Feedback interactions in the control of citric acid cycle activity in rat heart mitochondria.

Factors regulating the citric acid cycle have been investigated in rat heart mitochondria oxidizing pyruvate plus malate or acetylcarnitine plus malate as substrates. Effects caused by changing the NAD oxidation-reduction state, the intramitochondrial ATP:ADP ratio, and the respiratory rate were studied in five different metabolic states produced by additions of ADP, oligomycin, or uncouplers. The accumulations of cycle intermediates and the mitochondrial content of CoA derivatives and pyridine nucleotides were measured in extracts prepared from the whole incubation medium or after rapid separation of the mitochondria from the medium. These data, together with measurements of substrate utilization, were used to calculate flux through the individual steps of the citric acid cycle. Pyruvate dehydrogenase was found not to be rate limiting for citric acid cycle activity. Flux through citrate synthase in State 3 was approximately the same (100 nmoles per min per mg) whether pyruvate or acetylcarnitine were used to generate acetyl-CoA, whereas acetyl-CoA levels were higher with pyruvate as substrate. Flux in State 4 with either substrate was diminished by 75 to 85% relative to State 3 and was associated during early incubation times with elevated levels of both NADH and acetyl-CoA, consonant with regulation of pyruvate dehydrogenase by product inhibition. A comparison of flux through citrate synthase and changes in the levels of intramitochondrial malate, citrate, ATP, and the NAD oxidation-reduction state showed that increased flux associated with a State 4 to 3 transition could be accounted for largely by an increased availability of oxalacetate to citrate synthase rather than by ATP inhibition. On the other hand, comparisons between states in which phosphate acceptor was not rate limiting for electron transport (State 3, uncoupled or uncoupled plus oligomycin) showed that oxalacetate availability remained high because of the highly oxidized state of the pyridine nucleotides, and indicated a regulation of citrate synthase under conditions of low acetylCoA availability by an energy-linked process dependent on