David Pamela, Baumann Marc, Wikström Mårten, Finel Moshe
Helsinki Bioenergetics Group, Biotechnology Institute, Biocenter 2, University of Helsinki, Helsinki, Finland.
Biochim Biophys Acta. 2002 Feb 15;1553(3):268-78. doi: 10.1016/s0005-2728(01)00248-1.
The NADH:ubiquinone oxidoreductase (NDH-1 or Complex I) of Escherichia coli is a smaller version of the mitochondrial enzyme, being composed of 13 protein subunits in comparison to the 43 of bovine heart complex I. The bacterial NDH-1 from an NDH-2-deficient strain was purified using a combination of anion exchange chromatography and sucrose gradient centrifugation. All 13 different subunits were detected in the purified enzyme by either N-terminal sequencing or matrix-assisted laser desorption/ionization time-of-flight mass spectral analysis. In addition, some minor contaminants were observed and identified. The activity of the enzyme was studied and the effects of phospholipid and dodecyl maltoside were characterized. Kinetic analyses were performed for the enzyme in the native membrane as well as for the purified NDH-1, using ubiquinone-1, ubiquinone-2 or decylubiquinone as the electron acceptors. The purified enzyme exhibited between 1.5- and 4-fold increase in the apparent K(m) for these acceptors. Both ubiquinone-2 and decylubiquinone are good acceptors for this enzyme, while affinity of NDH-1 for ubiquinone-1 is clearly lower than for the other two, particularly in the purified state.
大肠杆菌的NADH：泛醌氧化还原酶（NDH-1或复合体I）是线粒体酶的一个较小版本，与牛心复合体I的43个蛋白质亚基相比，它由13个蛋白质亚基组成。使用阴离子交换色谱和蔗糖梯度离心相结合的方法，从一个缺乏NDH-2的菌株中纯化出细菌NDH-1。通过N端测序或基质辅助激光解吸/电离飞行时间质谱分析，在纯化的酶中检测到了所有13种不同的亚基。此外，还观察并鉴定了一些微量污染物。对该酶的活性进行了研究，并对磷脂和十二烷基麦芽糖苷的作用进行了表征。使用泛醌-1、泛醌-2或癸基泛醌作为电子受体，对天然膜中的酶以及纯化的NDH-1进行了动力学分析。纯化的酶对这些受体的表观K(m)增加了1.5至4倍。泛醌-2和癸基泛醌都是该酶的良好受体，而NDH-1对泛醌-1的亲和力明显低于对其他两者的亲和力，尤其是在纯化状态下。
