Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli.
The nicotinamide nucleotide transhydrogenase of Escherichia coli has been purified from cytoplasmic membranes by pre-extraction of the membranes with sodium cholate and Triton X-100, solubilization of the enzyme with sodium deoxycholate in the presence of 1 M potassium chloride, and centrifugation through a 1.1 M sucrose solution. The purified enzyme consists of two subunits, alpha and beta, of apparent Mr 50000 and 47000. During transhydrogenation between NADPH and 3-acetylpyridine adenine dinucleotide by both the purified enzyme reconstituted into liposomes and the membrane-bound enzyme, a pH gradient is established across the membrane as indicated by the quenching of the fluorescence of 9-aminoacridine. Treatment of transhydrogenase with N,N'-dicyclohexylcarbodiimide results in an inhibition of proton pump activity and transhydrogenation, suggesting that proton translocation and catalytic activities are obligatory linked. NADH protected the enzyme against inhibition by N,N'-dicyclohexylcarbodiimide, while NADP, and to a lesser extent NADPH, appeared to increase the rate of inhibition. [14C]Dicyclohexylcarbodiimide preferentially labelled the 50000-Mr subunit of the transhydrogenase enzyme. The presence of an allosteric binding site which reacts with NADH, but not with reduced 3-acetylpyridine adenine dinucleotide, has been demonstrated.
D000495:Allosteric Site; D002384:Catalysis; D003593:Cytoplasm / Q000201:enzymology; D004024:Dicyclohexylcarbodiimide / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004926:Escherichia coli / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D009243:NAD / Q000031:analogs & derivatives / Q000378:metabolism / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000302:isolation & purification*; D009249:NADP / Q000494:pharmacology; D009250:NADP Transhydrogenases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D010084:Oxidation-Reduction / Q000187:drug effects; D010446:Peptide Fragments; D012995:Solubility; D013050:Spectrometry, Fluorescence
D010446:Peptide Fragments; D009243:NAD; D009249:NADP; D004024:Dicyclohexylcarbodiimide; C005941:3-acetylpyridine adenine dinucleotide; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases