Immunochemical and kinetic evidence for two different prostaglandin H-prostaglandin E isomerases in sheep vesicular gland microsomes.
Splenic lymphocytes from mice immunized with a partially purified prostaglandin (PG) H-PGE isomerase from sheep vesicular glands were fused with SP2/0-Ag14 myeloma cells. Two spleen cell-myeloma hybrids (hei-7 and hei-26) were selected and cloned. The mouse antibodies secreted by the two hybrids, IgG1 (hei-7) and IgG1 (hei-26), caused immunoprecipitation of a maximum of 45 and 22%, respectively, of the solubilized PGH-PGE isomerase activity of sheep vesicular gland; immunoprecipitation of activity by the two antibodies was additive. The antigens reactive with IgG1 (hei-7) and IgG1 (hei-26) were identified as proteins with Mr = 17,500 and 180,000, respectively, by Western transfer blotting or sodium dodecyl sulfate-polyacrylamide gel electrophoresis of immunoprecipitated 125I-labeled microsomes. The PGH-PGE isomerase activities precipitated by IgG1 (hei-7) and IgG1 (hei-26) exhibited different kinetic properties with respect to time course, Km for PGH2, and concentration dependence for GSH. No significant GSH-S-transferase activity was present in these immunoprecipitates. These data indicate that there are at least two different proteins in sheep vesicular gland microsomes capable of catalyzing GSH-dependent PGH-PGE isomerase reactions. IgG1 (hei-7), but not IgG1 (hei-26), caused coprecipitation of PGH synthase and PGH-PGE isomerase activities when incubated with intact right-side-out vesicular gland microsomes. Thus, the epitope for IgG1 (hei-7) is located on the cytoplasmic surface of those microsomal spheres which contain PGH synthase. This latter finding suggests that the isomerase reactive with IgG1 (hei-7) is involved in PGE synthesis in sheep vesicular glands.
D000818:Animals; D000911:Antibodies, Monoclonal; D005088:Exocrine Glands / Q000201:enzymology*; D005260:Female; D006651:Histocytochemistry; D007159:Immunologic Tests; D007163:Immunosorbent Techniques; D019746:Intramolecular Oxidoreductases; D007535:Isomerases / Q000032:analysis* / Q000276:immunology / Q000378:metabolism; D007700:Kinetics; D051379:Mice; D008813:Mice, Inbred ICR; D008861:Microsomes / Q000201:enzymology; D008970:Molecular Weight; D011450:Prostaglandin Endoperoxides, Synthetic / Q000378:metabolism; D044262:Prostaglandin H2; D000071876:Prostaglandin-E Synthases; D011451:Prostaglandin-Endoperoxide Synthases / Q000032:analysis; D011463:Prostaglandins H / Q000378:metabolism; D012756:Sheep
D000911:Antibodies, Monoclonal; D011450:Prostaglandin Endoperoxides, Synthetic; D011463:Prostaglandins H; D044262:Prostaglandin H2; D011451:Prostaglandin-Endoperoxide Synthases; D007535:Isomerases; D019746:Intramolecular Oxidoreductases; D000071876:Prostaglandin-E Synthases