Interrelationships between malate-aspartate shuttle and citric acid cycle in rat heart mitochondria

Control of substrate utilization was investigated using rat heart mitochondria incubated under conditions of state 4, state 3, oligomycin-inhibited and uncoupled respiration and conclusions drawn regarding regulation of the transport of reducing equivalents were verified using mitochondria supplemented with the extramitochondrial components of the malate aspartate shuttle.

Abstract

The control of substrate utilization was investigated using rat heart mitochondria incubated under conditions of state 4, state 3, oligomycin-inhibited and uncoupled respiration. A comparison of the changes in metabolite levels after addition of pyruvate or acetylcarnitine showed that cycle flux was controlled primarily at citrate synthase, which appeared to be regulated by the intramitochondrial oxalacetate concentration. A secondary control site located between α-ketoglutarate and succinate was revealed by addition of oligomycin which caused an increased flux through α-ketoglutarate dehydrogenase and thereby a decreased efflux of α-ketoglutarate from the mitchondria. The increased α-ketoglutarate dehydrogenase activity was caused by the fall of the ATP/ADP ratio, which by increasing the availability of GDP for substrate level phosphorylation produced diminished product inhibition by succinyl CoA. Because transport of NADH into mitochondria by the malate-aspartate shuttle requires a stoichiometric influx of malate and glutamate and efflux of aspartate and α-ketoglutarate from the mitochondria, alterations in the rate of efflux of α-ketoglutarate can significantly alter flux through the shuttle and the rate of utilization of cytosolic NADH. Studies with mitochondria oxidizing glutamate and malate in the presence and absence of acetylcarnitine or octanoate showed that α-ketoglutarate efflux could be strongly effected by the intramitochondrial ATP/ADP ratio. Rates of α-ketoglutarate efflux were also increased by extramitochondrial malate (half maximal stimulation at 0.6 mM). Glutamate transamination was inhibited by uncoupling agents and subsequent studies, measuring intramitochondrial aspartate levels, showed that this was due to an inhibition of aspartate efflux from the mitochondria in the uncoupled state. Conclusions drawn regarding regulation of the transport of reducing equivalents were verified using mitochondria supplemented with the extramitochondrial components of the malate aspartate shuttle.