Regulation of Oxalacetate Metabolism in Liver Mitochondria EVIDENCE FOR NICOTINAMIDE ADENINE DINUCLEOTIDE-MALATE DEHYDROGENASE EQUILIBRIUM AND THE ROLE OF PHOSPHOENOLPYRUVATE CARBOXYKINASE IN THE CONTROL OF OXALACETATE METABOLISM IN INTACT GUINEA PIG AND RAT LIVER MITOCHONDRIA

Abstract The metabolism of malate was investigated in liver mitochondria from both the rat and the guinea pig. The oxidation of this intermediate was found to proceed in an inhibited and nonlinear manner in guinea pig liver mitochondria and to a lesser extent this situation held also for rat liver mitochondria. This inhibition was reversed by glutamate and partially by pyruvate addition. Analyses of the mitochondrial suspensions throughout an ADP-induced respiratory cycle showed increasing levels of oxalacetate during the transition from State 4 to State 3, with further increases occurring throughout State 3. Inhibition of malate oxidation was associated with markedly elevated levels of oxalacetate. In guinea pig liver mitochondria with malate as substrate, a large amplitude oxidation of the respiratory chain-linked pyridine nucleotides was observed during State 3 which persisted beyond the apparent transition into State 4. A significant release of protons to the medium was noted in State 4, whereas in State 3, lower H+ to ADP and ADP to oxygen ratios were obtained with guinea pig as compared to rat liver mitochondria. Factors increasing the rate of oxalacetate removal in mitochondria were noted to also increase the rate of malate oxidation and the formation of reduced pyridine nucleotides during the transition from State 3 to State 4. These same factors also decreased the rate of proton release in State 4, and increased the ratios of H+ to ADP and ADP to oxygen. P-enolpyruvate carboxykinase was found to function in a reversible manner in guinea pig liver mitochondria. Significant amounts of intramitochondrial oxalacetate could be formed from added P-enolpyruvate, provided that the ratio of ATP:ADP was low. Using this technique to vary the level of oxalacetate within the matrix space, marked inhibition of malate oxidation was produced by increasing the concentration of oxalacetate. In addition, the mechanisms of oxalacetate removal appear different in liver mitochondria from the guinea pig as compared to the rat. The flow of carbon from oxalacetate to P-enolpyruvate limits, in part, the State 3 rate of respiration from malate in guinea pig liver mitochondria. Such a limitation arising from P-enolpyruvate synthesis is observed despite the markedly elevated levels of oxalacetate which thereby result. Other factors such as the availability of ATP to form GTP by transphosphorylation also appear to control the rate and direction of the P-enolpyruvate carboxykinase reaction. Decreased rates of P-enolpyruvate formation inhibit the rate of malate oxidation in either State 3 or State 4. The metabolic consequences of mitochondrial P-enolpyruvate formation results in significantly altered kinetic parameters of malate metabolism in mitochondria from these two species. It is concluded that NAD+-malate dehydrogenase functions in an equilibrium manner in the intact liver mitochondria of both rat and guinea pig, and that the intramitochondrial pools of malate, oxalacetate, and the nicotinamide coenzymes freely interact in such a system. Alterations of any one factor produce direct and reciprocal changes in the other intermediates. Additionally, P-enolpyruvate carboxykinase although reversible, appears to function primarily as an oxalacetate-removing system. In guinea pig liver mitochondria, the presence of this enzyme results in altered control mechanisms of oxalacetate metabolism and in marked differences in the regulation of the citric acid cycle. These observations have important implications for the mechanisms of hepatic gluconeogenesis in both the rat and the guinea pig.