Moriyama S, Dickhoff W W, Plisetskaya E M
School of Fisheries HF-15, University of Washington, Seattle 98195, USA.
Gen Comp Endocrinol. 1995 Aug;99(2):221-9. doi: 10.1006/gcen.1995.1105.
Insulin-like growth factor-I (IGF-I) has been purified from plasma of adult rainbow trout, Oncorhynchus mykiss. Plasma samples were collected 48 hr following injection of recombinant tuna growth hormone at a dose of 0.5 microgram/g body weight. Acid-ethanol extract of plasma was fractionated by gel filtration on a Sephadex G-50 superfine column. Two-step ion-exchange chromatography on DEAE-52 and then Mono-S columns followed. Rainbow trout IGF-I was further purified by immunoaffinity chromatography with anti-recombinant coho salmon IGF-I (rsIGF-I) serum and HPLC on a reverse-phase C18 column. During purification, trout IGF-I was monitored by SDS-PAGE, immunoblotting with anti-IGF-I-serum, homologous radioimmunoassay (RIA) for salmon IGF-I, and sulfation bioassay. The trout IGF-I appeared on SDS-PAGE as a single band with a molecular weight of 7 kDa, the same size as rsIGF-I. The partial N-terminal amino acid sequence (residues 1-20) was identical to the predicted mature trout IGF-I cDNA sequence. Trout IGF-I cross-reacted with anti-rsIGF-I serum in immunoblotting and its dilution curve was parallel to the rsIGF-I standard curve in salmon RIA. In concentrations of 50 and 500 ng/ml, trout IGF-I significantly stimulated sulfation uptake by the cultured branchial cartilage of rainbow trout. This stimulatory effect of trout IGF-I was dose-dependent and similar in its biological potency to rsIGF-I.
胰岛素样生长因子-I（IGF-I）已从成年虹鳟鱼（Oncorhynchus mykiss）的血浆中纯化出来。在以0.5微克/克体重的剂量注射重组金枪鱼生长激素48小时后采集血浆样本。血浆的酸乙醇提取物通过在Sephadex G - 50超细柱上进行凝胶过滤进行分级分离。随后在DEAE - 52柱上进行两步离子交换色谱，然后在Mono - S柱上进行。虹鳟鱼IGF - I通过用抗重组银大麻哈鱼IGF - I（rsIGF - I）血清进行免疫亲和色谱和在反相C18柱上进行高效液相色谱进一步纯化。在纯化过程中，通过SDS - PAGE、用抗IGF - I血清进行免疫印迹、针对鲑鱼IGF - I的同源放射免疫测定（RIA）和硫酸化生物测定来监测鳟鱼IGF - I。鳟鱼IGF - I在SDS - PAGE上呈现为一条分子量为7 kDa的单带，与rsIGF - I大小相同。部分N端氨基酸序列（第1 - 20位残基）与预测的成熟鳟鱼IGF - I cDNA序列相同。鳟鱼IGF - I在免疫印迹中与抗rsIGF - I血清发生交叉反应，其稀释曲线在鲑鱼RIA中与rsIGF - I标准曲线平行。在浓度为50和500 ng/ml时，鳟鱼IGF - I显著刺激虹鳟鱼培养鳃软骨的硫酸化摄取。鳟鱼IGF - I的这种刺激作用具有剂量依赖性，并且其生物学活性与rsIGF - I相似。
