The NADH dehydrogenase of the respiratory chain of Escherichia coli. I. Properties of the membrane-bound enzyme, its solubilization, and purification to near homogeneity.
The NADH dehydrogenase of the Escherichia coli respiratory chain has been identified by the following properties: (a) its location in membrane vesicles; (b) its inhibition by AMP in a fashion similar to that of the NADH oxidase; (c) its specificity for NADH, but not NADPH, with the same Km for NADH as that of the NADH oxidase; (d) its sensitivity when membrane-bound to inhibition by dicoumarol, rotenone, and 2-heptyl-4-hydroxyquinoline-N-oxide, which are also inhibitors for the NADH oxidase. The NADH-dehydrogenase of the cytosol fraction (assayed as NADH-dichlorphenolindophenol reductase activity) differs substantially from the membrane-bound activity both in substrate specificity and in the inhibitors of the reaction. The respiratory chain NADH dehydrogenase was extracted from isolated membrane vesicle preparations by solubilization in Triton X-100, and was purified in buffers containing that detergent. The purification employed chromatography on DEAE-cellulose, precipitation by 30% ethanol, and chromatography on hydroxyalapatite and DEAE-agarose. The most highly purified preparations of the enzyme were homogeneous in migration on polyacrylamide gels containing Triton X-100, at pH 9.5, where one band accounted for all of the protein and activity. Electrophoresis on polyacrylamide gels containing sodium dodecul sulfate showed 1 band of molecular weight 38,000, which accounted for over 75% of the protein on the gel. Because of requirements for either Triton X-100 or phospholipid for activity of the purified enzyme, it is difficult to estimate the level of purification achieved over isolated membrane vesicles. However, we estimate that the enzyme was purified some 30-fold over membrane vesicles, or some 300-fold over whole cells.
D000249:Adenosine Monophosphate / Q000494:pharmacology; D002455:Cell Division; D002462:Cell Membrane / Q000201:enzymology; D004589:Electrophoresis, Disc; D004926:Escherichia coli / Q000201:enzymology* / Q000378:metabolism; D005947:Glucose / Q000378:metabolism; D007700:Kinetics; D007773:Lactates / Q000378:metabolism; D007785:Lactose / Q000378:metabolism; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification / Q000378:metabolism*; D011092:Polyethylene Glycols / Q000494:pharmacology; D013386:Succinates / Q000378:metabolism
D007773:Lactates; D013386:Succinates; D011092:Polyethylene Glycols; D000249:Adenosine Monophosphate; D009247:NADH, NADPH Oxidoreductases; D005947:Glucose; D007785:Lactose