Involvement of membrane charges in constituting the active form of NADPH oxidase in guinea pig polymorphonuclear leukocytes.
NADPH oxidase activity in a membrane fraction prepared from phorbol 12-myristate 13-acetate (PMA)-stimulated guinea pig polymorphonuclear leukocytes (PMNL) was inhibited by positively charged myristylamine. The inhibitory effect of myristylamine was significantly suppressed by simultaneous addition of a negatively charged fatty acid, such as myristic acid. However, the suppression by myristylamine was not sufficiently restored when myristic acid was added later. On the other hand, pretreatment of PMA-stimulated PMNL with glutaraldehyde, a protein crosslinking reagent, stabilized NADPH oxidase activity against inhibition by myristylamine, but not against that by p-chloromercuribenzenesulfonic acid. In a cell-free system of reconstituted plasma membrane and cytosolic fractions prepared from unstimulated PMNL, arachidonic acid-stimulated NADPH oxidase activity was also inhibited by myristylamine. During the activation of NADPH oxidase by PMA in intact PMNL and by arachidonic acid in the cell-free system, cytosolic activation factor(s) translocated to plasma membranes. The bound cytosolic activation factor(s) was released from the membranes by myristylamine, accompanied by a loss of NADPH oxidase activity. It is plausible from these results that the inhibitory effect of alkylamine on NADPH oxidase is due to induction of the decoupling and/or dissociation of the cytosolic activation component(s) from the activated NADPH oxidase complex by increments of positive charges in the membranes, and that the glutaraldehyde treatment prevents the dissociation of component(s).
Ohtsuka
T
T
Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
Hiura
M
M
Ozawa
M
M
Okamura
N
N
Nakamura
M
M
Ishibashi
S
S
D000588:Amines / Q000494:pharmacology; D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000494:pharmacology; D002458:Cell Fractionation; D002462:Cell Membrane / Q000201:enzymology; D004789:Enzyme Activation; D005976:Glutaral / Q000494:pharmacology; D006168:Guinea Pigs; D007700:Kinetics; D009247:NADH, NADPH Oxidoreductases / Q000097:blood*; D019255:NADPH Oxidases; D009504:Neutrophils / Q000187:drug effects / Q000201:enzymology* / Q000502:physiology; D013755:Tetradecanoylphorbol Acetate
D000588:Amines; D001095:Arachidonic Acids; D016718:Arachidonic Acid; D009247:NADH, NADPH Oxidoreductases; D019255:NADPH Oxidases; D013755:Tetradecanoylphorbol Acetate; C020514:N-tetradecylamine; D005976:Glutaral