从可溶性成分中重组大肠杆菌琥珀酸氧化酶。

Reconstitution of escherichia coli succinoxidase from soluble components.

Reddy T L, Hendler R W

J Biol Chem. 1978 Nov 10;253(21):7972-9.

Abstract

The membrane-bound succinoxidase of Escherichia coli was fractionated with deoxycholate into three soluble components, viz. succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase complex, and a factor identified as a phospholipid-containing component. 2. The dehydrogenase and cytochrome oxidase complexes were partially purified by filtration on Amicon membranes, Sepharose 4B chromatography, and sucrose gradient centrifugation. 3. Reconstitution of membranous succinoxidase, which catalyzes the oxidation of succinate by molecular oxygen by an integrated CN(-)-sensitive pathway, was achieved by mixing the soluble succinate dehydrogenase.cytochrome b1 complex with the soluble cytochrome oxidase complex in the presence of deoxycholate and then removing the detergent by gel filtration on Sephadex G-75. The phospholipid-containing factor stimulated the formation of succinoxidase by about 100% over that observed with the two complexes. 4. Isopycnic sucrose gradient centrifugation of succinate dehydrogenase.cytochrome b1 complex, cytochrome oxidase, and the reconstituted succinoxidase gave buoyant densities (p value) as 1.167, 1.229, and 1.194, respectively. 5. Electron microscopic evidence is presented for the vesicular nature of the reconstituted succinoxidase.

摘要

用脱氧胆酸盐将大肠杆菌的膜结合琥珀酸氧化酶分离成三种可溶性成分，即琥珀酸脱氢酶 - 细胞色素b1复合物、细胞色素氧化酶复合物以及一种被鉴定为含磷脂的成分。2. 通过在Amicon膜上过滤、琼脂糖4B柱层析和蔗糖梯度离心对脱氢酶和细胞色素氧化酶复合物进行部分纯化。3. 通过在脱氧胆酸盐存在下将可溶性琥珀酸脱氢酶 - 细胞色素b1复合物与可溶性细胞色素氧化酶复合物混合，然后通过Sephadex G - 75凝胶过滤去除去污剂，实现了膜状琥珀酸氧化酶的重建，该酶通过完整的对氰化物敏感的途径催化琥珀酸被分子氧氧化。含磷脂的成分使琥珀酸氧化酶的形成比两种复合物单独存在时观察到的情况增加了约100%。4. 对琥珀酸脱氢酶 - 细胞色素b1复合物、细胞色素氧化酶和重建的琥珀酸氧化酶进行等密度蔗糖梯度离心，得到的浮力密度（p值）分别为1.167、1.229和1.194。5. 提供了重建的琥珀酸氧化酶呈囊泡状的电子显微镜证据。