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

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. beta-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 beta-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 beta-methyleneaspartate showed a similar one-to-one relationship between inhibition of oxygen comsumption 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 beta-methyleneaspartate (D,L-beta-methyl-D,L-aspartate, gamma-methyl-D,L-glutamate, and alpha-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 beta-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 beta-methyleneaspartate-mediated inhibition of respiration with glucose as substrate. The content of ATP and phosphocreatine declined steadily in slices incubated with glucose and beta-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.(ABSTRACT TRUNCATED AT 400 WORDS)

Cisplatin-Induced Toxicity Is Associated with Platinum Deposition in Mouse Kidney Mitochondria in Vivo and with Selective Inactivation of the α-Ketoglutarate Dehydrogenase Complex in LLC-PK1 Cells

“…Our previous studies showed that AOAA protects mice and LLC-PK 1 cells against cisplatin (10). AOAA forms an oxime with α-keto acids and with the coenzyme at the active site of many PLP-containing enzymes, inactivating them (52). The present work provides strong evidence that mitAspAT, acting at least in part as a β-lyase, contributes to the bioactivation of cisplatin to a renal toxicant.…”

“…The Pt-S compound also apparently slowly inactivates mitAspAT. We found a significant decline in total AspAT activity 24 h after LLC-PK 1 cells had been exposed to cisplatin for 3 h. Inhibition of AspAT would lead to disruption of the malate-aspartate shuttle and impeded passage of reducing equivalents across the mitochondrial membrane (52). In summary, inhibition of mitAspAT, KGDHC and aconitase may contribute to the cisplatin-induced mitochondrial dysfunction of energy metabolism in kidney cells.…”

Mitochondrial aspartate aminotransferase catalyses cysteine S-conjugate β-lyase reactions

“…mitAspAT is an important component of the malate\ aspartate shuttle for the passage of reducing equivalents across the mitochondrial membrane (cf. [33], and references cited therein). In kidney, this shuttle is thought to control Na + reabsorption [34].…”

The Transcriptome and Metabolic Gene Signature of Protoplasmic Astrocytes in the Adult Murine Cortex

“…Because NADH is impermeable to the mitochondrial membrane, shuttle systems transfer reducing equivalents into the mitochondria without physically moving them over the membrane by using membrane permeable substrates, such as glutamate and malate, as energy carriers. Classically there are described two such shuttle systems, the malate-aspartate shuttle (MAS) and the glycerol-phosphate shuttle (Fitzpatrick et al, 1983;Cheeseman and Clark, 1988;Lai et al, 1989;Berg et al, 2002). The malate-aspartate shuttle has been reported to be active in neurons, whereas the glycerol-phosphate shuttle has been reported as less active in the brain.…”