Use of β‐Methylene‐D,L‐Aspartate to Assess the Role of Aspartate Aminotransferase in Cerebral Oxidative Metabolism

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Abstract
Several inhibitors of aspartate aminotransferase, a key enzyme of the malate‐aspartate shuttle, were investigated for their effects on cerebral oxidative metabolism in vitro. β‐Methylene‐D,L‐aspartate (2 mM), aminooxyacetate (0.1 mM), and D,L‐vinylglycine (20 mM) all significantly reduced the activity of aspartate aminotransferase and the rate of oxygen consumption of rat cerebral cortex slices respiring on glucose. In the presence of β‐methyleneaspartate, a one‐to‐one correlation was found between the degree of inhibition of tissue respiration and the degree of inhibition of transaminase activity. Slices of rat liver incubated in the presence of glucose and β‐methyleneaspartate showed a similar oneto‐one relationship between inhibition of oxygen cornsumption and inhibition of aspartate aminotransferase activity, whereas with rat kidney cortex slices, the inhibition of aspartate aminotransferase activity was greater than the inhibition of oxygen consumption. Structural analogs of β‐methyleneaspartate (D,L‐β‐methyl‐D,L‐aspartate, ‐γ‐methyl‐D,L‐glutamate, and α‐methyl‐D,L‐didehydroglutamate) that did not inhibit the activity of aspartate aminotransferase similarly did not inhibit the rate of oxygen consumption by cerebral cortex slices. In the presence of β‐methyleneaspartate, pyruvate oxidation by cerebral cortex slices was inhibited to almost the same extent as was glucose oxidation, and the oxidation of succinate was decreased by approximately 20%. The artificial electron acceptor phenazine methosulfate (0.1 mM) only partially overcame the β‐methyleneaspartate‐mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and β‐methyleneaspartate. At 1 h the concentration of lactate and the lactate/ pyruvate ratio, an indicator of the cytoplasmic redox state, increased threefold, whereas the concentrations of malate, citrate, and aspartate decreased. The findings are interpreted in the context of the hypothesis that enzymes common to the malate‐aspartate shuttle and the tricarboxylic acid cycle are physically complexed in brain, so that inhibition of aspartate aminotransferase, a component of the complex, impedes the flow of carbon through both metabolic pathways. The operation of the malateaspartate shuttle may provide a link between cerebral glycolysis (a continued need for NAD+) and the tricarboxylic acid cycle (supply of oxaloacetate) that is vulnerable to several metabolic disturbances that impair brain function.