Energy-linked nicotinamide nucleotide transhydrogenase. Properties of proton-translocating mitochondrial transhydrogenase from beef heart purified by fast protein liquid chromatography.

Persson, Bengt L.; Enander, Karin; Tang, HL; Rydström, Jan

doi:10.1016/s0021-9258(17)39776-4

Cited by 41 publications

(1 citation statement)

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“…Proton Pump Activity. Initial relative rates of proton pumping catalyzed by freshly prepared membrane vesicles obtained from transformed TGI or JM109 cells were assayed fluorimetrically as quenching of ACMA fluorescence (using excitation at 418 nm and emission at 480 nm) or as quenching of quinacrine fluorescence (using excitation at 430 nm and emission at 505 nm) (Persson et al, 1984; Clarke & Bragg, 1985a). All activity-dependent quenching was sensitive to uncouplers, e.g., CCCP.…”

Section: Methodsmentioning

confidence: 99%

Site-directed mutagenesis of tyrosine residues at nicotinamide nucleotide binding sites of Escherichia coli transhydrogenase

Olausson

Hultman²,

Holmberg³

et al. 1993

Biochemistry

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Nicotinamide nucleotide transhydrogenase (E.C.1.6.1.1) from Escherichia coli was investigated with respect to the role of specific conserved tyrosine residues of putative substrate-binding regions. The enzyme from E. coli is made up of two subunits, alpha (510 residues) and beta (462 residues). The corresponding enzyme from bovine mitochondria is a single polypeptide (1043 residues) whose N-terminal region corresponds to the alpha subunit and whose C-terminal region corresponds to the beta subunit. Tyrosines 245 and 1006 of the mitochondrial enzyme have been shown to react selectively with 5'-(p-fluorosulfonylbenzoyl)adenosine with inactivation of the enzyme. In E. coli these residues correspond to tyrosine 226 of the alpha subunit and tyrosine 431 of the beta subunit. In addition, tyrosine 315 of the beta subunit is of interest since mutation of an adjacent residue (glycine 314) leads to inactivation [Ahmad, S., Glavas, N. A., & Bragg, P. D. (1992) Eur. J. Biochem. 207, 733-739]. In order to assess the role of the aforementioned conserved tyrosine residues in the mechanism and structure of transhydrogenases, these were replaced by site-specific mutagenesis, using the cloned and overexpressed E. coli transhydrogenase genes [Clarke, D. M., & Bragg, P. D. (1985) J. Bacteriol. 162, 367-373]. Phenylalanine mutants of all three tyrosine residues showed approximately 50% activity or more with regard to catalytic activity assayed as reduction of 3-acetylpyridine-NAD+ by NADPH. These mutants were also active in proton pumping assayed as quenching of 9-methoxy-6-chloro-2-aminoacridine or quinacrine fluorescence.(ABSTRACT TRUNCATED AT 250 WORDS)

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Section: Methodsmentioning

confidence: 99%