Role of specific aminotransferases in de novo glutamate synthesis and redox shuttling in the retina
Role of specific aminotransferases in de novo glutamate synthesis and redox shuttling in the retina
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DOI：
10.1002/jnr.10064
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发表时间：
2001-12-01
影响因子：
4.2
通讯作者：
Hutson, S
中科院分区：
文献类型：
--
作者：
LaNoue, KF;Berkich, DA;Hutson, S
在这项研究中，利用转氨酶抑制剂来确定不同的转氨酶在视网膜中为谷氨酸从头合成提供氮的相对重要性。氨基乙酸酯抑制所有转氨酶，可阻断(HCO3)- c -14 -从头合成谷氨酸的60%以上。加巴巴丁抑制神经元胞质支链氨基酸转氨化或l -系统底物类似物2-氨基-双环-(2,2,1)-庚烷-2-羧酸转运支链氨基酸，可使谷氨酸的总新合成降低30%，表明支链氨基酸可能占转氨化反应中谷氨酸氮贡献的一半。l -环丝氨酸是一种丙氨酸转氨酶抑制剂，当添加5 mM丙酮酸时，对谷氨酸合成的抑制作用小于15%，而在添加0.2 mM丙酮酸时，对谷氨酸合成的抑制作用为47%。尽管高水平的丙酮酸降低了l -环丝氨酸的抑制效果，但结果表明，在生理条件下，丙氨酸和支链氨基酸可能是离体视网膜中谷氨酸氮的主要来源。l -环丝氨酸的结果也被用来评估苹果酸/天冬氨酸穿梭的活性。在这种穿梭中，胞质天冬氨酸（在线粒体中合成）产生胞质草酰乙酸，通过苹果酸脱氢酶氧化胞质NADH。由于l -环丝氨酸抑制细胞质内而非线粒体天冬氨酸转氨酶，因此l -环丝氨酸应阻止天冬氨酸的利用而不是其产生，从而增加C-14-天冬氨酸水平。相反，l -环丝氨酸导致c -14-天冬氨酸显著下降。结果表明，视网膜Muller细胞的穿梭活性可能较低。低苹果酸/天冬氨酸穿梭活性可能是视网膜Muller细胞有氧糖酵解率高的分子基础。(C) 2001 Wiley-Liss, Inc。
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 (HCO3)-C-14 - 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 C-14- aspartate. Instead, L-cycloserine caused a significant decline in C-14-aspartate. The results suggest the possibility that shuttle activity is low in retinal Muller cells. Low malate/aspartate shuttle activity may be the molecular basis for the high rate of aerobic glycolysis in retinal Muller cells. (C) 2001 Wiley-Liss, Inc.