albumin' resulted in a parallel decrease in the apparent Km. The apparent Km for the citrate transporter was increased after coupled β-oxidation of L-palmitoylcarnitine or octanoate without a detectable increase in acid insoluble CoA. Inhibition of β-oxidation of L-palmitoylcarnitine by the D-derivative prevented the rise in the apparent Km. Preincubation with ATP resulted in an increase in this apparent Km. When L-palmitoylcarnitine oxidation occurred without ATP accumulation (hexokinase, glucose, ADP, and inorganic phosphate) the apparent Km for the citrate transporter increased two- to threefold.Therefore, the apparent Km for the citrate transporter varied directly with the acid insoluble CoA content. In addition, this Km was increased as a result of β-oxidation of fatty acids but the mechanism was not solely attributable to a rise in acid insoluble CoA or ATP. The physiological implications of these findings are discussed.