Reconstitution of escherichia coli succinoxidase from soluble components.
1. 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.
D002462:Cell Membrane / Q000201:enzymology; D003580:Cytochromes / Q000302:isolation & purification; D003840:Deoxycholic Acid; D003576:Electron Transport Complex IV / Q000302:isolation & purification; D004926:Escherichia coli / Q000201:enzymology*; D008854:Microscopy, Electron; D009097:Multienzyme Complexes / Q000302:isolation & purification; D012995:Solubility; D013385:Succinate Dehydrogenase / Q000302:isolation & purification
D003580:Cytochromes; D009097:Multienzyme Complexes; D003840:Deoxycholic Acid; D013385:Succinate Dehydrogenase; D003576:Electron Transport Complex IV