Studies on Cytochrome c Modified by [PtdienNO_3]Cl

For the first time, [PtdienNO_3]Cl was used as a stable reagent to modify ferricytochrome c and the reaction products were separated and purified with the CM-52 cation exchange chromatography. Five components were obtained, corresponding to the native cytochrome c single-labeled, dual-labeled, and triple-labeled derivatives as shown by the analysis of the molar ratio of the two metal atoms (Pt and Fe). The reduction potentials of these proteins were measured by differential pulse voltammetry. His-33 and Trp-59 were identified by~1HNMR as the binding sites of the platinum complex in the modified cytochrome c derivatives. Trp-59 was a conserved amino acid connected with the heme through hydrogen bond, which had not been modified by other transition metal complexes. The platinummodified cytochrome c derivatives might be valuable in exploring the role of the aromatic amino acids, especially Trp-59, in electron transfer.     

抗活化血小板单克隆抗体与尿激酶杂合分子的纤溶作用

用双功能团试剂将尿激酶(UK)B链和抗人活化血小板α-颗粒膜蛋白GMP-140单克隆抗体(SZ-51)的Fab片段共价偶联。偶联的杂合分子UK-SZ-51保留了原抗体的结合专一性,它在体外的溶栓效率较尿激酶提高约5倍,其溶栓作用对血浆中纤维蛋白原的含量无影响。     

慈菇蛋白酶抑制剂的抑制特性及其活性中心的探讨

本文采用亲和层析分离纯化了结晶慈菇蛋白酶抑制剂A和B,抑制剂A和B均为双头多功能蛋白酶抑制剂,抑制剂A能同时等当量抑制胰蛋白酶和胰凝乳蛋白酶,对激肽释放酶的抑制作用较弱,抑制剂B能当量抑制2克分子的胰蛋白酶,对激肽释放酶的抑制活力高于抑制剂A,但对胰凝乳蛋白酶的抑制作用远比抑制剂A弱。化学修饰以及胰蛋白酶与胰凝乳蛋白酶对抑制剂A的竞争性结合表明:抑制剂A和B的两个活性中心均为Lys和Arg残基,其中Lys活性中心专一抑制胰蛋白酶,而由Arg活性中心构成的活性区域则表现为多功能,能抑制多种蛋白酶,从抑制剂A和B的结构特征推测,两活性中心应分别为Lys-Ser(44—45)及Arg-Tyr-Lys(76—78),在抑制剂A中还存在一疏水性残基参与对胰凝乳蛋白酶的抑制,此残基位于由Arg活性中心所构成的活性区域中。     

Urokinase片断的二级、三级结构预测及构效关系研究

urokinase(简称UK)属于絲氨酸蛋白酶,其一级结构已经测定。去掉前135个残基后得到的低分子量UK(LUK)氨基酸残基的顺序与糜胰蛋白酶、胰蛋白酶等絲氨酸蛋白酶十分类似。从二硫桥的配置来看LUK与糜胰蛋白酶比较接近,而UK对Lys侧链处肽键的专一性则和胰蛋白酶相近。在这一类酶中,起到活性中心作用的氨基酸为Ser195,His57及Asp102.在UK中此三个氨基酸都保留了下来。蛋白水解酶中起专一性作用的残基主要是     

THE STRUCTURAL BASIS OF THE POOR FIBRIN SPECIFICITY OF UROKINASE(Ⅰ)——KNOWLEDGE-BASED PREDICTION OF KRINGLE STRUCTURES OF UROKINASE AND ITS RELATED PROTEINS

The Kringle-1 structure of plasminogen (PGK-1), the Kringle-2 structure of tissue plasminogen activator (PAK-2) and the Kringle structure of prourokinase (UKK) has been modeled on the basis of the three-dimensional structure of Kringle-1 of prothrombin (PTK-1) at 2.8 resolution. The predicted three-dimensional structure of these Kringles shows that the binding site of PGK-1 is characterized by an apparent dipolar site, the polar parts of which are separated by a hydrophobic region. PAK-2 possesses the anionic center but has not a cationic binding center which might be provided by a guanidinium group from Arg-69 located adjacent to the Arg-71 position. UKK possesses neither the anionic binding center nor the cationic center which are probably the main reason for the poor fibrin specificity of urokinase.     

