Purification and characterization of recombinant human cyclooxygenase-2
| Autor: | M.D. Percival, Brian P. Kennedy, Gary P. O'Neill, C.J. Vincent, Marc Ouellet, J. Yergey |
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| Rok vydání: | 1994 |
| Předmět: |
Glycosylation
Linolenic acid Protein Conformation Size-exclusion chromatography Detergents Biophysics Vaccinia virus Heme Biology Biochemistry law.invention Substrate Specificity chemistry.chemical_compound law Humans Molecular Biology chemistry.chemical_classification Arachidonic Acid Molecular mass Recombinant Proteins Molecular Weight Oxygen Enzyme chemistry Peroxidases Prostaglandin-Endoperoxide Synthases Spectrophotometry Recombinant DNA Fatty Acids Unsaturated Oxygenases Arachidonic acid Baculoviridae |
| Zdroj: | Archives of biochemistry and biophysics. 315(1) |
| ISSN: | 0003-9861 |
| Popis: | 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 μmol O 2 /min/mg with arachidonic acid which is within the range of values reported for ovine Cox-1. The K m values of arachidonic acid for hCox-2 and ovine Cox-1 are 0.9 and 2.7 μM, 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 γ-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% β-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. |
| Databáze: | OpenAIRE |
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