Taylor Nicolas L, Heazlewood Joshua L, Day David A, Millar A Harvey
Biochemistry and Molecular Biology, School of Biomedical and Chemical Sciences, The University of Western Australia, 35 Stirling Highway, Crawley, Western Australia 6009, Australia.
Plant Physiol. 2004 Feb;134(2):838-48. doi: 10.1104/pp.103.035675. Epub 2004 Feb 5.
Lipoic acid-dependent pathways of alpha-keto acid oxidation by mitochondria were investigated in pea (Pisum sativum), rice (Oryza sativa), and Arabidopsis. Proteins containing covalently bound lipoic acid were identified on isoelectric focusing/sodium dodecyl sulfate-polyacrylamide gel electrophoresis separations of mitochondrial proteins by the use of antibodies raised to this cofactor. All these proteins were identified by tandem mass spectrometry. Lipoic acid-containing acyltransferases from pyruvate dehydrogenase complex and alpha-ketoglutarate dehydrogenase complex were identified from all three species. In addition, acyltransferases from the branched-chain dehydrogenase complex were identified in both Arabidopsis and rice mitochondria. The substrate-dependent reduction of NAD(+) was analyzed by spectrophotometry using specific alpha-keto acids. Pyruvate- and alpha-ketoglutarate-dependent reactions were measured in all three species. Activity of the branched-chain dehydrogenase complex was only measurable in Arabidopsis mitochondria using substrates that represented the alpha-keto acids derived by deamination of branched-chain amino acids (Val [valine], leucine, and isoleucine). The rate of branched-chain amino acid- and alpha-keto acid-dependent oxygen consumption by intact Arabidopsis mitochondria was highest with Val and the Val-derived alpha-keto acid, alpha-ketoisovaleric acid. Sequencing of peptides derived from trypsination of Arabidopsis mitochondrial proteins revealed the presence of many of the enzymes required for the oxidation of all three branched-chain amino acids. The potential role of branched-chain amino acid catabolism as an oxidative phosphorylation energy source or as a detoxification pathway during plant stress is discussed.
在豌豆（Pisum sativum）、水稻（Oryza sativa）和拟南芥中研究了线粒体通过硫辛酸依赖性途径氧化α-酮酸的过程。通过使用针对该辅因子产生的抗体，在等电聚焦/十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分离的线粒体蛋白质上鉴定出了含有共价结合硫辛酸的蛋白质。所有这些蛋白质均通过串联质谱法进行了鉴定。在这三个物种中均鉴定出了来自丙酮酸脱氢酶复合体和α-酮戊二酸脱氢酶复合体的含硫辛酸酰基转移酶。此外，在拟南芥和水稻线粒体中均鉴定出了来自支链脱氢酶复合体的酰基转移酶。使用特定的α-酮酸通过分光光度法分析了NAD(+)的底物依赖性还原。在所有三个物种中均测定了丙酮酸和α-酮戊二酸依赖性反应。仅在拟南芥线粒体中使用代表由支链氨基酸（缬氨酸、亮氨酸和异亮氨酸）脱氨基产生的α-酮酸的底物时，才能检测到支链脱氢酶复合体的活性。完整的拟南芥线粒体对支链氨基酸和α-酮酸依赖性的氧消耗速率在使用缬氨酸和缬氨酸衍生的α-酮酸α-酮异戊酸时最高。对拟南芥线粒体蛋白质经胰蛋白酶消化产生的肽段进行测序，揭示了所有三种支链氨基酸氧化所需的许多酶的存在。本文讨论了支链氨基酸分解代谢在植物应激期间作为氧化磷酸化能量来源或解毒途径的潜在作用。
