Ymer S I, Herington A C
Biochem J. 1986 Aug 1;237(3):813-20. doi: 10.1042/bj2370813.
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.
已知催乳素（PRL）的特异性受体存在于乳腺的质膜和细胞内膜上。然而，我们现在报告在怀孕和未怀孕兔子乳腺的高速（200,000g）胞质制剂中检测到一种可溶性催乳素特异性结合蛋白并对其进行了表征。该结合蛋白不能通过聚乙二醇沉淀检测到；相反，使用微型凝胶过滤技术分离结合的和游离的125I标记的人生长激素（hGH；一种有效的催乳素）。125I-hGH的特异性结合在21-23℃下10至14小时达到明显的平衡。它取决于乳腺蛋白浓度，部分取决于Ca2+或Mg2+浓度。Scatchard分析显示出陡峭的曲线，高亲和力成分的KA约为3×10(10)M-1。用校准的Ultrogel AcA34柱对125I-hGH-胞质复合物或单独的胞质进行凝胶过滤，然后测量每个洗脱级分中125I-hGH的结合，表明结合蛋白的Mr为33,000-43,000。当使用125I-绵羊或-人PRL作为配体时，观察到相同大小的特异性结合蛋白，但125I-牛GH则未观察到。结合蛋白与抗[GH受体（兔肝）]单克隆抗体缺乏交叉反应，证实了明显的催乳素特异性。用辛二酸二琥珀酰亚胺酯将125I-hGH与胞质共价交联后的聚丙烯酰胺凝胶电泳显示Mr为35,000（非还原）和37,000（还原）的结合蛋白，结果与通过凝胶过滤获得的结果相当，表明不存在亚基间二硫键。这些研究表明在兔乳腺的胞质部分存在一种明显天然可溶的催乳素结合蛋白。这种结合蛋白与膜PRL受体之间的关系尚不清楚。