Synthesis and Expression of a Gene From Kringle-2 Domain of Tissue Plasminogen Activator in E. coli

The DNA fragment corresponding to the tissue plasminogen activator (tPA) sequence 174-262 (Kringle-2 domain) has been synthesized by using the solid phase phosphotriester method. The Kringle-2 domain of human tPA was expressed in Escherichia colt by secretion into the periplasmic space using the Lpp-Lac promoter and PIN-Ⅲ OmpA2 signal sequence. About two thirds of the expression product was secreted into the periplasmic space , and purified with ammonium sulfate fractionation, affinity chro-matography on Lysine-Sepharose, and FPLC-Mono Q exchange chromatography. The amino acid composition observed from the Kringle-2 purified from E. coli is identical with that expected for the 174-262 fragment of human tPA. Radio binding assay shows that the recombinant Kringle-2 domain possesses the activity of fibrin binding.     

THE STRUCTURE BASIS OF THE POOR FIBRIN SPECIFICITY OF UROKINASE(Ⅱ)——THE INHIBITION OF UROKINASE A CHAIN 149--157 ON THE FIBRIN STIMULATED ACTIVATION OF PLASMINOGEN BY TISSUE TYPE PLASMINOGEN ACTIVATOR

In view of the similarity of the charge distribution between fibrin A_α148--161 and Achain 149--157 of urokinase,the latter might compete with fibrin A_α148--161 when singlechain pro-urokinase is converted to double chain urokinase.To test this, the stretch of uro-kinase A chain 135--157 was separated from the low molecular weight urokinase, a competi-tive binding between this stretch and fibrin to tPA kringle-2 was shown by radio-bindingassay. The inhibition of the stretch on the fibrin stimulated activation of plasminogen wasdemonstrated in the caseinolytic system. The synthesized novapeptide urokinase A chain 149--157 (R-peptide) showed a significant inhibition on the activation of plasminogen in the pres-ence of fibrin. By contrasting finely with R-peptide, a synthesized novapeptide in which Arg154and Arg156 were replaced by Asp (D-peptide) did not show any inhibition effect on the fi-brin stimulated activation of plasminogen by tPA. These results suggest that the positivelycharged residues in the     

尿激酶对纤维蛋白低亲和性的结构基础——Ⅰ.尿激酶及相关环饼结构域的三维结构预测

根据凝血酶原(Prothrombin)环饼结构域-1(PTK-1)2.8晶体X光衍射结构,用残基置换和构型建造(Modeling)预测了纤溶酶原环饼结构域-1(PGK-1),组织型纤溶酶原激活因子环饼结构域-2(PAK-2)和尿激酶原环饼结构域(UKK)的三维结构。预测的三维结构显示PGK-1具有典型的配体结合部位,PAK-2在结构上不具有和配体结合的典型正电荷中心,但邻近的69位精氨酸残基能和配体的羧基生成离子对。UKK在结构上不能形成和配体结合的正负电荷中心,这可能是尿激酶对纤维蛋白低亲和性的主要原因。     

慈菇蛋白酶抑制剂的一级结构

慈菇蛋白酶抑制剂A和B经硫硫键还原及羧甲基化后,分别用内肽酶及溴化氰裂解,肽段经分离纯化后在气相蛋白质序列仪上进行序列分析,通过重叠肽段的拼接,阐明了全部一级结构.慈菇抑制剂A和B均由150个氨基酸残基组成,含有三对硫硫键.两者在结构上有91％的同源性,但与其它已知结构的丝氨酸蛋白酶抑制剂有明显不同.因而它是属于新的一族抑制剂.根据两者的结构特征.推测它们的活性中心均为Lys-Ser(44—45)和Arg-Tyr-Lys(77—79).在抑制剂A,B 13个残基差异中,其中第87位残基由Leu置换为Arg.这可能是引起两者抑制特性差异的主要原因.     

快速蛋白质液相色谱法分离鉴定脱氧寡核苷酸片段

由机器合成的反义核酸药物需要有效的纯度鉴定方法。用ＭＯＮＯ Ｑ柱在ｐＨ１２时以ＮａＣｌ的浓度梯度洗脱,可将１９至２１Ｎｔｓ的小片段寡核苷酸很好分开,因此快速蛋白质液相色谱法可用来分离鉴定反义核酸药物。