烙铁头(TRI MERESURUS MUCROSQUAMATUS)蛇毒中精氨酸酯酶的研究

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	烙铁头(TRI MERESURUS MUCROSQUAMATUS)蛇毒中精氨酸酯酶的研究
	朱毅; 朱毅
学位类型	硕士
导师	熊郁良
	1989
学位授予单位	中国科学院研究生院
学位授予地点	北京
关键词	蛇毒精氨酸酯酶纤维蛋白原溶解活性精氨酶酯酶纤维蛋白溶解活性
摘要	采用DEAE－sephadex A-50、Sephadex G-100、CM-sepharose cl-6B三步柱层析，从烙铁头(T. mucrosquamatus)蛇毒中纯化得到的精氨酸酯酶在聚丙烯酰胺凝胶电泳(pH 8.3)和SDS－聚丙烯酰胺凝胼电泳中均呈现单一的蛋白带。其分子量为29000，等电点pI为5.2；由225个氨基酸残基组成，其中Gly,Asp和Glu的含量较高。它是一个糖蛋白，含有0.5%的中性已糖和0.75%的唾液酸，对热及酸碱变化较稳定。在280nm波长处有典型的蛋白质吸收峰，此时的消光数E0.1%/1cm为1.332。该酶具有较强的精氨酸酯酶活性和纤维蛋白原溶解活性，无出血活性、酪蛋白水解活性以及粗毒中含有的其它酶活性。从烙铁头(T. mucrosquamatus)蛇毒中纯化的具纤维蛋白原溶解活性的精氨酸酯酶（MFAE）是一丝氨酸蛋白酶，其活性可被PMSF抑制而不受EDTA的影响。MFAE酶促反应的最适pH为8.4，最适温度55 ℃; pH7.6、37 ℃时水解BAEER的米氏常数K_m为20 * 10~(-3)M。该酶能降解纯化人纤维蛋白原的Bβ链以及纯化牛纤维蛋白原的Aα和Bβ链，并有一定的纤维蛋白溶解活性。它能明显延长兔血浆的凝血酶时间和复钙时间。纯化的MFAE无出血活性、凝血酶样活性及血小板聚集活性，对ADP、AA、TMVA、Melittin等诱导的血小板聚集也无抑制或解聚作用。本文还测定了它对凝血酶及胞浆毒特异性合成三肽底物的水解活性。
其他摘要	By means of DEAE-sephadex A-50, Sephadex G-100, CM-sepharose cl-6B column chromatography, an arginine ester hydrolase was purified from the venom of Trimeresurus mucrosquamatus, which showed single band on SDS-polyacrylamide gel electrophoresis and polyacrylamide gel electrophoresis at pH 8.3. This enzyme possessed both BAEE hydrolase and fibrinogenolytic activity, no caseinolytic, hemorrhagin activity and other enzymatic activies which contained in crude venom. Its molecular weight is approximately 29,000 and isoelectric point is 5.2. The enzyme consists of 225 amino acid residues, containing relatively large amounts of acidic amino acids and glycine. The enzyme is a glycoprotein which contains 0.5% neutral hexose and 0.75% sialic acid. This enzyme is stable to heat treatment and pH change. The extinction coefficient E0.1%/1cm at 280nm of this enzyme was 1.332. The arginine ester hydrolase purified from the venom of Trimeresurus mucrosquamatus (MFAE) showed obvious fibrinogenolytic activity, which could digest the Bβ-chain of purified human fibrinogen and the Aα-,Bβ-chain of purified bovine fibrinogen. The enzyme also had fibrinolytic activity, but no hemorrhagin, thrombin-like or platelet aggregative activity. It did not inhibit or dissociate the platelet aggregation induced by ADP, AA, TMVA and Melittin. MFAE was inhibited by PMSF not by EDTA. suggesting that the serine residues were essential for enzyme activity and the enzyme activity was not metal-ion dependent. The optimum pH and optimum temperature for this enzyme was 8.4 and 55 ℃ respectively, the Michaelis-Mentenconstant(Km) of this enzyme for hydrolyzing BAEE was 20 * 10-3M. The hydrolysis of MFAE on synthetic chromogenic substrates (for assay of thrombin and plasmin activity) was also reported in the article.
语种	中文
文献类型	学位论文
条目标识符	http://ir.kiz.ac.cn/handle/152453/6232
专题	其他
推荐引用方式 GB/T 7714	朱毅,朱毅. 烙铁头(TRI MERESURUS MUCROSQUAMATUS)蛇毒中精氨酸酯酶的研究[D]. 北京. 中国科学院研究生院,1989.

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