Camp P J, Randall D D
Department of Biochemistry, University of Missouri, Columbia, Missouri 65211.
Plant Physiol. 1985 Mar;77(3):571-7. doi: 10.1104/pp.77.3.571.
The pyruvate dehydrogenase complex has been purified 76-fold, to a specific activity of 0.6 mumoles per minute per milligram protein, beginning with isolated pea (Pisum sativum L. var Little Marvel) chloroplasts. Purification was accomplished by rate zonal sedimentation, polyethyleneglycol precipitation, and ethyl-agarose affinity chromatography. Characterization of the substrates as pyruvate, NAD(+), and coenzyme-A and the products as NADH, CO(2), and acetyl-CoA, in a 1:1:1 stoichiometry unequivocally established that activity was the result of the pyruvate dehydrogenase complex. Immunochemical analysis demonstrated significant differences in structure and organization between the chloroplast pyruvate dehydrogenase complex and the more thoroughly characterized mitochondrial complex. Chloroplast complex has a higher magnesium requirement and a more alkaline pH optimum than mitochondrial complex, and these properties are consistent with light-mediated regulation in vivo. The chloroplast pyruvate dehydrogenase complex is not, however, regulated by ATP-dependent inactivation. The properties and subcellular localization of the chloroplast pyruvate dehydrogenase complex are consistent with its role of providing acetyl-CoA and NADH for fatty acid synthesis.
从分离的豌豆（小惊奇品种，Pisum sativum L. var Little Marvel）叶绿体开始，丙酮酸脱氢酶复合体已被纯化了76倍，比活性达到每分钟每毫克蛋白质0.6微摩尔。纯化过程通过速率区带沉降、聚乙二醇沉淀和乙基琼脂糖亲和层析完成。底物丙酮酸、NAD⁺和辅酶A以及产物NADH、CO₂和乙酰辅酶A以1:1:1的化学计量比明确表明该活性是丙酮酸脱氢酶复合体的作用结果。免疫化学分析表明，叶绿体丙酮酸脱氢酶复合体与特征更明确的线粒体复合体在结构和组织上存在显著差异。叶绿体复合体比线粒体复合体对镁的需求更高，最适pH值更偏碱性，这些特性与体内光介导的调节作用一致。然而，叶绿体丙酮酸脱氢酶复合体不受ATP依赖性失活的调节。叶绿体丙酮酸脱氢酶复合体的特性和亚细胞定位与其为脂肪酸合成提供乙酰辅酶A和NADH的作用相一致。
