Abstract
Prostaglandin H synthase oxidizes arachidonic acid to prostaglandin G2 (PGG2) via its cyclooxygenase activity and reduces PGG2 to prostaglandin H2 by its peroxidase activity. The purpose of this study was to determine if endogenously generated PGG2 is the preferred substrate for the peroxidase compared with exogenous PGG2. Arachidonic acid and varying concentrations of exogenous PGG2 were incubated with ram seminal vesicle microsomes or purified prostaglandin H synthase in the presence of the reducing cosubstrate, aminopyrine. The formation of the aminopyrine cation free radical (AP.+) served as an index of peroxide reduction. The simultaneous addition of PGG2 with arachidonic acid did not alter cyclooxygenase activity of ram seminal vesicle microsomes or the formation of the AP.+. This suggests that the formation of AP.+, catalyzed by the peroxidase, was supported by endogenous endoperoxide formed from arachidonic acid oxidation rather than by the reduction of exogenous PGG2. In addition to the AP.+ assay, the reduction of exogenous versus endogenous PGG2 was studied by using [5,6,8,9,11,12,14,15-2H]arachidonic acid and unlabeled PGG2 as substrates, with gas chromatography-mass spectrometry techniques to measure the amount of reduction of endogenous versus exogenous PGG2. Two distinct results were observed. With ram seminal vesicle microsomes, little reduction of exogenous PGG2 was observed even under conditions in which all of the endogenous PGG2 was reduced. In contrast, studies with purified prostaglandin H synthase showed complete reduction of both exogenous and endogenous PGG2 using similar experimental conditions. Our findings indicate that PGG2 formed by the oxidation of arachidonic acid by prostaglandin H synthase in microsomal membranes is reduced preferentially by prostaglandin H synthase.
Highlights
Prostaglandin H synthase oxidizes arachidonic acid prostaglandin G2 (PGG2)’ by the cyclooxygenase portion of to prostaglandin GZ (PGG2)via itscyclooxygenase ac- prostaglandin H synthase (1, 2)
Inthestudy arachidonic acid did notalter cyclooxygenase activity presented here we asked thequestion: Is the PGG, generated of ram seminalvesicle microsomesor the formationof from arachidonic acid by the cyclooxygenase activity of prosthe AP”. This suggests that the formation of AP’+, taglandin H synthase reduced preferentially over exogenous catalyzed by the peroxidase, was supportedby endog- PGG,? To answer this questionwe measured the conversion enous endoperoxide formed from arachidonic acid ox-of octadeuterated arachidonic acid to &-PGH2 by purified pros
In addition to the A P ” assay, the reduction of taglandin H synthase and prostaglandin H synthase present exogenous versus endogenous PGGz was studied by in ram seminal vesicle microsomes in the presence and abusing [5,6,8,9,11,12,14,15-2H]arachidoniaccidand sence of exogenous &-PGG
Summary
These conditions, PGG, and PGH, decompose almost exclusively to Metabolism of Arachidonic Acid by Prostaglandin H Syn-. Similar results were obtained with measured with a Clark-type electrode, and concentrations of endogenously formed PGG, were calculated based on the incorporationof 2 mol of oxygen into 1 mol of arachidonic acid during the formation dopw dg PGHZ of PGG, (Scheme 1).After 90 s the reactions were stopped by the addition of 4 ml of ice-cold ethanol, and 0.05 pCi of 15-[3H]hydrox-. PGG, is reduced to prostaglandin H, by the pera n internal standard for derivatization and GC-MS procedures, and oxidase of prostaglandin H synthase, and PGH, decomposes samples were evaporatedtodrynessandreconstitutedin a small nonenzymatically to prostaglandin E,.
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