Rabinovich S E, Lobareva L S, Paskhina T S
Biokhimiia. 1990 Sep;55(9):1675-89.
A procedure for obtaining tissue kallikrein (EC 3.4.21.35) from large specimens of human urea (100 l) has been developed. The isolation procedure included primary extraction of the protein with chitosan (a crustacean chitin deacylated by alkaline treatment), desorption from chitosan with 1 M NH3, affinity chromatography on contrical-Sepharose, ion-exchange chromatography on DEAE-Sepharose and gel filtration on Sephadex G-100. This method permits to obtain tissue kallikrein preparations purified 1080-fold (with respect to AcPheArg-OEt esterase) and 1360-fold (with respect to kininogenase) with 33 and 40% yields, respectively. Tissue kallikrein preparations were homogeneous as could be judged from the results of electrophoresis performed in 12% PAAG in the presence of 0.1% SDS as well as from the presence of one N-terminal amino acid identified as isoleucine. Purified tissue kallikrein had specific activities of 133 mumol/min/mg protein (with respect to AcPheArg-OEt hydrolysis) and 8.8 mumol/min/mg protein (with respect to D-Val-Leu-Arg-pNa hydrolysis) and liberated 462 micrograms equiv. of bradykinin/min/mg protein from heated human blood plasma used as a kininogen source. The protein exhibited the highest stability at pH 8.0-9.0; the pH optimum is at pH 8.0 with AcPheArg-OMe as substrate. The enzyme revealed a high thermostability and was fully inactivated only after 1-hour heating in a boiling water bath. The identity of the urine enzyme to tissue kallikrein could be confirmed by the resistance of the enzyme activity to SIT, high sensitivity to the inhibiting effect of aprotinin (Ki = 0.94 x 10(-10) M) and by an exceedingly low value of the second order inhibition constant for DPP (4.6 M-1 min-1). The fact that this value differs drastically from that for human blood plasma kallikrein (EC 3.4.21.34) which is equal to 360 M-1 min-1 points to marked differences in the structure of the active centers of the both kallikreins as well as to the uniqueness of the tissue kallikrein active center.
已开发出一种从大量人尿液样本（100升）中获取组织激肽释放酶（EC 3.4.21.35）的方法。分离过程包括用壳聚糖（一种经碱处理脱酰基的甲壳类几丁质）对蛋白质进行初步提取，用1M氨水从壳聚糖上解吸，在抗凝血酶原 - 琼脂糖上进行亲和层析，在DEAE - 琼脂糖上进行离子交换层析以及在葡聚糖G - 100上进行凝胶过滤。该方法分别以33%和40%的产率获得了纯化1080倍（相对于AcPheArg - OEt酯酶）和1360倍（相对于激肽原酶）的组织激肽释放酶制剂。从在0.1% SDS存在下于12%聚丙烯酰胺凝胶中进行的电泳结果以及鉴定出一个N端氨基酸为异亮氨酸来看，组织激肽释放酶制剂是均一的。纯化的组织激肽释放酶的比活性为133 μmol/min/mg蛋白质（相对于AcPheArg - OEt水解）和8.8 μmol/min/mg蛋白质（相对于D - Val - Leu - Arg - pNa水解），并且每分钟每毫克蛋白质从用作激肽原来源的加热人血浆中释放出462微克当量的缓激肽。该蛋白质在pH 8.0 - 9.0时表现出最高稳定性；以AcPheArg - OMe为底物时，最适pH为8.0。该酶具有高耐热性，仅在沸水浴中加热1小时后才完全失活。尿液酶与组织激肽释放酶的一致性可通过酶活性对大豆胰蛋白酶抑制剂（SIT）的抗性、对抑肽酶抑制作用的高敏感性（Ki = 0.94×10⁻¹⁰ M）以及二肽基肽酶（DPP）的二级抑制常数极低的值（4.6 M⁻¹ min⁻¹）来证实。该值与人类血浆激肽释放酶（EC 3.4.21.34）的该值（等于360 M⁻¹ min⁻¹）有极大差异，这表明两种激肽释放酶活性中心的结构存在显著差异，也表明组织激肽释放酶活性中心的独特性。
