title: Comparison of recombinant cyclooxygenase-2 to native isoforms: aspirin labeling of the active site.
abstract: 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 H synthase) was expressed in insect cells and purified to > 98% purity. 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.
authors.affiliation: Research Department, CIBA Pharmaceuticals Division, Summit, NJ 07901, USA.
authors.forename: L P
authors.initials: LP
authors.lastname: Wennogle
authors.forename: H
authors.initials: H
authors.lastname: Liang
authors.forename: J C
authors.initials: JC
authors.lastname: Quintavalla
authors.forename: B R
authors.initials: BR
authors.lastname: Bowen
authors.forename: J
authors.initials: J
authors.lastname: Wasvary
authors.forename: D B
authors.initials: DB
authors.lastname: Miller
authors.forename: A
authors.initials: A
authors.lastname: Allentoff
authors.forename: W
authors.initials: W
authors.lastname: Boyer
authors.forename: M
authors.initials: M
authors.lastname: Kelly
authors.forename: P
authors.initials: P
authors.lastname: Marshall
mesh_terms: D000595:Amino Acid Sequence; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D001241:Aspirin / Q000378:metabolism*; D001665:Binding Sites; D002417:Cattle; D015971:Gene Expression Regulation, Enzymologic; D006031:Glycosylation; D006801:Humans; D007700:Kinetics; D008969:Molecular Sequence Data; D010449:Peptide Mapping; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011993:Recombinant Fusion Proteins / Q000302:isolation & purification / Q000378:metabolism; D012694:Serine / Q000378:metabolism
chemical_list: D000596:Amino Acids; D011993:Recombinant Fusion Proteins; D012694:Serine; D011451:Prostaglandin-Endoperoxide Synthases; D001241:Aspirin