Hutson S M, Fenstermacher D, Mahar C
Department of Physiology, Milton S. Hershey Medical Center, Pennsylvania State University 17033.
J Biol Chem. 1988 Mar 15;263(8):3618-25.
Oxidative decarboxylation and transamination of 1-14C-branched chain amino and alpha-keto acids were examined in mitochondria isolated from rat heart. Transamination was inhibited by aminooxyacetate, but not by L-cycloserine. At equimolar concentrations of alpha-ketoiso[1-14C]valerate (KIV) and isoleucine, transamination was increased by disrupting the mitochondria with detergent which suggests transport may be one factor affecting the rate of transamination. Next, the subcellular distribution of the aminotransferase(s) was determined. Branched chain aminotransferase activity was measured using two concentrations of isoleucine as amino donor and [1-14C]KIV as amino acceptor. The data show that branched chain aminotransferase activity is located exclusively in the mitochondria in rat heart. Metabolism of extramitochondrial branched chain alpha-keto acids was examined using 20 microM [1-14C]KIV and alpha-ketoiso[1-14C]caproate (KIC). There was rapid uptake and oxidation of labeled branched chain alpha-keto acid, and, regardless of the experimental condition, greater than 90% of the labeled keto acid substrate was metabolized during the 20-min incubation. When a branched chain amino acid (200 microM) or glutamate (5 mM) was present, 30-40% of the labeled keto acid was transaminated while the remainder was oxidized. Provision of an alternate amino acceptor in the form of alpha-keto-glutarate (0.5 mM) decreased transamination of the labeled KIV or KIC and increased oxidation. Metabolism of intramitochondrially generated branched chain alpha-keto acids was studied using [1-14C]leucine and [1-14C]valine. Essentially all of the labeled branched chain alpha-keto acid produced by transamination of [1-14C]leucine or [1-14C]valine with a low concentration of unlabeled branched chain alpha-keto acid (20 microM) was oxidized. Further addition of alpha-ketoglutarate resulted in a significant increase in the rate of labeled leucine or valine transamination, but again most of the labeled keto acid product was oxidized. Thus, catabolism of branched chain amino acids will be favored by a high concentration of mitochondrial alpha-ketoglutarate and low intramitochondrial glutamate.
在从大鼠心脏分离出的线粒体中，对1-14C支链氨基酸和α-酮酸的氧化脱羧作用及转氨作用进行了研究。氨基氧乙酸可抑制转氨作用，但L-环丝氨酸无此作用。在等摩尔浓度的α-酮异[1-14C]戊酸（KIV）和异亮氨酸存在时，用去污剂破坏线粒体可使转氨作用增强，这表明转运可能是影响转氨速率的一个因素。接下来，测定了转氨酶的亚细胞分布。使用两种浓度的异亮氨酸作为氨基供体、[1-14C]KIV作为氨基受体来测量支链转氨酶活性。数据显示，大鼠心脏中的支链转氨酶活性仅存在于线粒体中。使用20微摩尔[1-14C]KIV和α-酮异[1-14C]己酸（KIC）研究了线粒体外支链α-酮酸的代谢。标记的支链α-酮酸有快速摄取和氧化现象，且无论实验条件如何，在20分钟孵育期间，超过90%的标记酮酸底物被代谢。当存在支链氨基酸（200微摩尔）或谷氨酸（5毫摩尔）时，30 - 40%的标记酮酸发生转氨作用，其余部分则被氧化。以α-酮戊二酸（0.5毫摩尔）形式提供替代氨基受体可减少标记KIV或KIC的转氨作用并增加氧化作用。使用[1-14C]亮氨酸和[1-14C]缬氨酸研究了线粒体内生成的支链α-酮酸的代谢。基本上，由[1-14C]亮氨酸或[1-14C]缬氨酸与低浓度未标记支链α-酮酸（20微摩尔）转氨作用产生的所有标记支链α-酮酸都被氧化。进一步添加α-酮戊二酸会使标记亮氨酸或缬氨酸的转氨速率显著增加，但同样，大部分标记酮酸产物被氧化。因此，高浓度的线粒体α-酮戊二酸和低浓度的线粒体内谷氨酸有利于支链氨基酸的分解代谢。
