Role of specific aminotransferases in de novo glutamate synthesis and redox shuttling in the retina

Abstract

In this study aminotransferase inhibitors were used to determine the relative importance of different aminotransferases in providing nitrogen for de novo glutamate synthesis in the retina. Aminooxyacetate, which inhibits all aminotransferases, blocked de novo glutamate synthesis from H(14)CO(3)(-) by more than 60%. Inhibition of neuronal cytosolic branched chain amino acid transamination by gabapentin or branched chain amino acid transport by the L-system substrate analog, 2-amino-bicyclo-(2,2,1)-heptane-2-carboxylic acid, lowered total de novo synthesis of glutamate by 30%, suggesting that branched chain amino acids may account for half of the glutamate nitrogen contributed by transamination reactions. L-cycloserine, an inhibitor of alanine aminotransferase, inhibited glutamate synthesis less than 15% when added in the presence of 5 mM pyruvate but 47% in the presence of 0.2 mM pyruvate. Although high levels of pyruvate blunted the inhibitory effectiveness of L-cycloserine, the results indicate that, under physiological conditions, alanine as well as branched chain amino acids are probably the predominant sources of glutamate nitrogen in ex vivo retinas. The L-cycloserine results were also used to evaluate activity of the malate/aspartate shuttle. In this shuttle, cytosolic aspartate (synthesized in mitochondria) generates cytosolic oxaloacetate that oxidizes cytosolic NADH via malate dehydrogenase. Because L-cycloserine inhibits cytosolic but not mitochondrial aspartate aminotransferase, L-cycloserine should prevent the utilization of aspartate but not its generation, thereby increasing levels of (14)C-aspartate. Instead, L-cycloserine caused a significant decline in (14)C-aspartate. The results suggest the possibility that shuttle activity is low in retinal Müller cells. Low malate/aspartate shuttle activity may be the molecular basis for the high rate of aerobic glycolysis in retinal Müller cells.

Inhibition of Mitochondrial Pyruvate Transport by Zaprinast Causes Massive Accumulation of Aspartate at the Expense of Glutamate in the Retina

“…In the retina, Ͼ60% of glutamate de novo synthesis depends on transaminases such as alanine transaminase, aspartate transaminase, and branched amino acids transaminase (49). However, only aspartate and not alanine or branched amino acids increase with Zaprinast.…”

“…Glutamate may also decarboxylate into GABA, but Zaprinast does not increase GABA content in the retina. Therefore, the amino group of aspartate most likely comes from glutamate transamination (49).…”

Effective disposal of nitrogen waste in blood‐fedAedes aegyptimosquitoes requires alanine aminotransferase

“…Moreover, it was shown that when mosquitoes or dissected tissues are exposed to an ammonia challenge in vivo or in vitro, ALAT plays an important role in both mosquito whole body (4,6) and tissues (8) contributing to the homeostasis of amino acid metabolism. Recent studies indicate that administration of high doses of L-cycloserine, a well-characterized inhibitor of ALAT (12)(13)(14)(15)(16), affects blood-fed mosquito survival and motor activity, suggesting an interference with ammonia and carbohydrate metabolism in a time-dependent manner (17). To compare this finding, we reduced A. aegypti ALAT through RNAi gene silencing and examined the outcome of blood-fed female mosquitoes injected with double-stranded RNA (dsRNA) against ALAT.…”

Transamination Is Required for α-Ketoisocaproate but Not Leucine to Stimulate Insulin Secretion*

“…Indeed, aminooxyacetic acid inhibits insulin secretion stimulated by glucose and other nutrients (18,22). All of the available inhibitors of BCATm preferentially block other transaminases (23)(24)(25). In this study, we used BCATm Ϫ/Ϫ mice to determine the precise role of this enzyme in KICSIS and LSIS, therefore resolving the catabolism versus transamination dispute that has lasted for more than 30 years.…”