PMID:3891338
| Citation |
Clarke, DM and Bragg, PD (1985) Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli. Eur. J. Biochem. 149:517-23 |
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| Abstract |
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. |
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| Keywords |
Allosteric Site; Catalysis; Cytoplasm/enzymology; Dicyclohexylcarbodiimide/pharmacology; Electrophoresis, Polyacrylamide Gel; Escherichia coli/enzymology; Hydrogen-Ion Concentration; Kinetics; NAD/analogs & derivatives; NAD/metabolism; NAD/pharmacology; NADH, NADPH Oxidoreductases/isolation & purification; NADP/pharmacology; NADP Transhydrogenases/antagonists & inhibitors; NADP Transhydrogenases/isolation & purification; Oxidation-Reduction/drug effects; Peptide Fragments; Solubility; Spectrometry, Fluorescence |
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