NBD-Cl modification of essential residues in mitochondrial nicotinamide nucleotide transhydrogenase from bovine heart.
Modification of mitochondrial nicotinamide nucleotide transhydrogenase (NADPH: NAD+ oxidoreductase, EC 1.6.1.1) with 4-chloro-7-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl), followed by measurement of the absorption or fluorescence of the transhydrogenase-NBD adducts, resulted in a biphasic labelling of approx. 4-6 sulfhydryls, presumably cysteine residues. Of these 1-2 (27%) were fast-reacting and 3-4 (73%) slow-reacting sulfhydryls. In the presence of substrates, e.g., NADPH, the labelling was monophasic and all sulfhydryls were fast-reacting, suggesting that the modified sulfhydryls are predominantly localized peripheral to the NAD(P)(H)-binding sites. The rates of modification allowed the calculation of the rate constants for each phase of the labelling. Both in the absence and in the presence of a substrate, e.g., NADPH, the extent of labelling essentially parallelled the inhibition of transhydrogenase activity. Attempts to reactivate transhydrogenase by reduction of labelled sulfhydryls were not successful. Photo-induced transfer of the NBD adduct in partially inhibited transhydrogenase, from the sulfhydryls to reactive NH2 groups of amino-acid residue(s), identified as lysine residue(s), was parallelled by an inhibition of the residual transhydrogenase activity. It is suggested that a lysine localized close to the fast-reacting NBD-Cl-reactive sulfhydryl groups is essential for activity.
D009327:4-Chloro-7-nitrobenzofurazan / Q000494:pharmacology*; D000818:Animals; D002417:Cattle; D055598:Chemical Phenomena; D002621:Chemistry; D003545:Cysteine; D007700:Kinetics; D008239:Lysine; D008929:Mitochondria, Heart / Q000201:enzymology*; D009243:NAD / Q000494:pharmacology; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors*; D009249:NADP / Q000494:pharmacology; D009250:NADP Transhydrogenases / Q000037:antagonists & inhibitors*; D010069:Oxadiazoles / Q000494:pharmacology*; D010777:Photochemistry; D013050:Spectrometry, Fluorescence; D013053:Spectrophotometry; D013438:Sulfhydryl Compounds
D010069:Oxadiazoles; D013438:Sulfhydryl Compounds; D009243:NAD; D009249:NADP; D009247:NADH, NADPH Oxidoreductases; D009250:NADP Transhydrogenases; D009327:4-Chloro-7-nitrobenzofurazan; D008239:Lysine; D003545:Cysteine