Purification and characterization of prolactin receptors from rat ovary.
Prolactin receptors were purified to homogeneity by two affinity chromatography steps using concanavalin A-Sepharose and human growth hormone (hGH)-Sepharose. The purified receptors showed specificity and high affinity for lactogenic hormones and a binding capacity of 20 nmol/mg of protein. Sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis analysis revealed that purified receptors were composed of two major protein bands of Mr = 41,000 and 88,000, which were identified as radioactive bands by binding of 125I-hGH to blotted renatured receptors and by autoradiogram of free and 125I-radiolabeled purified receptors. Autoradiographic analysis of SDS-polyacrylamide gel electrophoresis of cross-linked 125I-hGH-receptor or hGH-125I-iodinated receptor complexes showed two radioactive bands of Mr = 63,000 and 106,000. Analysis of the free receptors by high performance liquid chromatography using Superose 12 revealed two peaks of binding activity for 125I-hGH eluting in the positions of Mr approximately 150,000 and 250,000. After cross-linking with 125I-hGH, SDS-polyacrylamide gel electrophoresis analysis revealed that both peak fractions contained two binding species with Mr = 63,000 and 106,000. Chromatography of 125I-hGH-receptor complexes showed two radioactive fractions with approximate Mr approximately 180,000 and 300,000. The treatment of 125I-iodinated receptors with SDS and reductant resulted in the dissociation of the higher Mr form into the lower Mr form upon gel filtration. Chromatofocusing of free receptors showed three isoforms with pI 4.0, 5.0, and 5.3. These results indicate that detergent-solubilized prolactin receptors appear to be aggregated forms of holoreceptor containing two binding species of Mr = 41,000 +/- 2,000 and 88,000 +/- 3,000.
D000818:Animals; D002846:Chromatography, Affinity; D002850:Chromatography, Gel; D005260:Female; D007163:Immunosorbent Techniques; D007525:Isoelectric Focusing; D007700:Kinetics; D017830:Octoxynol; D010053:Ovary / Q000032:analysis*; D011092:Polyethylene Glycols; D051381:Rats; D011919:Rats, Inbred Strains; D011956:Receptors, Cell Surface / Q000302:isolation & purification*; D011981:Receptors, Prolactin; D012995:Solubility
D011956:Receptors, Cell Surface; D011981:Receptors, Prolactin; D011092:Polyethylene Glycols; D017830:Octoxynol