Studies on the reduction of endogenously generated prostaglandin G2 by prostaglandin H synthase.
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.
Eling
T E
TE
Laboratory of Molecular Biophysics, National Institute of Environmental Health Sciences, Research Triangle Park, North Carolina 27709.
Glasgow
W C
WC
Curtis
J F
JF
Hubbard
W C
WC
Handler
J A
JA
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism*; D002851:Chromatography, High Pressure Liquid; D015232:Dinoprostone / Q000378:metabolism; D008401:Gas Chromatography-Mass Spectrometry; D008297:Male; D008861:Microsomes / Q000201:enzymology; D010084:Oxidation-Reduction; D011451:Prostaglandin-Endoperoxide Synthases / Q000378:metabolism*; D011462:Prostaglandins G / Q000378:metabolism*; D012669:Seminal Vesicles / Q000201:enzymology; D012756:Sheep
D001095:Arachidonic Acids; D011462:Prostaglandins G; D016718:Arachidonic Acid; C038291:prostaglandin G2; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone