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.