大鼠肝脏支链α-酮酸脱氢酶复合体的纯化、拆分及重组

Purification, resolution, and reconstitution of rat liver branched-chain alpha-keto acid dehydrogenase complex.

作者信息

Ono K, Hakozaki M, Kimura A, Kochi H

出版信息

J Biochem. 1987 Jan;101(1):19-27. doi: 10.1093/oxfordjournals.jbchem.a121891.

DOI:10.1093/oxfordjournals.jbchem.a121891

Abstract

Branched-chain alpha-keto acid dehydrogenase (BCKADH) was solubilized as an enzyme complex from rat liver mitochondria by sonic treatment. Dehydrogenase (E1) and dihydrolipoyltransacylase (E2) components of the complex were purified in an associated form and resolved into individual components in the presence of 1 M NaCl, while lipoamide dehydrogenase (E3) component was dissociated from the complex during purification. Analysis by gel electrophoresis in dodecyl sulfate revealed the E1 comprised two different subunits with apparent molecular weights of 36,000 and 45,500, presumably in an equal molar ratio, while E2 consisted of a single subunit with an apparent molecular weight of 51,000. The BCKADH complex was reconstituted by combining E1, E2, and E3, and the formation of the complex was confirmed by analysis by sucrose density gradient centrifugation. The reconstituted enzyme complex oxidized not only alpha-ketoisovalerate (KIV), alpha-ketoisocaproate (KIC), and alpha-keto-beta-methylvalerate (KMV), but also pyruvate and alpha-ketoglutarate. Apparent Km values were 10-12 microM for the branched-chain alpha-keto acids, 2.2 mM for pyruvate, and 2.5 mM for alpha-ketoglutarate.

摘要

通过超声处理，从大鼠肝脏线粒体中以酶复合物的形式溶解支链α-酮酸脱氢酶（BCKADH）。该复合物的脱氢酶（E1）和二氢硫辛酰胺转乙酰酶（E2）组分以相关形式纯化，并在1 M NaCl存在下分解为单个组分，而硫辛酰胺脱氢酶（E3）组分在纯化过程中从复合物中解离。十二烷基硫酸钠凝胶电泳分析表明，E1由两个不同的亚基组成，表观分子量分别为36,000和45,500，推测摩尔比相等，而E2由一个表观分子量为51,000的单个亚基组成。通过将E1、E2和E3组合来重建BCKADH复合物，并通过蔗糖密度梯度离心分析确认复合物的形成。重建的酶复合物不仅氧化α-酮异戊酸（KIV）、α-酮异己酸（KIC）和α-酮-β-甲基戊酸（KMV），还氧化丙酮酸和α-酮戊二酸。支链α-酮酸的表观Km值为10 - 12 μM，丙酮酸为2.2 mM，α-酮戊二酸为2.5 mM。