Purification and properties of the membrane-bound NADH dehydrogenase from hydrocarbon-grown Acinetobacter calcoaceticus

Abstract

A single NADH dehydrogenase (EC 1.6.99.3) species which seems to be connected with the respiratory chain was localized in the membrane fraction of hydrocarbon-grown Acinetobacter calcoaceticus. The enzyme was completely solubilized by Triton X-100 and purified by chromatography on DEAE-cellulose, dextran blue-Sepharose 6B and Phenylsepharose 4B. SDS-polyacrylamide gel electrophoresis of the purified enzyme showed one band of molecular weight 34 000. The purified enzyme is inactive in the absence of detergent, needs added FMN for activity and is strictly specific for the electron donor NADH. During the course of purification, 0.4 μmol phospholipid per mg protein are retained most of which is cardiolipin with traces of phosphatidylethanolamine and phosphatidylglycerol also being found. The results of lipid substitution experiments using phospholipids purified from A. calcoaceticus are indicative of a specificity for phosphatidylglycerol but the possibility of a dual phospholipid requirement cannot be ruled out.