Decavanadate inhibits the cell-free activation of neutrophil NADPH oxidase without affecting tyrosine phosphorylation.
NADPH oxidase was activated by arachidonate in a cell-free system consisting of membrane and cytosol fractions prepared from guinea pig neutrophils. Vanadate apparently inhibited the NADPH oxidase activity in the cell-free system (IC50=2 microM) without phosphotyrosine accumulation. The pH dependency and stability of the inhibitory effect observed for vanadate solution indicated that decavanadate, an isopolyanion of vanadate, was responsible for the inhibition. Pervanadate (vanadyl hydroperoxide) also inhibited the oxidase activity but at a higher concentration (IC50=0.2 mM). Decavanadate lowered the Vmax but did not affect the Km value of NADPH oxidase for NADPH. Decavanadate inhibited the activation process of NADPH oxidase but not the oxidase activity itself. Decavanadate-pretreatment of membrane and cytosol fractions irreversibly decreased the abilities of both fractions to activate NADPH oxidase in the cell-free system. Translocation of p47-phox, one of the cytosolic activation factors of NADPH oxidase, from cytosol to membrane, was little affected by decavanadate. These results suggest that decavanadate inhibits the activation of NADPH oxidase in the cell-free system without affecting the phosphotyrosine phosphatase, and that decavanadate can bind to both the membrane and cytosolic activation factors when they are in a dormant state, but not to the active oxidase complex.
Okamura
N
N
Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
Sakai
T
T
Nishimura
Y
Y
Sakai
M
M
Araki
S
S
Yamaguchi
M
M
Ishibashi
S
S
D000595:Amino Acid Sequence; D000818:Animals; D001692:Biological Transport; D002474:Cell-Free System; D002478:Cells, Cultured; D004789:Enzyme Activation; D005260:Female; D006168:Guinea Pigs; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008969:Molecular Sequence Data; D019255:NADPH Oxidases / Q000378:metabolism*; D009504:Neutrophils / Q000187:drug effects / Q000378:metabolism; D010750:Phosphoproteins / Q000378:metabolism; D010766:Phosphorylation; D014443:Tyrosine / Q000378:metabolism*; D014638:Vanadates / Q000494:pharmacology*
D010750:Phosphoproteins; D014638:Vanadates; D014443:Tyrosine; D019255:NADPH Oxidases; C060505:neutrophil cytosolic factor 1