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Comparison of recombinant cyclooxygenase‐2 to native isoforms: aspirin labeling of the active site
Author(s) -
Lawrence P. Wennogle,
Hongbin Liang,
Joseph Quintavalla,
Benjamin R. Bowen,
James Wasvary,
Donna B. Miller,
Albin Allentoff,
William F. Boyer,
Michele A. Kelly,
Paul Marshall
Publication year - 1995
Publication title -
febs letters
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.593
H-Index - 257
eISSN - 1873-3468
pISSN - 0014-5793
DOI - 10.1016/0014-5793(95)00930-8
Subject(s) - enzyme , biochemistry , recombinant dna , active site , chemistry , gene isoform , enzyme assay , protease , peptide , cyclooxygenase , enzyme kinetics , glycosylation , gene
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.