Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system.
Human prostaglandin G/H synthase 1 and 2 were expressed in the baculovirus expression system and purified to high levels. Both enzymes were glycosylated. PGHS-1 appeared to be homogeneous by SDS-PAGE analysis but two closely migrating bands were detected in PGHS-2 preparation which were evidently due to heterogeneity in glycosylation. The amino-acid sequence of the N-termini of both isoforms indicated that the signal sequences were efficiently cleaved by the insect cells. The recombinant human PGHS-1 and PGHS-2 possessed both cyclooxygenase and peroxidase activities. Both had high affinities for arachidonate as substrate and underwent self-inactivation during catalysis. The recombinant isoforms were not pharmacologically identical, since some NSAIDs were selective inhibitors of either PGHS-1 or PGHS-2. This is the first report of high levels of expression and purification of human PGHS isoforms. The recombinant enzymes are invaluable in developing potent PGHS-2 selective inhibitors that may be efficacious anti-inflammatory drugs with no or low levels of toxicity.
Barnett
J
J
Institute of Organic Chemistry, Syntex Discovery Research, Palo Alto, CA 94303.
Chow
J
J
Ives
D
D
Chiou
M
M
Mackenzie
R
R
Osen
E
E
Nguyen
B
B
Tsing
S
S
Bach
C
C
Freire
J
J
D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D016718:Arachidonic Acid / Q000378:metabolism; D016367:Baculoviridae; D001483:Base Sequence; D002460:Cell Line; D003001:Cloning, Molecular; D016861:Cyclooxygenase Inhibitors / Q000494:pharmacology; D017931:DNA Primers; D006031:Glycosylation; D006801:Humans; D007527:Isoenzymes / Q000037:antagonists & inhibitors / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D008969:Molecular Sequence Data; D011451:Prostaglandin-Endoperoxide Synthases / Q000235:genetics* / Q000302:isolation & purification / Q000378:metabolism; D011994:Recombinant Proteins / Q000037:antagonists & inhibitors / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism; D018411:Spodoptera
D000894:Anti-Inflammatory Agents, Non-Steroidal; D016861:Cyclooxygenase Inhibitors; D017931:DNA Primers; D007527:Isoenzymes; D011994:Recombinant Proteins; D016718:Arachidonic Acid; D011451:Prostaglandin-Endoperoxide Synthases