Arámburo C, Montiel J L, Perera G, Navarrete S, Sánchez R
Departamento de Fisiología, Universidad Nacional Autónoma de México.
Gen Comp Endocrinol. 1990 Oct;80(1):59-67. doi: 10.1016/0016-6480(90)90148-f.
It has been suggested that the functional diversity of growth hormone (GH) is related to its molecular complexity. Here we report a characterization of charge and mass variants of chicken growth hormone (cGH) through a variety of electrophoretic systems [nondenaturing (ND-PAGE), denaturing (SDS-PAGE), under reducing and nonreducing conditions, isoelectrofocusing (IEF), and bidimensional electrophoresis] followed by Western blot and immunostaining with a specific antibody directed against pure cGH. We also report the biological properties of two charge variants on two homologous assays. The studies were carried out with purified cGH and with fresh chicken pituitary extracts. Three charge variants were obtained by ND-PAGE (Rf = 0.23, 0.30, and 0.35), which showed the same molecular weight (26 kDa), while in IEF eight isoforms were observed, the most conspicuous being those with pI = 6.86, 7.5, 7.9, 8.05, and 8.18. In SDS-PAGE under reducing conditions four immunoreactive bands were observed: the monomer (26 kDa), a dimer (52 kDa), a fragment (16 kDa), and a minor band at 22 kDa. Higher MW variants were found under nonreducing conditions. Bidimensional analysis also showed several charge variants for the monomer and the dimer. Bioactivity of two charge variants (0.23 and 0.3) was evaluated with a lipolytic and an antilipolytic assay on chicken adipose tissue explants. It was shown that variant 0.23 was mainly lipolytic, in a dose-dependent response, but lacked antilipolytic effect. On the other hand, variant 0.30 did not show lipolytic effect but presented a clear antilipolytic activity.
有人提出生长激素（GH）的功能多样性与其分子复杂性有关。在此，我们通过多种电泳系统[非变性（ND-PAGE）、变性（SDS-PAGE），分别在还原和非还原条件下、等电聚焦（IEF）以及双向电泳]对鸡生长激素（cGH）的电荷和质量变体进行了表征，随后进行了蛋白质印迹分析，并使用针对纯cGH的特异性抗体进行免疫染色。我们还报告了两种电荷变体在两种同源检测中的生物学特性。研究使用了纯化的cGH和新鲜的鸡垂体提取物。通过ND-PAGE获得了三种电荷变体（Rf = 0.23、0.30和0.35），它们显示出相同的分子量（26 kDa），而在IEF中观察到了八种同工型，最明显的是那些pI = 6.86、7.5、7.9、8.05和8.18的同工型。在还原条件下的SDS-PAGE中观察到四条免疫反应带：单体（26 kDa）、二聚体（52 kDa）、一个片段（16 kDa）以及一条22 kDa的次要带。在非还原条件下发现了更高分子量的变体。双向分析也显示了单体和二聚体的几种电荷变体。使用鸡脂肪组织外植体的脂解和抗脂解检测评估了两种电荷变体（0.23和0.3）的生物活性。结果表明，变体0.23主要具有脂解作用，呈剂量依赖性反应，但缺乏抗脂解作用。另一方面，变体0.30未显示脂解作用，但呈现出明显的抗脂解活性。
