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.
Johnson
J L
JL
Division of Bioorganic Chemistry, Cayman Chemical Company, Ann Arbor, Michigan 48108, USA.
Wimsatt
J
J
Buckel
S D
SD
Dyer
R D
RD
Maddipati
K R
KR
D000595:Amino Acid Sequence; D000818:Animals; D000894:Anti-Inflammatory Agents, Non-Steroidal / Q000494:pharmacology; D005260:Female; D006801:Humans; D015151:Immunoblotting; D007527:Isoenzymes / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D008297:Male; D008969:Molecular Sequence Data; D010920:Placenta / Q000201:enzymology*; D011247:Pregnancy; D011451:Prostaglandin-Endoperoxide Synthases / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D011994:Recombinant Proteins / Q000187:drug effects; D012669:Seminal Vesicles / Q000201:enzymology; D017421:Sequence Analysis; D017386:Sequence Homology, Amino Acid; D012727:Sex Characteristics; D012756:Sheep
D000894:Anti-Inflammatory Agents, Non-Steroidal; D007527:Isoenzymes; D011994:Recombinant Proteins; D011451:Prostaglandin-Endoperoxide Synthases