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Titolo:
The p67(phox) activation domain regulates electron flow from NADPH to flavin in flavocytochrome b(558)
Autore:
Nisimoto, Y; Motalebi, S; Han, CH; Lambeth, JD;
Indirizzi:
Emory Univ, Sch Med, Dept Biochem, Atlanta, GA 30322 USA Emory Univ Atlanta GA USA 30322 Med, Dept Biochem, Atlanta, GA 30322 USA Aichi Med Univ, Dept Biochem, Nagakute, Aichi 4801195, Japan Aichi Med Univ Nagakute Aichi Japan 4801195 agakute, Aichi 4801195, Japan
Titolo Testata:
JOURNAL OF BIOLOGICAL CHEMISTRY
fascicolo: 33, volume: 274, anno: 1999,
pagine: 22999 - 23005
SICI:
0021-9258(19990813)274:33<22999:TPADRE>2.0.ZU;2-3
Fonte:
ISI
Lingua:
ENG
Soggetto:
RESPIRATORY BURST OXIDASE; CHRONIC GRANULOMATOUS-DISEASE; CELL-FREE SYSTEM; NEUTROPHIL CYTOCHROME-B; IN-VITRO; EFFECTOR REGION; INSERT REGION; BINDING SITE; RAC-BINDING; COMPLEX;
Tipo documento:
Article
Natura:
Periodico
Settore Disciplinare:
Life Sciences
Citazioni:
53
Recensione:
Indirizzi per estratti:
Indirizzo: Lambeth, JD Emory Univ, Sch Med, Dept Biochem, Atlanta, GA 30322 USA EmoryUniv Atlanta GA USA 30322 iochem, Atlanta, GA 30322 USA
Citazione:
Y. Nisimoto et al., "The p67(phox) activation domain regulates electron flow from NADPH to flavin in flavocytochrome b(558)", J BIOL CHEM, 274(33), 1999, pp. 22999-23005

Abstract

An activation domain in p67(phox) (residues within 199-210) is essential for cytochrome b(558)-dependent activation of NADPH superoxide (O-2(.-)) generation in a cell-free system (Han, C.-H., Freeman, J. L. R., Lee, T., Motalebi, S. A., and Lambeth, J. D. (1998) J. Biol. Chem. 273, 16663-16668). Todetermine the steady state reduction flavin in the presence of highly absorbing hemes, 8-nor-8-S-thioacetamido-FAD ("thioacetamido-FAD") was reconstituted into the flavocytochrome, and the fluorescence of its oxidized form was monitored. Thioacetamido-FAD-reconstituted cytochrome showed lower activity (7% versus 100%) and increased steady state flavin reduction (28 versus<5%) compared with the enzyme reconstituted with native FAD, Omission of p67(phox) decreased the percent steady state reduction of the flavin to 4%, but omission of p47(phox) had little effect. The activation domain on p67(phox) was critical for regulating flavin reduction, since mutations in this region that decreased O-2(.-) generation also decreased the steady state reduction of flavin, Thus, the activation domain on p67(phox) regulates the reductive half-reaction for FAD, This reaction is comprised of the binding of NADPH followed by hydride transfer to the flavin, Kinetic deuterium isotope effects along with K-m values permitted calculation of the K-d for NADPH, (R)-NADPD but not (S)-NADPD showed kinetic deuterium isotope effects on Vand V/K of about 1.9 and 1.5, respectively, demonstrating stereospecificity for the R hydride transfer. The calculated K-d for NADPH was 40 mu M in the presence of wild type p67(phox) and was similar to 55 mu M using the weakly activating p67(phox)(V205A), Thus, the activation domain of p67(phox) regulates the reduction of FAD but has only a small effect on NADPH binding,consistent with a dominant effect on hydride/electron transfer from NADPH to FAD.

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Documento generato il 12/07/20 alle ore 06:50:44