Kikuchi M, Fukuyama K, Epstein W L
Department of Dermatology, University of California, San Francisco 94143-0536.
Biochim Biophys Acta. 1988 May 12;965(2-3):176-84. doi: 10.1016/0304-4165(88)90054-2.
Bradykinin-hydrolyzing enzyme was purified 200-fold from a soluble fraction of cornified cells from 2-day-old rat epidermis. The enzyme has an Mr of 80,000 as identified by SDS polyacrylamide gel electrophoresis and HPLC gel filtration. The isoelectric point of the enzyme is 5.05. The enzyme hydrolyzed Phe5-Ser6 of bradykinin and seven bradykinin-related peptides, and Tyr5-Ser6 of Tyr5-bradykinin. Production of bradykinin fragments, Arg-Pro-Pro-Gly-Phe and Ser-Pro-Phe-Arg, proceeded in a stoichiometric fashion. Km and Vmax values for bradykinin were 33 microM and 22.2 mumol/min per mg, respectively. The enzyme did not hydrolyze azocasein, denatured hemoglobin or synthetic substrates for other epidermal proteinases. The enzyme activity was enhanced by reducing agents and inhibited by sulfhydryl-blocking agents and divalent cations. Diisopropyl fluorophosphate and phenylmethylsulfonyl fluoride had no effects. The enzyme has a pH optimum of 7.0-7.5 and is stable at 4 degrees C for 1 month, but loses activity completely at 60 degrees C for 10 min. The epidermal endopeptidase differs in several properties from endooligopeptidase A purified from brain which hydrolyzes Phe5-Ser6 of bradykinin.
从2日龄大鼠表皮的角质化细胞可溶性部分中纯化出缓激肽水解酶，纯化倍数为200倍。通过SDS聚丙烯酰胺凝胶电泳和高效液相色谱凝胶过滤鉴定，该酶的分子量为80,000。该酶的等电点为5.05。该酶可水解缓激肽的Phe5-Ser6以及七种缓激肽相关肽，还有Tyr5-缓激肽的Tyr5-Ser6。缓激肽片段Arg-Pro-Pro-Gly-Phe和Ser-Pro-Phe-Arg的产生呈化学计量方式进行。缓激肽的Km和Vmax值分别为33μM和每毫克22.2μmol/分钟。该酶不水解偶氮酪蛋白、变性血红蛋白或其他表皮蛋白酶的合成底物。还原剂可增强该酶的活性，而巯基阻断剂和二价阳离子则可抑制其活性。二异丙基氟磷酸酯和苯甲基磺酰氟无作用。该酶的最适pH为7.0 - 7.5，在4℃下可稳定保存1个月，但在60℃下10分钟会完全丧失活性。这种表皮内肽酶在几个特性上与从脑中纯化的内寡肽酶A不同，后者可水解缓激肽的Phe5-Ser6。
