Purification and properties of reconstitutively active nicotinamide nucleotide transhydrogenase of Escherichia coli

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, α and β, 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. Copyright © 1985, Wiley Blackwell. All rights reserved