Expression and selective inhibition of the constitutive and inducible forms of human cyclo-oxygenase.
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 mumol of O2/mg with a Km of 13.8 microM for arachidonate and Vmax. of 1500 nmol of O2/nmol of enzyme, whereas hCOX-2 had a specific activity of 12.2 mumol of O2/mg with a Km of 8.7 microM for arachidonate and a Vmax. 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.
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prostaglandins mefenamic acid steroidal O2 arachidonic acid arachidonate O2 arachidonate O2 Indomethacin NS-398 Dup-697 NS-398 Dup-697 indomethacin hCOX-1 hCOX-2 cyclo-oxygenase cyclo-oxygenase COX-1 COX-2 cytokines Constitutive and inducible forms of human cyclo-oxygenase hCOX-1 hCOX-2 hCOX-1 hCOX-2 hCOX-1 hCOX-2 hCOX-2 hCOX-2 constitutive and inducible forms of human cyclo-oxygenase