Arámburo C, Navarrette S, Montiel J L, Sánchez R, Berghman L R
Departamento de Fisiología, Universidad Nacional Autónoma de México, D.F.
Gen Comp Endocrinol. 1991 Oct;84(1):135-46. doi: 10.1016/0016-6480(91)90073-f.
It has been shown that chicken growth hormone (cGH) exhibits functional and molecular heterogeneity. Mass and charge variants have been described in fresh pituitary extracts and in pure preparations of the hormone. In an attempt to further study the molecular heterogeneity of cGH we have purified the glycosylated variant of this hormone by affinity chromatography and analyzed it by different electrophoretic methods. Purification was achieved by homogeneizing chicken pituitaries in a protease inhibitor solution (0.5 mM PMSF and aprotinin, 50 KIU/ml); the supernatant of the alkaline extract (pH 9.5) was precipitated with 0.15 M ammonium sulfate and metaphosphoric acid, pH 4.0. The supernatant from this step was further precipitated with 80% ammonium sulfate, pH 6.5. After dialysis and lyophilization, the extract was chromatographed in a Con A-Sepharose column. The fraction eluted with 10 mM alpha-methylmannoside (which contained the glycoproteins) was passed through an immunoaffinity column (anticGH). Glycosylated cGH (G-cGH) was obtained pure after this step. Pure G-cGH was analyzed by nondenaturing electrophoresis (ND-PAGE), SDS-PAGE, isoelectrofocusing (IEF), and bidimensional electrophoresis (2D-PAGE) followed by Western blot and staining either with a specific antibody or with peroxidated Con A. Results showed that monomeric G-cGH has a MW of 29 kDa (under reducing conditions) and is heterogeneous, showing at least three important charge variants with pIs 6.5, 6.7, and 7.2. Mass variants of G-cGH were also detected under nonreducing conditions. Bidimensional analysis revealed that the charge variants had a similar MW (29 kDa).
研究表明，鸡生长激素（cGH）存在功能和分子异质性。在新鲜垂体提取物和该激素的纯制剂中已描述了质量和电荷变体。为了进一步研究cGH的分子异质性，我们通过亲和色谱法纯化了该激素的糖基化变体，并采用不同的电泳方法对其进行了分析。通过在蛋白酶抑制剂溶液（0.5 mM苯甲基磺酰氟和抑肽酶，50 KIU/ml）中匀浆鸡垂体来实现纯化；碱性提取物（pH 9.5）的上清液用0.15 M硫酸铵和pH 4.0的偏磷酸沉淀。该步骤的上清液再用80%硫酸铵（pH 6.5）沉淀。透析和冻干后，提取物在伴刀豆球蛋白A-琼脂糖柱上进行色谱分离。用10 mMα-甲基甘露糖苷洗脱的部分（含有糖蛋白）通过免疫亲和柱（抗cGH）。经过这一步骤后获得了纯的糖基化cGH（G-cGH）。通过非变性电泳（ND-PAGE）、SDS-PAGE、等电聚焦（IEF）和双向电泳（2D-PAGE）对纯G-cGH进行分析，随后进行蛋白质印迹，并使用特异性抗体或过氧化物酶标记的伴刀豆球蛋白A进行染色。结果表明，单体G-cGH在还原条件下的分子量为29 kDa，具有异质性，显示出至少三种重要的电荷变体，其等电点分别为6.5、6.7和7.2。在非还原条件下也检测到了G-cGH的质量变体。双向分析表明，电荷变体具有相似的分子量（29 kDa）。
