On the mechanism of inhibition of the neutrophil respiratory burst oxidase by a peptide from the C-terminus of the large subunit of cytochrome b558.
A peptide (RGVHFIF) from near the carboxyl terminus (residues 559-565) of gp91-phox, the large subunit of cytochrome b558, was previously shown to inhibit activation of the respiratory burst oxidase [Kleinberg, M. E., Malech, H. L., & Rotrosen, D. (1990) J. Biol. Chem. 265, 15577-15583]. The peptide has been proposed to compete with gp91-phox binding to p47-phox, one of the cytosolic oxidase components. In the present studies, we have used a semirecombinant system consisting of recombinant cytosolic factors (p47-phox, p67-phox, and Rac1) along with isolated plasma membrane to investigate the mechanism by which the peptide inhibits oxidase activation. In an in vitro translocation model, the peptide inhibited arachidonate-activated translocation of both p47-phox and p67-phox to the plasma membrane. The kinetic mechanism of inhibition was examined. Inhibition was noncompetitive or mixed with respect to not only Rac and p67-phox but also to p47-phox. We suggest that the peptide, rather than competing for cytochrome-p47-phox interactions, inhibits indirectly, perhaps by binding to and altering the conformation of cytochrome b558.
Uhlinger
D J
DJ
Department of Biochemistry, Emory University School of Medicine, Atlanta, Georgia 30322.
Tyagi
S R
SR
Lambeth
J D
JD
D000328:Adult; D001692:Biological Transport; D002474:Cell-Free System; D003573:Cytochrome b Group / Q000378:metabolism*; D003600:Cytosol / Q000378:metabolism; D019204:GTP-Binding Proteins / Q000378:metabolism; D006801:Humans; D066298:In Vitro Techniques; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000037:antagonists & inhibitors* / Q000378:metabolism; D009252:NADPH Dehydrogenase / Q000378:metabolism; D019255:NADPH Oxidases; D009504:Neutrophils / Q000378:metabolism*; D010446:Peptide Fragments / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D016897:Respiratory Burst / Q000502:physiology*; D020744:rac GTP-Binding Proteins
D003573:Cytochrome b Group; D010446:Peptide Fragments; D010750:Phosphoproteins; C079789:neutrophil cytosol factor 67K; C036888:cytochrome b558; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1; C022709:superoxide-forming enzyme; D009252:NADPH Dehydrogenase; D019204:GTP-Binding Proteins; D020744:rac GTP-Binding Proteins