COMPARISON OF RECOMBINANT CYCLOOXYGENASE-2 TO NATIVE ISOFORMS - ASPIRIN LABELING OF THE ACTIVE-SITE

The search for isoform-specific enzyme inhibitors has been the focus o f much recent research effort, Towards this goal, human recombinant cy clooxygenase-2 (EC 1.14.99.1, prostaglandin Il synthase) was expressed in insect cells and purified to >98% parity, Recombinant enzyme was c haracterized both by physical methods and activity measurements and sh own to be fully active with kinetic properties similar to native COX-2 and COX-1, After detergent extraction, the enzyme had hydrodynamic pr operties indistinguishable from native bovine COX-1 and corresponded t o the enzyme dimer as measured with size-exclusion chromatography. Pep tide mapping via Lys-C protease identified a site of N-linked glycosyl ation and the aspirin covalent modification site, In the presence of h eme, aspirin-specifically acetylated Ser-516, The enzyme will be suita ble for biophysical studies and may lead to isoform-specific enzyme in hibitors.