Cavari B, Le Bail P Y, Levavi-Sivan B, Melamed P, Kawauchi H, Funkenstein B
National Institute of Oceanography, Israel Oceanographic & Limnological Research, Tel Shikmona, Haifa.
Gen Comp Endocrinol. 1994 Sep;95(3):321-9. doi: 10.1006/gcen.1994.1129.
Growth hormone (GH) polypeptide was purified from pituitary glands of the gilthead sea bream (Sparus aurata) by a two-step procedure involving gel filtration on Sephadex G-100 and reverse-phase high-performance liquid chromatography (rpHPLC). At each stage of purification, fractions were monitored by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) and by immunoblotting using anti-bonito GH antiserum. The molecular weight of the sea bream GH was estimated by SDS-PAGE to be 21 kDa when electrophoresed in the absence of beta-mercaptoethanol (nonreduced conditions) and 22 kDa when electrophoresed under reduced conditions (in the presence of 1% beta-mercaptoethanol). Pituitary RNA was used to direct cell-free translation. When specific immunoisolation from 35S-labeled proteins was conducted, using antisera against Sparus or tilapia GH, a larger prehormone was immunoprecipitated. The size of the pre-GH was estimated to be 27-28 kDa under reduced conditions and 26-27 kDa under nonreduced conditions, in agreement with the calculated molecular weight of Sparus pre-GH of 26,296 based on the deduced amino acid sequence of Sparus GH cDNA. The specificity of the immunoprecipitation reaction was demonstrated by the ability of recombinant tilapia GH to compete with the radioactively labeled translation product. No such competition was found after the addition of BSA. Our results demonstrate that the sea bream GH is similar in its size to other purified fish GHs and provide direct evidence for the synthesis of GH as a prepeptide, thus supporting the conclusions presented earlier by GH cDNA cloning.
通过两步法从金头鲷（Sparus aurata）的垂体中纯化生长激素（GH）多肽，这两步法包括在Sephadex G - 100上进行凝胶过滤和反相高效液相色谱（rpHPLC）。在纯化的每个阶段，通过SDS - 聚丙烯酰胺凝胶电泳（SDS - PAGE）和使用抗鲣鱼GH抗血清的免疫印迹法监测各组分。在不存在β - 巯基乙醇（非还原条件）下进行电泳时，通过SDS - PAGE估计金头鲷GH的分子量为21 kDa，在还原条件下（存在1%β - 巯基乙醇）电泳时为22 kDa。垂体RNA用于指导无细胞翻译。当使用针对金头鲷或罗非鱼GH的抗血清对35S标记的蛋白质进行特异性免疫分离时，一种较大的前体激素被免疫沉淀。在还原条件下，前GH的大小估计为27 - 28 kDa，在非还原条件下为26 - 27 kDa，这与根据金头鲷GH cDNA推导的氨基酸序列计算出的金头鲷前GH分子量26,296一致。重组罗非鱼GH与放射性标记的翻译产物竞争的能力证明了免疫沉淀反应的特异性。添加牛血清白蛋白（BSA）后未发现这种竞争。我们的结果表明，金头鲷GH的大小与其他纯化的鱼类GH相似，并为GH以前体肽形式合成提供了直接证据，从而支持了早期通过GH cDNA克隆得出的结论。
