Abstract
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.
Highlights
From the Molecular Endocrinology Section, Endocrinology and Reproduction Research Branch, National Institute of Child Health and HumanDeuelopment, Bethesda, Maryland 20892
Since the binding sites for lactohigh affinity for lactogenic hormones and a binding genic hormones have been identified in various tissues includcapacity of 20 nmolfmg of protein.Sodiumdodecyl ing mammary gland, liver, ovary, kidney, prostate, and brain sulfate (SDS)-polyacrylamide gel electrophoresis anal-(8-ll), itis necessary to characterize thereceptor in order to ysis revealed that purified receptors wceormeposed of two major protein bandsof M, = 41,000 and 88,000, which were identifiedas radioactive bandsby binding of 12'I-hGH to blotted renatured receptors and by autoradiogram of free and '261-radiolabeled purified receptors
Autoradiographic analysisof SDS-polyacrylamide gel electrophoresis of cross-linked 12'I-hGH-receptor or hGH-12'I-iodinated receptor complexes understandthe signal transmissionmechanism across the membranes as the initial evenotf hormone action
Summary
Autoradiographic analysisof SDS-polyacrylamide gel electrophoresis of cross-linked 12'I-hGH-receptor or hGH-12'I-iodinated receptor complexes understandthe signal transmissionmechanism across the membranes as the initial evenotf hormone action. After cross-linking with I2'I-hGH, SDS-polyacrylam- linking reagents,we identified forovarian membranelactogen ide gel electrophoresis analysis reveatlheadt both peak receptor a M , = 80,000 species containing a binding unit of fractionscontainedtwobindingspecieswith. For these treatment of Iz5I-iodinated receptors with SDS and studies, the purified free and complexed prolactin receptors reductant resulted in thedissociation of the higherM, form into the loweMr , form upon gel filtration. Chromatofocusing of free receptorsshowed three isoforms with PI 4.0, 5.0, and 5.3 These results indicate that detergent-solubilized prolactin receptors appear to be aggregated formsof holoreceptor containing two binding species of M , =41,000 2 2,000 and88,000f3,000.
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