Dumas R, Joyard J, Douce R
Laboratoire de Physiologie Cellulaire Végétale (Unité mixte CNRS Rhône-Poulenc Agrochimie, UM 41), Lyon, France.
Biochem J. 1989 Sep 15;262(3):971-6. doi: 10.1042/bj2620971.
Acetohydroxyacid reductoisomerase was purified over 400-fold to a specific activity of 62 nkat.mg-1, with 2-aceto-2-hydroxybutyrate as substrate, from the stroma of spinach leaf chloroplasts. The enzyme was not intrinsically membrane bound. The native enzyme was a tetramer with a subunit Mr of 59,000. The activity was optimum between pH 7.5 and 8.5. The apparent Km for 2-acetolactate was 25 microM and for 2-aceto-2-hydroxybutyrate was 37 microM. The enzyme required Mg2+ and the Vmax. was attained at physiological Mg2+ concentrations. NADP+ competitively inhibited the reaction when NADPH was the varied substrate. The native enzyme eluted from Mono-Q ion-exchange resins as three distinct peaks of activity. This elution pattern was preserved when the peaks were combined, dialysed and re-chromatographed. Each form exhibited identical Mr of 59,000 after SDS/polyacrylamide gel electrophoresis (PAGE), whereas they were easily distinguishable from each other after PAGE under non-denaturing conditions. These results provide evidence for the existence of multiple forms of acetohydroxyacid reductoisomerase in chloroplasts isolated from spinach leaves.
以2-乙酰-2-羟基丁酸为底物，从菠菜叶叶绿体基质中纯化出乙酰羟酸还原异构酶，纯化倍数超过400倍，比活性为62 nkat·mg-1。该酶并非固着于膜上。天然酶为四聚体，亚基分子量为59,000。酶活性在pH 7.5至8.5之间最佳。2-乙酰乳酸的表观Km为25 μM，2-乙酰-2-羟基丁酸的表观Km为37 μM。该酶需要Mg2+，在生理Mg2+浓度下达到最大反应速度（Vmax）。当以NADPH作为可变底物时，NADP+竞争性抑制该反应。天然酶从Mono-Q离子交换树脂上洗脱下来时呈现出三个不同的活性峰。当将这些峰合并、透析并重新层析时，这种洗脱模式得以保留。在SDS/聚丙烯酰胺凝胶电泳（PAGE）后，每种形式的分子量均为59,000，而在非变性条件下进行PAGE后，它们彼此易于区分。这些结果为从菠菜叶中分离出的叶绿体中存在多种形式的乙酰羟酸还原异构酶提供了证据。
