Regulation of Citrate Synthesis in Isolated Rat Liver Mitochondria

Abstract

Abstract The regulation of the fate of acetyl units derived from the β-oxidation of l(-)-palmitylcarnitine was studied using isolated rat liver mitochondria. Citrate synthesis from acetyl-CoA was dependent upon the concentration of l-malate, the immediate precursor of oxalacetate in this system. The independence of the rate of citrate formation on the means used to stimulate respiration, e.g. ADP for coupled mitochondria and p-trifluoromethoxphenyl hydrazone of carbonyl cyanide (FCCP) for uncoupled mitochondria, indicated that the mitochondrial citrate synthase was insensitive to the energy charge of the mitochondria. Various concentrations of externally added ATP inhibited both the rates of oxygen consumption and citrate formation but caused little alteration in the ratio of these two rates indicating that citrate synthase was insensitive to the added nucleotide. An effort was made to assess the effect of changes in the intramitochondrial ATP level on the formation of citrate. Using 2-methyl-1,4-naphthoquinone plus rotenone to stimulate respiration and to maintain a high intramitochondrial ATP level, linear rates of citrate formation and oxygen consumption were obtained. Addition of oligomycin caused a marked fall in ATP, no change in the rate of oxygen consumption, and no change in the rate of citrate formation suggesting that changes in the intramitochondrial ATP level did not effect a release of an inhibition of the citrate synthase. Addition of FCCP to this system caused a precipitous fall in ATP and an immediate and complete inhibition of citrate formation indicating an interruption in energized substrate transport into the mitochondria. Inclusion of the inhibitor, malonate, in the incubation medium of a citrate-synthesizing system prevented the formation of citrate when the mitochondria were coupled. Malonate seemed to compete with l-malate for entry into the mitochondria. Malonate did not prevent citrate formation in uncoupled mitochondria indicating that a relatively low level of uncoupler enhanced the permeability of the mitochondria for anionic substrates. Positive correlations were observed between the oxidation-reduction state of the mitochondrial pyridine nucleotides or the rate of respiration and the rate of citrate formation in an uncoupler titration and in a potassium concentration using valinomycin-treated mitochondria. It is concluded that the rate of citrate synthesis in isolated liver mitochondria depends mainly on the availability of the two substrates of the citrate synthase reaction, acetyl-CoA and oxalacetate.