Abstract
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.
Highlights
IgGl(hei-7) and IgGl(hei-26), caused immunoprecipi- epithelial cells of the collecting tubule [4,5,6]
When IgGl(hei-2), IgGl(hei-7), and IgGl(hei-26) were immobilized by attachment to attenuatedS. aureus cells conjugated with rabbit anti-mouseIgG and the antibody-S. aureus complexes incubated with solubilized sheep vesicular gland microsomes, the three antibodycomplexes precipitated 9, 28, and 15%,respectively, of the initial solubilized PGH-PGE
An IgGl(hei-7)-asu.reus suspension prepared from 0.4 mlof hei-7 medium was mixed with 90 pl (180 pg) of solubilized sheep vesicular gland microsomes (PGH-PGE isomerase activity of25.3 nmol/l5 s), and the samples were incubated on ice for 15 min
Summary
IgGl(hei-7) and IgGl(hei-26), caused immunoprecipi- epithelial cells of the collecting tubule [4,5,6]. IgGl(hei-2), IgG,(hei-7), andIgGl(hei-26)) when incubated with rabbit anti-mouse IgG second antibody and solubilized sheep vesicular gland microsomalprotein precipitateda maximum of 10, 45, and 22% of the total solubilized PGH-PGE Aureus cells conjugated with rabbit anti-mouseIgG and the antibody-S. aureus complexes incubated with solubilized sheep vesicular gland microsomes, the three antibodycomplexes precipitated 9, 28, and 15%,respectively, of the initial solubilized PGH-PGE
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