Nitroarachidonic acid, a novel peroxidase inhibitor of prostaglandin endoperoxide H synthases 1 and 2 |
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Authors: | Trostchansky Andrés Bonilla Lucía Thomas Christopher P O'Donnell Valerie B Marnett Lawrence J Radi Rafael Rubbo Homero |
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Institution: | From the ‡Departamento de Bioquímica and ;the §Center for Free Radical and Biomedical Research, Facultad de Medicina, Montevideo, Uruguay, ;the ¶Department of Infection, Immunity and Biochemistry, Cardiff University, Cardiff CF4 4XN, United Kingdom, and ;the ‖Department of Biochemistry, Vanderbilt University School of Medicine, Nashville, Tennessee 37232 |
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Abstract: | Prostaglandin endoperoxide H synthase (PGHS) catalyzes the oxidation of arachidonate to prostaglandin H(2). We have previously synthesized and chemically characterized nitroarachidonic acid (AANO(2)), a novel anti-inflammatory signaling mediator. Herein, the interaction of AANO(2) with PGHS was analyzed. AANO(2) inhibited oxygenase activity of PGHS-1 but not PGHS-2. AANO(2) exhibited time- and concentration-dependent inhibition of peroxidase activity in both PGHS-1 and -2. The plot of k(obs) versus AANO(2) concentrations showed a hyperbolic function with k(inact) = 0.045 s(-1) and K(i)(*app) = 0.019 μM for PGHS-1 and k(inact) = 0.057 s(-1) and K(i)(*app) = 0.020 μM for PGHS-2. Kinetic analysis suggests that inactivation of PGHS by AANO(2) involves two sequential steps: an initial reversible binding event (described by K(i)) followed by a practically irreversible event (K(i)(*app)) leading to an inactivated enzyme. Inactivation was associated with irreversible disruption of heme binding to the protein. The inhibitory effects of AANO(2) were selective because other nitro-fatty acids tested, such as nitrooleic acid and nitrolinoleic acid, were unable to inhibit enzyme activity. In activated human platelets, AANO(2) significantly decreased PGHS-1-dependent thromboxane B(2) formation in parallel with a decrease in platelet aggregation, thus confirming the biological relevance of this novel inhibitory pathway. |
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Keywords: | Arachidonic Acid Cyclooxygenase (COX) Pathway Enzyme Inhibitors Lipid Oxidation Nitric Oxide Platelet |
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