Control of Citric Acid Cycle Activity in Rat Heart Mitochondria

Abstract

Rat heart mitochondria were incubated in state 3 (with ADP) or state 4 (without ADP) in the presence of [3-14C]pyruvate, [3-14C]pyruvate plus unlabeled malate, or unlabeled pyruvate plus uniformly labeled [14C]malate. The net accumulation or loss of each intermediate in the citric acid cycle was determined by specific enzyme assays. In addition, the incorporation of radioactivity into cycle intermediates was measured after resolution of mitochondrial extracts by means of ion exchange column chromatography. In some experiments, mitochondrial suspensions were filtered through Millipore filters to determine the extramitochondrial content of metabolites. In the absence of malate, pool sizes of the citric acid cycle intermediates were very low in comparison to flux through the cycle. Recycling of 14C caused the specific activity of cycle intermediates to exceed that of the substrate, [3-14C]pyruvate. In the presence of a large pool of unlabeled malate, relatively large accumulations of 14C intermediates occurred during the oxidation of 14C-pyruvate in both state 3 and state 4. With malate added, 14C does not recycle but is diluted into the large malate pool. Thus, calculations of flux through the various steps in the cycle are made possible by comparing pyruvate disappearance with accumulation of radioactivity into the various intermediates and the final accumulation of radioactivity into malate. Rates of pyruvate utilization and oxygen consumption were linear under all conditions. Accumulations of citrate, α-ketoglutarate, and succinate were linear over the 8 min of incubation in state 4 and were largely extramitochondrial. In state 3, the accumulations of citrate and α-ketoglutarate, but not succinate, reached a constant maximum within 8 min. Both total accumulation and loss of citrate to the medium were lower in state 3 than in state 4. On the other hand, the accumulations of α-ketoglutarate and succinate were higher in state 3 than in state 4 and were mainly extramitochondrial. The accumulation of fumarate was not affected by the respiratory state of the mitochondria and was formed directly from the added malate. Addition of pyruvate alone or pyruvate plus malate to the mitochondria in both state 3 and state 4 resulted in a fall of aspartate and a stoichiometric rise of glutamate. It is suggested that the increased flux through each of the steps of the citric acid cycle observed in state 3 is mediated by coordinated interactions at several sites and that this effect is exerted mainly by changes in the ratio of NADH to NAD. The evidence indicates that the accumulations of citrate, α-ketoglutarate, and succinate in the extramitochondrial space occur by anion exchange reactions with the added malate.