Ahsan M N, Watabe S
Laboratory of Aquatic Molecular Biology and Biotechnology, Graduate School of Agricultural and Life Sciences, University of Tokyo, Japan.
J Protein Chem. 2001 Jan;20(1):49-58. doi: 10.1023/a:1011005104727.
Two isoforms of anchovy trypsin (aT-I and aT-II) were purified from the visceral extracts by (NH4)2SO4 fractionation followed by affinity chromatography, gel filtration, and ion-exchange chromatography. The homogeneity of the purified preparation was evidenced by both native- and SDS-PAGE, and further by gelatin zymography. Identities of aT-I and aT-II as trypsins were established by N-terminal amino acid sequencing, which matched exactly to the corresponding stretches of their respective amino acid sequences obtained by molecular cloning [Ahsan et al. (2000), Marine Biotechnol., in press]. Both isoforms were completely inhibited by serine protease inhibitors as well as by specific trypsin inhibitors. The purified anchovy trypsins showed considerably higher catalytic efficiencies (kcat/Km) than bovine trypsin as measured toward benzoyl-arginine p-nitroanilide (BAPA) and benzoyl-arginine ethyl ester (BAEE) at 25 degrees C; in particular, aT-II was 35 times more efficient than its mammalian counterpart against BAPA. This was due mainly to a dramatic decrease of Km values for anchovy trypsins, which are indicative of an evolutionary response toward increased substrate binding at suboptimal temperatures in the marine environment.
通过硫酸铵分级沉淀，随后进行亲和色谱、凝胶过滤和离子交换色谱，从内脏提取物中纯化出两种凤尾鱼胰蛋白酶同工型（aT-I和aT-II）。纯化制剂的均一性通过天然聚丙烯酰胺凝胶电泳和十二烷基硫酸钠聚丙烯酰胺凝胶电泳得以证明，明胶酶谱法进一步证明了这一点。通过N端氨基酸测序确定了aT-I和aT-II作为胰蛋白酶的身份，其与通过分子克隆获得的各自氨基酸序列的相应片段完全匹配[Ahsan等人（2000年），《海洋生物技术》，即将出版]。两种同工型均被丝氨酸蛋白酶抑制剂以及特异性胰蛋白酶抑制剂完全抑制。在25℃下，以对硝基苯甲酰精氨酸（BAPA）和苯甲酰精氨酸乙酯（BAEE）为底物测定时，纯化的凤尾鱼胰蛋白酶显示出比牛胰蛋白酶更高的催化效率（kcat/Km）；特别是，aT-II对BAPA的催化效率比其哺乳动物对应物高35倍。这主要是由于凤尾鱼胰蛋白酶的Km值显著降低，这表明在海洋环境中，在次优温度下，对增加底物结合的一种进化反应。
