Binding and structural characteristics of a soluble lactogen-binding protein from rabbit mammary-gland cytosol.
Specific receptors for prolactin (PRL) are known to be present on plasma membranes and intracellular membranes of mammary gland. We now report, however, the detection and characterization of a soluble lactogen-specific binding protein in high-speed (200,000 g) cytosolic preparations from pregnant- and non-pregnant-rabbit mammary gland. The binding protein was not detectable by poly(ethylene glycol) precipitation; instead, bound and free 125I-labelled human growth hormone (hGH; a potent lactogen) was separated using a mini-gel filtration technique. Specific binding of 125I-hGH reached an apparent equilibrium between 10 and 14 h at 21-23 degrees C. It was dependent on mammary-gland protein concentration and, partially, on Ca2+ or Mg2+ concentrations. Scatchard analysis revealed steep curvilinear plots, the high-affinity component having a KA of approximately 3 X 10(10) M-1. Gel filtration on calibrated Ultrogel AcA34 columns of 125I-hGH-cytosol complexes or of cytosol alone, followed by measurement of 125I-hGH binding in each eluted fraction, indicated that the binding protein had an Mr of 33,000-43,000. A specific binding protein of the same size was observed when 125I-ovine or -human PRL, but not 125I-bovine GH, was used as ligand. The apparent lactogenic specificity was confirmed by a lack of cross-reactivity of the binding protein with an anti-[GH receptor (rabbit liver)] monoclonal antibody. Polyacrylamide-gel electrophoresis of 125I-hGH covalently cross-linked to cytosol with disuccinimidyl suberate revealed binding proteins of Mr 35,000 (non-reduced) and 37,000 (reduced), results comparable with those obtained by gel filtration and indicating an absence of inter-subunit disulphide bonds. These studies have shown the presence of an apparently naturally soluble lactogen-binding protein in the cytosolic fraction of rabbit mammary gland. The relationship between this binding protein and the membrane PRL receptor is not yet known.
D000818:Animals; D000911:Antibodies, Monoclonal; D002118:Calcium / Q000494:pharmacology; D002850:Chromatography, Gel; D003600:Cytosol / Q000378:metabolism; D005260:Female; D013006:Growth Hormone / Q000378:metabolism; D008274:Magnesium / Q000494:pharmacology; D008321:Mammary Glands, Animal / Q000378:metabolism*; D011247:Pregnancy; D011485:Protein Binding / Q000187:drug effects; D011817:Rabbits; D018000:Receptors, Peptide; D011981:Receptors, Prolactin / Q000187:drug effects / Q000276:immunology / Q000378:metabolism*; D011986:Receptors, Somatotropin / Q000378:metabolism
D000911:Antibodies, Monoclonal; D018000:Receptors, Peptide; D011981:Receptors, Prolactin; D011986:Receptors, Somatotropin; C028202:placental lactogen receptor; D013006:Growth Hormone; D008274:Magnesium; D002118:Calcium