Murthy K K, Scicli A G
Hypertension Research Division, Henry Ford Hospital, Detroit, MI.
Biochim Biophys Acta. 1988 Feb 17;964(2):276-84. doi: 10.1016/0304-4165(88)90176-6.
A non-kallikrein arginine esterase (esterase I) has been purified from dog urine and characterized. The enzyme was purified by a three-step procedure, including ion exchange chromatography on DEAE-Sephacel, affinity chromatography on p-aminobenzamidine-Sepharose, and final gel filtration on Ultrogel AcA-54. The purified preparation gave three protein bands on polyacrylamide gel electrophoresis, all of which had esterolytic activity. The enzyme has a specific activity of 601 esterase units/mg protein. It has negligible kininogenase activity. Esterase I gave two closely migrating protein bands on reduced sodium dodecyl sulfate-polyacrylamide gel electrophoresis with molecular weights of 34,000 and 33,300. Esterase I is a glycoprotein with a pH optimum of 9.5 and a pI of 4.62. The enzyme is strongly inhibited by a host of inhibitors including aprotinin, leupeptin, antipain, soybean trypsin inhibitor, lima bean trypsin inhibitor, and DPhe-Phe-Arg-chloromethyl ketone (I50 in the 10(-9)-10(-8) M range). However, p-aminobenzamidine, N alpha-p-tosyl-lysyl chloromethyl ketone and phenylmethylsulfonyl fluoride were weak inhibitors, with I50 values in the 10(-5)-10(-7) M range. The enzyme preferentially hydrolyzes Pro-Arg bonds. Among fluorogenic substrates used in this study, butyloxycarbonyl-Val-Pro-Arg-methylcoumarinamide (alpha-thrombin substrate) was found to be the best, with a Km of 1.7 microM and a kcat/Km of 6.3 s.microM-1. However, esterase I does not convert fibrinogen to fibrin nor activate plasminogen to plasmin. Esterase I is immunologically distinct from dog urinary kallikrein, having no cross-reactivity with antibodies against dog kallikrein.
已从狗尿中纯化并鉴定出一种非激肽释放酶精氨酸酯酶（酯酶I）。该酶通过三步法进行纯化，包括在DEAE - Sephacel上进行离子交换色谱、在对氨基苯甲脒 - 琼脂糖上进行亲和色谱以及最后在Ultrogel AcA - 54上进行凝胶过滤。纯化后的制剂在聚丙烯酰胺凝胶电泳上产生三条蛋白带，所有这些蛋白带都具有酯解活性。该酶的比活性为601酯酶单位/毫克蛋白。其激肽原酶活性可忽略不计。酯酶I在还原型十二烷基硫酸钠 - 聚丙烯酰胺凝胶电泳上产生两条迁移距离相近的蛋白带，分子量分别为34,000和33,300。酯酶I是一种糖蛋白，最适pH为9.5，pI为4.62。该酶受到多种抑制剂的强烈抑制，包括抑肽酶、亮抑肽酶、抗蛋白酶、大豆胰蛋白酶抑制剂、利马豆胰蛋白酶抑制剂以及二苯丙氨酰 - 苯丙氨酰 - 精氨酸 - 氯甲基酮（半数抑制浓度在10⁻⁹ - 10⁻⁸ M范围内）。然而，对氨基苯甲脒、Nα - 对甲苯磺酰基 - 赖氨酰氯甲基酮和苯甲基磺酰氟是弱抑制剂，半数抑制浓度值在10⁻⁵ - 10⁻⁷ M范围内。该酶优先水解脯氨酸 - 精氨酸键。在本研究中使用的荧光底物中，发现丁氧羰基 - 缬氨酸 - 脯氨酸 - 精氨酸 - 甲基香豆素酰胺（α - 凝血酶底物）是最佳底物，Km为1.7微摩尔，kcat/Km为6.3秒·微摩尔⁻¹。然而，酯酶I不会将纤维蛋白原转化为纤维蛋白，也不会将纤溶酶原激活为纤溶酶。酯酶I在免疫学上与狗尿激肽释放酶不同，与抗狗激肽释放酶的抗体没有交叉反应。
