Crystallization of bovine heart mitochondrial respiratory complex I

复合物I（NADH-泛醌氧化还原酶）是线粒体呼吸链的第一个酶。它催化两个电子从NADH转移到奎宁，并使大约四个质子跨膜转移。线粒体酶含有46个不同的亚基，是已知最大的膜蛋白复合体之一，分子量约为1000 kDa。为了阐明这种大的膜蛋白复合物的反应机理，三维结构是必不可少的，为了制备这种膜复合物的晶体，必须从牛心肌线粒体膜中纯化天然酶。NADH-Q1氧化还原酶活性在线粒体膜上被杀粉蝶菌素A或鱼藤酮特异性抑制，但当酶发生变性时，对这些抑制剂的敏感性降低。酶用脱氧胆酸盐和DDM和DM增溶，并应用于阴离子交换琼脂糖色谱。新方法只需要两天。纯化后的酶活力比文献报道的酶高2倍以上，对抑制剂的敏感性高（>93%）。不同批次的酶制剂含有约200个磷脂分子，表明这些磷脂是稳定该酶所必需的。将DM溶液中的纯化蛋白与PC混合，然后通过透析除去DM。酶在某些条件下形成2D晶格，但面积很小。在气相扩散法下获得了呈现该酶颜色的晶体状物质。

Complex I (NADH-ubiquinone oxidoreductase) is the first enzyme of the mitochondrial respiratory chains. It catalyzes the transfer of two electrons from NADH to quinine, coupled to the translocation of about four protons across the membrane. The mitochondrial enzyme contain 46 different subunits and is one of the largest known membrane protein complex, the molecular mass of about 1000kDa. To elucidate the mechanism of this big membrane protein complex reactions, the three dimensional structure is indispensable.For making the crystals of this membrane complex, the native enzyme must be purified from bovine heart mitochondrial membrane. NADH-Q1 oxidoreducutase activities are inhibited by piericidin A or rotenone specifically in the mitochondrial membrane, but the sensitivity to these inhibitors is decreased if some denaturalization occur.The method for purification of this enzyme were established. Enzymes were solubilized with deoxycholate and DDM and DM, and applied to anionic exchange sepharose chromatography. The new methods needs only two days. The purified enzyme had 2-fold higher activities than reported one, high sensitivities for inhibitors (>93%). The enzyme preparation from different batch contained about 200 molecules of phospholipids, suggested these phospholipids were essential to stabilize this enzyme. The purified protein in DM solution is mixed with PC, then DM was removed by dialysis. Enzymes form 2D lattices, but small area, under some conditions. The crystal-like masses, which exhibit color of this enzyme, were obtained under vapor-diffusion methods.