Purification and properties of prostaglandin H-E isomerase from the cytosol of human brain: identification as anionic forms of glutathione S-transferase.
Prostaglandin H-E isomerase (EC 5.3.99.3) was purified from human brain cytosol. Purification was by ammonium sulfate fractionation, diethylaminoethyl-Sepharose chromatography, gel filtration on a BioGel P-100 column, GSH-agarose chromatography, and MonoQ chromatography. The activity was eluted in two peaks from the MonoQ column, which were designated peaks 1 and 2. The molecular weights of peaks 1 and 2, determined by gel filtration, were 42,000 and 44,000, respectively. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, peak 1 showed two bands at the molecular weights of 24,500 and 25,000, and peak 2 showed a single band at the molecular weight of 25,000, results suggesting that both were dimeric proteins. The pI values of both enzymes were approximately 5.4. The enzymes catalyzed selective conversion of prostaglandin H2 to prostaglandin E2. The Km values for prostaglandin H2 of peaks 1 and 2 were 147 and 308 microM, respectively, and the Vmax values were 380 and 720 nmol/min/mg of protein, respectively. GSH was required for the catalysis of both enzymes, and no other sulfhydryl compounds could support the reaction. A part of glutathione S-transferase (EC 2.5.1.18) was copurified with peaks 1 and 2 of prostaglandin H-E isomerase. Prostaglandin H-E isomerase activity of peak 2 enzyme was competitively inhibited by 1-chloro-2,4-dinitrobenzene, a substrate of glutathione S-transferase. These results suggested that prostaglandin H-E isomerases in human brain cytosol were identical with anionic forms of glutathione S-transferase.
Ogorochi
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T
Ujihara
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M
Narumiya
S
S
D000818:Animals; D001921:Brain / Q000201:enzymology*; D002845:Chromatography; D003600:Cytosol / Q000201:enzymology; D015232:Dinoprostone; D004591:Electrophoresis, Polyacrylamide Gel; D005978:Glutathione / Q000494:pharmacology; D005982:Glutathione Transferase; D006801:Humans; D019746:Intramolecular Oxidoreductases; D007526:Isoelectric Point; D007535:Isomerases / Q000037:antagonists & inhibitors / Q000302:isolation & purification* / Q000378:metabolism; D007700:Kinetics; D008251:Macaca; D013058:Mass Spectrometry; D008970:Molecular Weight; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism; D044262:Prostaglandin H2; D000071876:Prostaglandin-E Synthases; D011458:Prostaglandins E / Q000378:metabolism; D011463:Prostaglandins H / Q000378:metabolism; D011817:Rabbits; D051381:Rats; D013347:Subcellular Fractions / Q000201:enzymology; D013552:Swine
D011450:Prostaglandin Endoperoxides, Synthetic; D011458:Prostaglandins E; D011463:Prostaglandins H; D044262:Prostaglandin H2; D005982:Glutathione Transferase; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases; D005978:Glutathione; D015232:Dinoprostone