Purification of prolactin receptors from sow mammary gland and polyclonal antibody production.
After solubilization with Triton X-100 or 3-[(3-cholamidopropyl)-dimethylammonio]-1-propane sulfonate (CHAPS), prolactin receptors from mammary crude membranes of primiparous lactating sows (pretreated with bromocriptine) have been purified by affinity chromatography using ovine prolactin or a monoclonal antibody against rabbit prolactin receptor. Comparative analysis of these two methods of purification demonstrated that use of an immunoaffinity step allowed a great improvement of receptor yield (40%) compared to the hormone affinity method (10%). In addition, partially purified fractions obtained by immunoaffinity appeared more homogeneous and had much higher specific activity. Affinity labelling of prolactin receptors from crude membranes or solubilized extracts with iodinated ovine prolactin, followed by electrophoretic analysis (SDS-PAGE) and autoradiography, revealed one binding unit of approximately 45 kDa. When partially purified receptor preparations were labelled with 125I, submitted to an additional affinity chromatography and analyzed by SDS-PAGE, prolactin receptors appeared as a single form having a molecular weight of 42-45 kDa, which is not associated with itself or other subunits by disulfide linkages. Partially purified fractions were used to produce anti-prolactin receptor serum from goats. These polyclonal antibodies were able to completely inhibit the binding of lactogenic hormones in sow and rabbit mammary membranes. They were also able to recognize hormone-receptor complexes, but more specifically in sow mammary gland. These antisera could inhibit prolactin binding to its receptors in several organs of various species, suggesting that prolactin receptors shared numerous antigenic similarities between species and particularly between sow and rabbit. These similarities appeared to be located essentially on the part of the molecule more specifically involved in the recognition of the hormone.
D000345:Affinity Labels; D000818:Animals; D000911:Antibodies, Monoclonal; D001971:Bromocriptine / Q000494:pharmacology; D002846:Chromatography, Affinity; D005260:Female; D007774:Lactation; D008321:Mammary Glands, Animal / Q000032:analysis* / Q000276:immunology; D008970:Molecular Weight; D011247:Pregnancy; D011388:Prolactin / Q000378:metabolism*; D011981:Receptors, Prolactin / Q000276:immunology / Q000302:isolation & purification* / Q000378:metabolism; D012995:Solubility
D000345:Affinity Labels; D000911:Antibodies, Monoclonal; D011981:Receptors, Prolactin; D001971:Bromocriptine; D011388:Prolactin