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 of much recent research effort, Towards this goal, human recombinant cyclooxygenase-2 (EC 1.14.99.1, prostaglandin Il synthase) was expressed in insect cells and purified to >98% parity, Recombinant enzyme was characterized both by physical methods and activity measurements and shown to be fully active with kinetic properties similar to native COX-2 and COX-1, After detergent extraction, the enzyme had hydrodynamic properties indistinguishable from native bovine COX-1 and corresponded to the enzyme dimer as measured with size-exclusion chromatography. Peptide mapping via Lys-C protease identified a site of N-linked glycosylation and the aspirin covalent modification site, In the presence of heme, aspirin-specifically acetylated Ser-516, The enzyme will be suitable for biophysical studies and may lead to isoform-specific enzyme inhibitors.