Studies on thrombolytic enzyme nattokinase

纳豆激酶（NK）是从纳豆的盐水提取物中制备的，如先前报道的（H。Sumi等人，Experientia 43：1110，1987;血栓形成。血啊62：5409，1989）。摩尔重量和pI分别约为20，000和8.6。NK不仅能消化纤维蛋白（含或不含纤溶酶原），而且能消化纤溶酶底物H-D-Val-Leu-Lys-pNA（S-2251）。纯化的NK不与纤溶酶滴定剂对硝基苯基胍基苯甲酸酯反应，而与N-反式肉桂酰咪唑（一种木瓜蛋白酶或胰凝乳蛋白酶滴定剂）反应，每分子活性为53.0%。NK是一种丝氨酸蛋白酶，由275个氨基酸残基组成，N-末端为Ala，不含S-S分子键，这一点与以前已知的纤维蛋白溶解酶，包括尿激酶，组织纤溶酶原激活物（t-PA）和纤溶酶。它不具有一般纤溶酶分子所具有的Kringle结构 ...更多信息 es.用Ouchterlony法和ELISA法从枯草杆菌中分离出蛋白酶（枯草杆菌蛋白酶），用WBCLT法检测纳豆中富含聚谷氨酸的部分，发现纳豆中富含聚谷氨酸的部分能增强血浆纤溶作用。以48，000 NK单位/kg b.w.将从高NK纳豆产品制备的含有20，000 NK单位和113 mg/g聚谷氨酸的粗材料饲喂给15只雄性Wister大鼠。每天3个月。给药后或与未给药组相比，血浆ELT显著缩短（p<0.01），血浆酰胺水解（pyro-Glu-Gly-Arg-pNA和H-D-Val-Leu-Lys-pNA，p<0.01）增加。肾和肺PA活性升高（p<0.1），而APTT和Re-Ca^<++>T无变化。制备NK的肠溶包衣胶囊（2.13CU/mg蛋白质）。EFA从给药后第1天至第8天逐渐升高。与给药前相比，NK给药第1天的FDP在统计学上更高（p<0.001）。t-PA抗原的显着增加也被证明在长期（NK胶囊的临床应用正在进行中，在大学伦理委员会的许可下）。这些结果表明，NK或富含NK的纳豆不仅可用于治疗栓塞，而且可用于预防疾病，因为它们是安全的，并且是由酵母生产的。少

Nattokinase (NK) was prepared from the saline extract of natto as reported previously (H. Sumi et al., Experientia 43: 1110, 1987; Thromb. Haemostas. 62: 5409, 1989). The mol. wt. and pI were about 20,000 and 8.6, respectively. NK not only digested fibrin (with or without plasminogen) but also the plasmin substrate H-D-Val-Leu-Lys-pNA (S-2251). While not reacting with the plasmin titrator p-nitrophenyl-guanidnobenzoate, purified NK reacted with N-trans-cinnamoylidazole, a papain or chymotrypsin titrator, 53.0 % active per molecule.The amino acid sequence of the purified preparation was determined from 9 peptides obtained by Lys-endopeptidase treatment ; NK was found to be a serine protease consisting of 275 residues with Ala at the N-terminal, and possessing no S-S molecular bond, in which it differs from previously known fibrinolytic enzymes, including urokinase, tissue plasminogen activator (t-PA) and plasmin. It had no "Kringle structure" seen in ordinary fibrinolytic enzyme molecul … More es. NK antibodies prepared in rabbits failed to respond to proteases (subtilisin) thus far isolated from Bacillus subtilis by Ouchterlony and ELISA methods.By WBCLT method, the poly-Glu rich fraction of natto was found to potentiate the plasma fibrinolysis. A crude material, containing 20,000 NK units and 113 mg/g poly-Glu, prepared from a high-NK natto product, was fed to 15 male Wister rats at 48,000 NK units/kg b.w. daily for 3 months. After dosing or in comparison with the non-dosed group, plasma ELT shortened significantly (p<0.01),and plasma amidolysis (pyro-Glu-Gly-Arg-pNA and H-D-Val-Leu-Lys-pNA,p<0.01) increased. Renal and pulmonary PA activities (p<0.1) increased, whereas APTT and Re-Ca^<++>T unchanged. The enteric coated capsules of NK (2.13 CU/mg of protein) were prepared. EFA was gradually increased from the 1st day to the 8th day after administration. FDP was statistically higher (p<0.001) on the 1st day of NK administration in comparison with pre-administration. The significant increase of t-PA antigen (p<0.005) was also proved over the long te rm (Clinical applications of NK capsule are now in progress under the permission of University Committee for Ethics).These results demonstrate that NK or NK-rich natto will be useful not only for the treatment of embolism but also for prevention of the disease,since they are safety and also canbemass-produced. Less