BOVINE ENTEROKINASE - PURIFICATION, SPECIFICITY, AND SOME MOLECULAR-PROPERTIES
BOVINE ENTEROKINASE - PURIFICATION, SPECIFICITY, AND SOME MOLECULAR-PROPERTIES
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DOI：
10.1021/bi00634a011
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发表时间：
1977-01-01
期刊：
影响因子：
2.9
通讯作者：
NEURATH, H
中科院分区：
文献类型：
--
作者：
ANDERSON, LE;WALSH, KA;NEURATH, H
肠激酶（EC 3.4.4.8）从牛大肠杆菌的内容物中分离，并以41%的产率纯化1200倍。分离过程采用DEAE层析、固定化对氨基苯甲脒亲和层析和凝胶过滤。所得纯酶在十二烷基硫酸钠凝胶电泳上显示单一条带，对应于145，000的MW。分子中由二硫键连接的两条链的分子量分别为57，000和82，000。纯化的酶表现出针对酶原底物的活化肽中的聚阴离子氨基酸序列的限制性特异性。在测试的丝氨酸蛋白酶的酶原中，包括几种参与凝血的酶，只有天然的和胍化的胰蛋白酶原被肠激酶激活，而乙酰化的胰蛋白酶原则不被激活。牛肠激酶的部分热变性会对胰蛋白酶原的激活和苯甲酰基-L-精氨酸乙酯（BzArgOEt）的水解产生不同的反应，进一步表明二级位点在胰蛋白酶原的结合中很重要。一个敏感的测定肠激酶（纳摩尔范围）开发使用氚BzArgOEt。
Enterokinase (EC 3.4.4.8) was isolated from the contents of bovine duodena and purified 1200-fold in 41% yield. The isolation procedure employed DEAE chromatography, affinity chromatography on immobilized p-aminobenzamidine and gel filtration. The resultant pure enzyme exhibits a single band on sodium dodecyl sulfate gel electrophoresis corresponding to a MW of 145,000. Two chains in the molecule, connected by disulfide linkages, have MW of 57,000 and 82,000, respectively. The purified enzyme exhibits a restricted specificity which is directed toward the polyanionic amino acid sequence in the activation peptide of the zymogen substrate. Of the zymogens of the serine proteases tested, including several of those involved in blood coagulation, only native and guanidinated trypsinogen are activated by enterokinase, whereas acetylated trypsinogen is not. Partial heat denaturation of bovine enterokinase causes a differential response toward the activation of trypsinogen and the hydrolysis of benzoyl-L-arginine ethyl ester (BzArgOEt), further suggesting that secondary sites are important in the binding of trypsinogen. A sensitive assay for enterokinase (nanomole range) was developed using tritiated BzArgOEt.