Purification and characterization of recombinant human cyclooxygenase-2.
Recombinant human cyclooxygenase-2 (hCox-2, Prostaglandin G/H synthase-2) has been purified from baculovirus-Sf9 and vaccina virus-Cos-7 cell expression systems. The detergent-solubilized, purified enzyme is heterogeneous in terms of its glycosylation. The vaccinia virus hCox-2 is a mixture of two glycoforms, whereas baculovirus hCox-2 comprises at least four species. The specific cyclooxygenase activities of both enzymes are 43 mumol O2/min/mg with arachidonic acid which is within the range of values reported for ovine Cox-1. The Km values of arachidonic acid for hCox-2 and ovine Cox-1 are 0.9 and 2.7 microM, respectively. Six other C-18 and C-20 fatty acids containing at least one 1,4-cis,cis-pentadiene moiety were also identified as substrates for hCox-2. Linoleic and gamma-linolenic acid were determined by mass spectrometry as being hydroxylated primarily at the C-9 and C-13 positions, whereas linolenic acid was hydroxylated primarily at the C-12 and C-16 positions. hCox-2 binds heme such that maximal activity is observed at a stoichiometry of 1.0 heme per enzyme subunit. The apparent molecular mass of hCox-2, determined by gel filtration chromatography in the presence of 2.0% beta-octylglucoside, is consistent with a dimeric structure. The results of this study indicate that the physical and catalytic properties of recombinant hCox-2 are very similar to that of the extensively studied ovine Cox-1.
Departments of Biochemistry and Molecular Biology and Medicinal Chemistry, Merck Frosst Centre for Therapeutic Research, Pointe-Claire-Dorval, Quebec, Canada.
M D
MD
Percival
M
M
Ouellet
C J
CJ
Vincent
J A
JA
Yergey
B P
BP
Kennedy
G P
GP
O'Neill
D016718:Arachidonic Acid / Q000378:metabolism; D016367:Baculoviridae / Q000235:genetics; D003902:Detergents / Q000494:pharmacology; D005231:Fatty Acids, Unsaturated / Q000378:metabolism; D006418:Heme / Q000378:metabolism; D006801:Humans; D008970:Molecular Weight; D010100:Oxygen / Q000378:metabolism; D010105:Oxygenases / Q000378:metabolism; D010544:Peroxidases / Q000378:metabolism; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D011487:Protein Conformation; D011994:Recombinant Proteins / Q000302:isolation & purification / Q000378:metabolism; D013053:Spectrophotometry; D013379:Substrate Specificity; D014616:Vaccinia virus / Q000235:genetics
D003902:Detergents; D005231:Fatty Acids, Unsaturated; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D006418:Heme; D010544:Peroxidases; D010105:Oxygenases; D011451:Prostaglandin-Endoperoxide Synthases; D010100:Oxygen