Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase

J. K. Gierse, S. D. Hauser, D. P. Creely, C. Koboldt, S. H. Rangwala, P. C. Isakson, K. Seibert

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Abstract

The enzyme cyclo-oxygenase catalyses the oxygenation of arachidonic acid, leading to the formation of prostaglandins. Recently two forms of cyclo-oxygenase have been described: a constitutive (COX-1) enzyme present in most cells and tissues, and an inducible (COX-2) isoenzyme observed in many cells in response to pro-inflammatory cytokines. Constitutive and inducible forms of human cyclo-oxygenase (hCOX-1 and hCOX-2) were cloned and expressed in insect cells, utilizing a baculovirus expression system. hCOX-1 had a specific activity of 18.8 μmol of O2/mg with a K(m) of 13.8 μM for arachidonate and V(max.) of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 μmol of O2/mg with a K(m) of 8.7 μM for arachidonate and a V(max.) of 1090 nmol of O2/nmol of enzyme. Indomethacin inhibited both hCOX-1 and hCOX-2, whereas NS-398 and Dup-697 selectively inhibited hCOX-2. Both NS-398 and Dup-697 exhibited time-dependent inactivation of hCOX-2, as did indomethacin on both enzymes. The competitive inhibitor of hCOX-1, mefenamic acid, also displayed competitive inhibition of hCOX-2. These results demonstrate the ability to generate selective non-steroidal anti-inflammatory drugs (NSAIDs), which could provide useful improvement therapeutically in the treatment of chronic inflammatory disease.

Original languageEnglish
Pages (from-to)479-484
Number of pages6
JournalBiochemical Journal
Volume305
Issue number2
DOIs
StatePublished - 1995

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