Inhibition Mechanism and Molecular Evolution of Amylase- and Protease-Inhibitors

基于我们先前发现大麦胰蛋白酶抑制剂在结构上与小麦-淀粉酶抑制剂、蓖麻种子秸秆蛋白和哺乳动物胰腺分泌型胰蛋白酶抑制剂的结构进化相关，我们研究了这些蛋白质的结构与功能关系和分子进化。对这些抑制剂的详细比较表明，它们是由编码一个约7kDa的蛋白酶抑制剂和一个淀粉酶抑制剂的两个祖先基因串联而成的，随后的突变使其中一个抑制位点消失或对一些未知的酶具有专一性。通过测定大麦抑制剂中的3个(5个)二硫键，部分证实了这种结构域结构。特别有趣的是，潜在的淀粉酶抑制域也与牛胰腺分泌性胰蛋白酶抑制物(KAZAL)同源，这表明不寻常的分化进化的…来自单一祖先基因的更多的蛋白酶和淀粉酶抑制剂的离子。植物种子含有许多功能已知和未知的低分子蛋白质。我们在大豆种子中鉴定出一种异常疏水的蛋白质，并测定了其完整的一级结构。该蛋白对哺乳动物三酰甘油脂肪酶的抑制作用很弱，与上述蛋白和Bowman-Birk型抑制物有微弱的部分序列相似性，这表明存在一个包括多种蛋白质的蛋白质超家族。它还显示出与哺乳动物荷尔蒙--大鼠催乳素--惊人的相似之处。另一种不同寻常的蛋白质，大豆富含天冬氨酸的多肽，已经被分离和测序。该蛋白质以天冬氨酸含量高而著称，序列分析表明，该蛋白质在C末端有一条长的聚(L-天冬氨酸)尾巴，与胰酶相互作用。翻译后修饰是蛋白质功能的重要体现，用普通分析系统很难检测到。我们构建了一种快速、灵敏的修饰氨基酸检测系统，并将其应用于本研究。较少

Based on our previous finding of an extraordinary dicergent evolution that Hordeum uulgare (barley) trypsin inhibitor is structurally related to Triticum (wheat) -amylase inhibitors, Ricinus communis(castor bean) seed strage protein, and mammalian pancreatic secretory trypsin inhibitors, we have investigated the structure-function relationship and molecular evelution of these proteins. Detailed comparison of these inhibitors suggested that they have arisen from fusion of two ancestral genes coding for a proteinase inhibitor and an -amylase inhibitor of about 7 kda in tandem, and subsequent mutation had made one of the inhibitory site silent or specific for some unknown enzymes. This domain structure was partially confirmed by determination of 3 (out of 5) disulfide bridges in the barley inhibitor. It is of particular interest that the potential -amylase inhibitory domain is also homologous to bovine pancreatic secretory trypsin inhibitor (kazal), indicating the unusual dicergent evolut … More ion of a proteinase- and an -amylase-inhibitors from a single ancestral gene.Plant seeds contain a number of low nolecular mass proteins of known and unknown function. We have identified an unusually hydrophobic protein in glycine max (soybean) seeds, and determined the complete primary structure. The protein showed a very weak inhibitory action on mammalian triacylglycerol lipases, and also a weak and partial sequence similarity to the above described proteins and to bowman-birk type inhibitors, which suggest the presence of a protein superfamily including a wide variety of proteins. It also showed a marked and unexpected similarity to a mammalian hormone, rat prolaction. Another unusual protein, soybean aspartic acid-rich polypeptide, has been isolated and sequence. The protein is remarkable for its high content of aspartic acid, and sequence analysis showed that the protein has a long poly(L-aspartic acid) tail at the C-terminus which interacts with trypsin.Post-translational modifications, important for manifestation of protein fonctions, is rather difficult to detect by the ordinary analytical systems. We have constructed a rapid and sensitive detection system for modified amino acids and applied to the present investigation. Less