玉米线粒体丙酮酸脱氢酶复合体的部分纯化及特性分析
Partial purification and characterization of the maize mitochondrial pyruvate dehydrogenase complex.
作者信息
Thelen JJ, Miernyk JA, Randall DD
出版信息
Plant Physiol. 1998 Apr;116(4):1443-50. doi: 10.1104/pp.116.4.1443.
Abstract
The pyruvate dehydrogenase complex was partially purified and characterized from etiolated maize (Zea mays L.) shoot mitochondria. Analysis by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed proteins of 40, 43, 52 to 53, and 62 to 63 kD. Immunoblot analyses identified these proteins as the E1beta-, E1alpha-, E2-, and E3-subunits, respectively. The molecular mass of maize E2 is considerably smaller than that of other plant E2 subunits (76 kD). The activity of the maize mitochondrial complex has a pH optimum of 7.5 and a divalent cation requirement best satisfied by Mg2+. Michaelis constants for the substrates were 47, 3, 77, and 1 &mgr;m for pyruvate, coenzyme A (CoA), NAD+, and thiamine pyrophosphate, respectively. The products NADH and acetyl-CoA were competitive inhibitors with respect to NAD+ and CoA, and the inhibition constants were 15 and 47 &mgr;m, respectively. The complex was inactivated by phosphorylation and was reactivated after the removal of ATP and the addition of Mg2+.
摘要
从黄化玉米（Zea mays L.）茎线粒体中对丙酮酸脱氢酶复合体进行了部分纯化和特性分析。十二烷基硫酸钠-聚丙烯酰胺凝胶电泳分析显示有40、43、52至53以及62至63 kDa的蛋白质。免疫印迹分析分别将这些蛋白质鉴定为E1β-、E1α-、E2-和E3-亚基。玉米E2的分子量明显小于其他植物E2亚基（76 kDa）。玉米线粒体复合体的活性在pH 7.5时最佳，二价阳离子需求以Mg2+最为满足。底物的米氏常数分别为：丙酮酸47 μM、辅酶A（CoA）3 μM、NAD+77 μM以及硫胺焦磷酸1 μM。产物NADH和乙酰辅酶A分别是NAD+和CoA的竞争性抑制剂，抑制常数分别为15 μM和47 μM。该复合体通过磷酸化失活，在去除ATP并添加Mg2+后重新激活。
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