Purification, characterization and selective inhibition of human prostaglandin G/H synthase 1 and 2 expressed in the baculovirus system

Biochim Biophys Acta. 1994 Nov 16;1209(1):130-9. doi: 10.1016/0167-4838(94)90148-1.

Abstract

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.

MeSH terms

  • Amino Acid Sequence
  • Animals
  • Anti-Inflammatory Agents, Non-Steroidal / pharmacology
  • Arachidonic Acid / metabolism
  • Baculoviridae
  • Base Sequence
  • Cell Line
  • Cloning, Molecular
  • Cyclooxygenase Inhibitors / pharmacology
  • DNA Primers
  • Glycosylation
  • Humans
  • Isoenzymes / antagonists & inhibitors
  • Isoenzymes / genetics*
  • Isoenzymes / isolation & purification
  • Isoenzymes / metabolism
  • Molecular Sequence Data
  • Prostaglandin-Endoperoxide Synthases / genetics*
  • Prostaglandin-Endoperoxide Synthases / isolation & purification
  • Prostaglandin-Endoperoxide Synthases / metabolism
  • Recombinant Proteins / antagonists & inhibitors
  • Recombinant Proteins / genetics
  • Recombinant Proteins / isolation & purification
  • Recombinant Proteins / metabolism
  • Spodoptera

Substances

  • Anti-Inflammatory Agents, Non-Steroidal
  • Cyclooxygenase Inhibitors
  • DNA Primers
  • Isoenzymes
  • Recombinant Proteins
  • Arachidonic Acid
  • Prostaglandin-Endoperoxide Synthases