N-V蛋白酶的溶栓机理及酶学特点研究

发布时间：2018-09-12 13:17

【摘要】：N-V蛋白酶(商品名:溶栓素)具有良好的溶栓作用。明确其生物学性质及溶解血栓的机理,对于它将来可能的临床应用意义重大。N-V蛋白酶提纯工艺的成熟,使提取产品的纯度达到了较高水平[中国国家知识产权局专利公开(公告)号:1500873];日本秋田大学生化系Toshihiro sugiyama教授对其进行氨基酸序列分析,测出了部分序列。提交SWISSPROT后得出结果认定为一种新的蛋白酶,并推测其可能是一种丝氨酸蛋白酶并且在氨基端有糖基化等保护(访问号:P8343);N-V蛋白酶可以溶解血栓等性质是进一步研究的工作基础,也是立题依据。为研究其溶栓作用的机理,本研究创新性地利用了电泳技术,使N-V蛋白酶的纯度提高到了97.57%,为进一步研究创造了有利条件。在研究N-V蛋白酶对牛纤溶酶原的激活作用过程中发现了N-V蛋白酶可以将S2251这一以往被认为是纤溶酶特异性底物水解,进而找到了一种可以对N-V蛋白酶活性进行准确测定的显色底物。通过平板法对比N-V蛋白酶和尿激酶的纤溶酶原激活作用时发现二者间的作用机制明显不同,结合N-V蛋白酶水解白蛋白、酪蛋白、牛纤维蛋白、人纤维蛋白的水解产物在SDS-PAGE上的结果可以初步判断:N-V蛋白酶没有发挥纤溶酶原激活剂的作用,而是直接将蛋白底物水解成氨基酸。经过N-V蛋白酶水解insulin-B链,对其产物衍生化后进行氨基酸分析证实了上述判断。作者还对N-V蛋白酶进行了糖蛋白鉴定、酶学分类和外切酶性质研究。得出以下结论:①N-V蛋白酶非糖蛋白类,在酶学分类上它属于丝氨酸蛋白酶类。②N-V蛋白酶是一种氨基肽酶。③N-V蛋白酶不是纤溶酶原激活剂,它在溶栓过程中发挥直接纤溶作用。④据N-V蛋白酶已知酶学特性,可以确认它是一种新的氨基肽酶。这使得丝氨酸蛋白酶家族的成员得到进一步丰富。⑤N-V蛋白酶具有良好的水解蛋白质性质,所以可以用于蛋白质氨基酸测定以及核酸提取时溶解蛋白质的工具酶。
[Abstract]:N-V protease (trade name: thrombolytic hormone) has good thrombolytic effect. It is of great significance to clarify its biological properties and the mechanism of thrombolysis, which is of great significance to the future clinical application of the purification process of .N-V protease. The purity of the extracted product reached a high level [patent disclosure (announcement) no.: 1500873] of the State intellectual property Office of China, and amino acid sequence analysis was carried out by Professor Toshihiro sugiyama of the Department of Biochemistry of Akita University, Japan, and some of the sequences were detected. After SWISSPROT was submitted, the results were identified as a new protease, and it was speculated that it might be a serine protease and protected by glycosylation at the amino end (visit No.: P8343). The properties of N-V protease that can dissolve thrombus are the basis of further study. It is also the basis for setting the subject. In order to study the mechanism of thrombolytic effect, the electrophoretic technique was used to improve the purity of N-V protease to 97.57, which created favorable conditions for further study. In the course of studying the activation of bovine plasminogen by N-V protease, it was found that N-V protease could hydrolyze S2251, which was previously regarded as plasminase-specific substrate. Furthermore, a color developing substrate which can accurately determine the activity of N-V protease was found. When the plasminogen activation of N-V protease and urokinase was compared by plate method, it was found that the mechanism of plasminogen activation between N-V protease and urokinase was obviously different, which combined with N-V protease to hydrolyze albumin, casein and bovine fibrin. The hydrolysis product of human fibrin on SDS-PAGE can be preliminarily judged that the protein substrate was directly hydrolyzed into amino acid instead of plasminogen activator. The insulin-B chain was hydrolyzed by N-V protease, and the results were confirmed by amino acid analysis. The identification of glycoprotein, enzymatic classification and exonuclease properties of N-V protease were also studied. The following conclusions are drawn: 1: 1N-V protease is a kind of aminopeptidase. 3N-V protease is not a plasminogen activator, and it belongs to serine protease class. 2N-V protease is a kind of aminopeptidase. It plays a direct fibrinolysis in thrombolytic process. According to the known enzymatic properties of N-V protease, it can be confirmed that it is a new aminopeptidase. This makes the members of the serine protease family further enriched. 5N-V protease has good hydrolytic protein properties, so it can be used for the determination of protein amino acids and the enzyme for dissolving protein in nucleic acid extraction.
【学位授予单位】：吉林大学
【学位级别】：博士
【学位授予年份】：2006
【分类号】：R341

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