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Involvement of membrane charges in constituting the active form of NADPH oxidase in guinea pig polymorphonuclear
1Department of Physiological Chemistry, Hiroshima University School of Medicine, Japan.
Abstract:
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).
Insights
Myristylamine inhibits NADPH oxidase in immune cells by disrupting the interaction between cytosolic factors and cell membranes. This disruption, caused by positive charges, reduces enzyme activity.
Area of Science:
- Biochemistry
- Cell Biology
- Immunology
Background:
- NADPH oxidase is a crucial enzyme in immune cells, responsible for generating reactive oxygen species.
- Its activity is regulated by the translocation and interaction of cytosolic and membrane-bound components.
Purpose of the Study:
- To investigate the mechanism by which myristylamine inhibits NADPH oxidase activity.
- To elucidate the role of charge interactions in the regulation of NADPH oxidase.
Main Methods:
- Studied NADPH oxidase activity in guinea pig polymorphonuclear leukocytes (PMNL) stimulated with phorbol 12-myristate 13-acetate (PMA).
- Utilized a cell-free system with reconstituted plasma membrane and cytosolic fractions.
- Examined the effects of myristylamine, myristic acid, and glutaraldehyde on enzyme activity.
Main Results:
- Myristylamine, a positively charged molecule, inhibited NADPH oxidase activity in both intact PMNL and cell-free systems.
- The inhibitory effect was partially reversed by negatively charged myristic acid.
- Glutaraldehyde pretreatment stabilized the enzyme activity against myristylamine inhibition, suggesting a role for protein interactions.
Conclusions:
- Myristylamine inhibits NADPH oxidase by causing the dissociation of cytosolic activation factors from the plasma membrane.
- This dissociation is likely mediated by the positive charge of myristylamine disrupting membrane-protein interactions.
- Glutaraldehyde stabilizes the complex by crosslinking proteins, preventing this dissociation.