Characterization of prostaglandin endoperoxide synthase from a human cell line.
Prostaglandin endoperoxide synthase has been purified from the recently established human lung tumor cell line, Lu-65. By gel filtration, the purified enzyme migrated with a relative molecular weight of 115,000, unlike the ovine enzyme, which migrated at 155,000. Two protein bands of 45,000 and 68,000 were seen when the purified Lu-65 enzyme was fractionated under reducing conditions by SDS-polyacrylamide gel electrophoresis; in contrast, purified ovine prostaglandin endoperoxide synthase showed the Mr 68,000 band under the same conditions. The purified Lu-65 enzyme showed both cyclooxygenase and hydroperoxidase activities, and metabolized [3H]arachidonic acid to 3H-labeled products that, when separated by reverse-phase HPLC, co-eluted with authentic prostaglandin D2 and prostaglandin E2. An apparent Km for arachidonic acid of 3 mM was measured for the purified enzyme, and the crude membrane-bound enzyme showed an apparent Km of 1.6 mM. Under the same conditions, an apparent Km of 17 microM was measured for the purified ovine enzyme.
D000818:Animals; D016718:Arachidonic Acid; D001095:Arachidonic Acids / Q000378:metabolism; D002460:Cell Line; D002850:Chromatography, Gel; D002851:Chromatography, High Pressure Liquid; D015232:Dinoprostone; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D007700:Kinetics; D008175:Lung Neoplasms / Q000201:enzymology; D008970:Molecular Weight; D015230:Prostaglandin D2; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis* / Q000378:metabolism; D011457:Prostaglandins D / Q000378:metabolism; D011458:Prostaglandins E / Q000378:metabolism; D012756:Sheep
D001095:Arachidonic Acids; D011457:Prostaglandins D; D011458:Prostaglandins E; D016718:Arachidonic Acid; D011451:Prostaglandin-Endoperoxide Synthases; D015232:Dinoprostone; D015230:Prostaglandin D2