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.
Kinetics of ATP and inorganic phosphate release during hydrolysis of ATP by rabbit skeletal actomyosin subfragment 1. Oxygen exchange between water and ATP or phosphate.