Structure elucidation of prothoracicotropic hormone from the silkworm

从家蚕成虫头中分离到两种促胸激素，22K-PTTH和4K-PTTH。22K-PTTH是一种激活家蚕前胸腺诱导蜕皮激素合成和释放的异质分子种，目前已分离出其中一种，并确定了其13个氨基酸残基的n端序列。可以激活另一种蚕蛾（Samia cynthia ricini）前胸腺的Bombyxin由两条肽链组成，A链和B链分别含有20和28个氨基酸残基，它们通过二硫键相互连接。它与脊椎动物胰岛素具有相当大的序列同源性（40%）。热溶酶消化蚕蛹毒素ii，蚕蛹毒素的各种分子形式之一，提供两个胱氨酸肽。对这些肽的测序显示，其中一个含有一种胱氨酸，另一个含有两种胱氨酸。前者建立了A(20)-B（22）的连接。对于后一种胱氨酸肽，有三种可能的结构具有不同的二硫键，它们都是合成的。采用反相高效液相色谱法分离，其中一种与天然热溶菌素肽共洗脱。由此建立了A(6)-A（11）和A(7)-B（10）的联系。这种二硫桥的模式与胰岛素的模式相同。这种与胰岛素的同源结构使得我们可以构建出蚕蛹素- ii的三维结构模型，在分子表面显示出相当大的差异。这种差异可能解释了生物活性的差异。该单克隆抗体针对与牛血清白蛋白偶联的A链n端合成十肽。利用该抗体进行免疫组化分析，发现蚕蚕素在中间4对神经分泌细胞中产生，转移到allata体并释放到血淋巴中。

Two kinds of prothoracicotropic hormones, 22K-PTTH and bombyxin(4K-PTTH), have been isolated from adult heads of the silkmoth, Bombyx mori. 22K-PTTH, that activates Bombyx prothoracic glands to induce synthesis and release of ecdysone, consists of many heterogeneous molecular species, one of which has been isolated and its N-terminal sequence of 13 amino acid residues determined.Bombyxin, that can activate prothoracic glands of another silkmoth, Samia cynthia ricini, consists of two peptide chains, A and B chains with 20 and 28 amino acid residues, respectively, which are connected by disulfide bonds to each other. It has a considerable sequence homology (40%) with vertebrate insulin. Thermolysin digestion of bombyxin-II, one of various molecular form of bombyxins, afforded two cystine peptides. Sequencing of the peptides revealed that one contained one cystine and the other two. Hte former established the connection fo A(20)-B(22). As to the latter cystine peptide, there were three possible structures with different disulfide bonds, which were all synthesized. They were separated on reverse phase HPLC, one of them was co-eluted with the natural thermolysin peptide. Thus, the connection of A(6)-A(11) and A(7)-B(10) was established. This mode of disulfide bridges is identical with that of insulin. Such homologous structure of bombyxin-II with insulin permitted us to construct a three dimensional structural model of bombyxin-II, which showed a considerable difference in the surface of the molecules. This difference might account for the difference of biological activity.Monoclonal antibldy was made against an N-terminal synthetic decapeptide of A chain coupled with bovine serum albumin. Immunohistochemistry using this antibody showed that bombyxin is produced in median 4 pairs of neurosecretory cells, transferred to corpora allata and released into the hemolymph.