Friedrich T, Kröger B, Bialojan S, Lemaire H G, Höffken H W, Reuschenbach P, Otte M, Dodt J
Department of Biotechnology, BASF Aktiengesellschaft, Ludwigshafen, Germany.
J Biol Chem. 1993 Aug 5;268(22):16216-22.
A thrombin-specific inhibitor with an apparent molecular mass of 11 kDa has been purified from the insect Rhodnius prolixus. Amino-terminal protein sequence analysis allowed the molecular cloning of the corresponding cDNA. The open reading frame codes for a protein of about 103 amino acid residues and displays an internal sequence homology of residues 6-48 with residues 57-101 indicating a two-domain structure. Based on the amino acid sequence the two domains exhibit high homology to protease inhibitors belonging to the Kazal-type family. Model building suggests that the first domain binds to the active site with residue His10 pointing into the specificity pocket. From gel filtration and tight-binding inhibition experiments the inhibitor appears to form 1:1 complexes with thrombin. Periplasma-directed heterologous expression of the rhodniin cDNA in Escherichia coli yields the intact thrombin inhibitor. Natural and recombinant rhodniin both display inhibition constants of about 2 x 10(-13) M.
已从昆虫长红猎蝽中纯化出一种表观分子量为11 kDa的凝血酶特异性抑制剂。氨基末端蛋白质序列分析使得相应cDNA的分子克隆成为可能。开放阅读框编码一个约103个氨基酸残基的蛋白质，并且显示出第6 - 48位残基与第57 - 101位残基的内部序列同源性，表明其具有双结构域结构。基于氨基酸序列，这两个结构域与属于Kazal型家族的蛋白酶抑制剂具有高度同源性。模型构建表明，第一个结构域通过His10残基指向特异性口袋与活性位点结合。从凝胶过滤和紧密结合抑制实验来看，该抑制剂似乎与凝血酶形成1:1复合物。在大肠杆菌中进行红猎蝽蛋白酶抑制剂cDNA的周质导向异源表达可产生完整的凝血酶抑制剂。天然和重组的红猎蝽蛋白酶抑制剂均显示出约2×10(-13) M的抑制常数。
