Purification of the reduced nicotinamide adenine dinucleotide dehydrogenase from membranes of Acholeplasma laidlawii.
The ethanol-extracted respiratory chain-linked NADH dehydrogenase of Acholeplasma laidlawii has been purified 25-35-fold. This purification involved delipidation of the ethanol-extracted minute non-sedimentable membrane fragments by detergent treatment and gel filtration on Bio-Gel P-200. Sodium deoxycholate-sucrose density gradient centrifugation was followed by dialysis of the active NADH dehydrogenase fractions which caused flocculation of 60% of the membrane proteins while the NADH dehydrogenase remained suspended. Poylacrylamide gel electrophoresis of the purified NADH dehydrogenase gave one major and two minor bands after staining with Coomassie Blue. The purified enzyme gave straight line kinetics in Lineweaver-Burk plots and a Km = 0.510 mM and V = 0.236 mumol/min. Fatty acid supplementation of A. laidlawii membranes had negligible effect on the membrane-bound or ethanol-extracted dehydrogenase, but substantiated the values of the Km and V. Purification, however, altered the constants by 2-4-fold, suggesting that alteration of the microenvironment or fragmentation of the dehydrogenase was significant. The purified dehydrogenase was very susceptible to a rapid inhibition was much slower (90 min) and less complete. Consideration of published purification procedures of NADH dehydrogenase strongly suggested that the purified A. laidlawii respiratory chian-linked NADH dehydrogenase was over 90% pure and certainly one of the most purified respiratory chain-linked bacterial NADH dehydrogenases.
D000128:Acholeplasma laidlawii / Q000201:enzymology*; D002413:Cations, Divalent; D002462:Cell Membrane / Q000201:enzymology; D005292:Ferricyanides / Q000494:pharmacology; D007501:Iron / Q000494:pharmacology; D007700:Kinetics; D008970:Molecular Weight; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification* / Q000378:metabolism
D002413:Cations, Divalent; D005292:Ferricyanides; D007501:Iron; D009247:NADH, NADPH Oxidoreductases