Nitric oxide inactivates NADPH oxidase in pig neutrophils by inhibiting its assembling process.
The effects of nitric oxide (NO) on superoxide (O-2) generation of the NADPH oxidase in pig neutrophils were studied. NO dose-dependently suppressed O-2 generation of both neutrophil NADPH oxidase and reconstituted NADPH oxidase. Effects of NO on NADPH-binding site and the redox centers including FAD and low spin heme in cytochrome b558 and the electron transfer rates from NADPH to heme via FAD were examined under anaerobic conditions. Both reaction rates and the Km value for NADPH were unchanged by NO. Visible and EPR spectra of cytochrome b558 showed that the structure of heme was unchanged by NO, indicating that NO does not affect the redox centers of the oxidase. In reconstituted NADPH oxidase system, NO did not inhibit O-2 generation of the oxidase when added after activation. The addition of NO to the membrane component or the cytosol component inhibited the activity by 24.0 +/- 5.3 or 37.4 +/- 7.1%, respectively. The addition of NO during the activation process or to the cytosol component simultaneously with myristate inhibited the activity by 74.0 +/- 5.2 or 70.0 +/- 8.3%, respectively, suggesting that cytosol protein(s) treated with myristate becomes susceptible to NO. Peroxynitrite did not interfere with O-2 generation.
Fujii
H
H
Department of Inflammation Research, Tokyo Metropolitan Institute of Medical Science, 18-22, Honkomagome 3-chome, Bunkyo-ku, Tokyo 113, Japan. fujii@rinshoken.or.jp
Ichimori
K
K
Hoshiai
K
K
Nakazawa
H
H
D000818:Animals; D003573:Cytochrome b Group / Q000378:metabolism; D004578:Electron Spin Resonance Spectroscopy; D005182:Flavin-Adenine Dinucleotide / Q000378:metabolism; D007700:Kinetics; D019814:Myristic Acid / Q000378:metabolism; D009249:NADP / Q000378:metabolism; D019255:NADPH Oxidases / Q000037:antagonists & inhibitors*; D009504:Neutrophils / Q000201:enzymology*; D009566:Nitrates / Q000378:metabolism; D009569:Nitric Oxide / Q000494:pharmacology*; D017510:Protein Folding; D013054:Spectrophotometry, Atomic; D013481:Superoxides / Q000378:metabolism; D013552:Swine
D003573:Cytochrome b Group; D009566:Nitrates; D019814:Myristic Acid; D013481:Superoxides; D005182:Flavin-Adenine Dinucleotide; C039642:peroxynitric acid; D009569:Nitric Oxide; D009249:NADP; C036888:cytochrome b558; D019255:NADPH Oxidases