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Studies on thrombolytic enzyme nattokinase

溶栓酶纳豆激酶的研究

基本信息

批准号：
02558013
负责人：
SUMI Hiroyuki
金额：
$ 4.42万
依托单位：
Department of Nutrition & Food Science , Okayama Prefectural Junior College
依托单位国家：
日本
项目类别：
Grant-in-Aid for Developmental Scientific Research (B)
财政年份：
1990
资助国家：
日本
起止时间：
1990 至 1993
项目状态：
已结题

项目摘要

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

纳豆激酶（NK）是从纳豆的盐水提取物中制备的，如先前报道(H. Sumi et al., Experientia 43: 1110,1987; Thromb。血液学杂志。62:5409,1989)。mol. wt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），还能消化纤溶酶底物H-D-Val-Leu-Lys-pNA （S-2251）。虽然不与纤溶酶滴定剂对硝基苯甲酰苯甲酸酯反应，但纯化的NK与n -反式肉桂酰唑反应，这是一种木瓜蛋白酶或凝乳胰蛋白酶滴定剂，每分子活性为53.0%。纯化产物的氨基酸序列由赖氨酸内肽酶处理得到的9个肽段确定；NK是一种丝氨酸蛋白酶，由275个残基组成，在n端有Ala，没有S-S分子键，这与以前已知的纤溶酶不同，包括尿激酶，组织纤溶酶原激活物（t-PA）和纤溶酶。它不具有普通纤溶酶分子的“克林格尔结构”。兔制备的NK抗体对迄今为止从枯草芽孢杆菌中分离到的蛋白酶（枯草杆菌素）没有反应。通过WBCLT方法，发现纳豆富含聚谷氨酸的部分具有增强血浆纤维蛋白溶解的作用。以高NK纳豆产品为原料，制备含有20,000 NK单位和113 mg/g聚谷氨酸的粗料，每天以48,000 NK单位/kg b.w.喂给15只雄性Wister大鼠，持续3个月。给药后或与未给药组比较，血浆ELT显著缩短（p<0.01），血浆酶解（p<0.01）升高（p<0.01）。肾和肺PA活性升高（p<0.1），而APTT和Re-Ca^<++>T不变。制备了蛋白含量为2.13 CU/mg的肠溶NK胶囊。给药后第1天至第8天EFA逐渐升高。与给药前相比，给药第1天FDP有统计学意义（p<0.001）。从长期看，t-PA抗原显著升高（p<0.005）（NK胶囊经大学伦理委员会批准，正在进行临床应用）。这些结果表明，NK或富含NK的纳豆不仅可用于治疗栓塞，还可用于预防疾病，因为它们安全且可批量生产。少