Isolation and identification of starfish gonad-stimulating substance.

对海星性腺刺激物质的分离方法进行了重新研究，建立了海星性腺刺激物质分离方法的两个系列（Ⅰ和Ⅱ）。I-1.神经收集I-2 GSS提取（在酸性乙醇中均质化），I-3。脱脂（用氯仿和乙醚分配），I-4。HPLC（在PrOH中用Poragel PN逐步洗脱的分配色谱法），I-5。凝胶渗透色谱法（Sephacryl S-200），I-6。等电强制电泳，I-7。正相分配色谱法（Licrosorb二醇凝胶在PrOH中梯度洗脱），I-8。反相分配色谱法（ODS凝胶在乙腈中梯度洗脱），和II-1，2，3。与第一个系列II-4相同。脱盐和凝胶渗透层析（Sephadex G-50），II-5。离子交换色谱法（ES-50 ZN凝胶），II-6。反相分配色谱法（ODS凝胶在乙腈中梯度洗脱），II-8。第二次反相分配层析（ODS凝胶，等度洗脱：0.1%三氟乙酸-10%prOH-18%乙腈），通过第一次反相层析分离了海盘车GSS，并对部分氨基酸序列进行了分析（从N端开始为AEKYVGMGSYMALVGR）。基于该序列合成肽。合成的多肽对GSS没有活性，也没有抑制活性，但其抗血清能与海星和星海星的天然GSS相互作用。尚未获得阳性DNA片段。上述抗合成肽IgG将用于检测可能的GSS DNA。

Procedure for starfish gonad-stimulating substance was reexamined and two series (I and II) of the procedure were established. I-1. nerve collection, I-2. GSS extraction (homogenization in acid ethanol), I-3. delipid (partition with chloroform and ether), I-4. desalting (partition chromatography with Poragel PN in PrOH step-wise elution), I-5. gel-permeation chromatography ( Sephacryl S-200), I-6. isoelectric forcusing electrophoresis, I-7. normal phase partition chromatography (Licrosorb diol gel in PrOH gradient elution), I-8. reverse phase partition chromatography (ODS gel in acetoniterile gradient elution), and II-1, 2, 3. the same as the first series, II-4. desalting and gel permiation chromatography (sephadex G-50), II-5. ion-exchange chromatography ( ES-50ZN gel), II-6. reverse phase partition chromatography (ODS gel in acetoniterile gradient elution), II-8. second reverse phase partition chromatography (ODS gel in isocratic elution: 0.1% trifluoroacetic acid-10% prOH-18% acetoniterile).Through the first series, GSS of Asterias amurensis was isolated and partial aminoacid sequence was analyzed (AEKYVGMGSYMALVGR from the N terminal). Based on the sequence a peptide was synthesized. Although the synthesized peptide neither showed the GSS activity nor inhibitory activity for GSS, its antiserum interacted with the native GSS of both Asterias and Asterina.DNA probes based on the aminoacid sequence of Asterias GSS were synthesized (sense and antisense). Positive DNA fragment have not been obtained yet. Above mentioned anti-synthesized peptide IgG will be used for detecting the possible GSS DNA.