Purification and characterization of prostaglandin H synthase-2 from sheep placental cotyledons

Recent identification of a second, inducible form of prostaglandin H synthase (PGHS-2) led to the hypothesis that constitutively expressed PGHS (PGHS-1) is involved in the homeostatic role of eicosanoids, whereas the inducible enzyme is responsible for their inflammatory actions. We report here the purification of PGHS-2 from near-term sheep placental cotyledons. The PGHS-2 from this tissue was purified in multimilligram quantities by a combination of anion-exchange, size-exclusion, and affinity chromatography. This enzyme is different from ovine seminal vesicle PGHS-1 and was characterized as PGHS-2 based on (a) chromatographic properties, (b) immunochemical reactivities with isoenzyme-specific antibodies, (c) amino acid microsequencing, (d) kinetics of reaction with arachidonic acid (Km = 2.1 +/- 0.2 microM vs 8.3 +/- 0.2 microM for ovine PGHS-1), and (e) different sensitivities for several non-steroidal antiinflammatory drugs. Since the first identification of PGHS, ram seminal vesicles served as a rich source of the enzyme (PGHS-1). Our studies establish the sheep placental cotyledons as a rich natural source of PGHS-2.