重组海蚯蚓纤溶酶的克隆、表达及活性鉴定北大核心CSCD

纤溶酶在溶栓治疗中起重要作用,能够溶解血凝块的主要成分纤维蛋白。采用RACE方法从海蚯蚓消化道组织中扩增出海蚯蚓纤溶酶编码序列,构建该基因原核表达载体,并进一步构建工程菌表达融合蛋白,经Ni2+树脂柱纯化后通过平板法检测该融合蛋白纤维蛋白酶原激活活性。结果获得海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并成功构建重组表达载体p ET-21a-AFE,表达纯化出融合蛋白,该融合蛋白能够激活纤维蛋白酶原而溶解纤维蛋白。总之,本研究获得了海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并初步证明其具有纤维蛋白酶原激活活性,为临床新型溶栓药物的开发提供实验基础。     

重组海蚯蚓纤溶酶的克隆、表达及活性鉴定

纤溶酶在溶栓治疗中起重要作用,能够溶解血凝块的主要成分纤维蛋白。采用RACE方法从海蚯蚓消化道组织中扩增出海蚯蚓纤溶酶编码序列,构建该基因原核表达载体,并进一步构建工程菌表达融合蛋白,经Ni2+树脂柱纯化后通过平板法检测该融合蛋白纤维蛋白酶原激活活性。结果获得海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并成功构建重组表达载体p ET-21a-AFE,表达纯化出融合蛋白,该融合蛋白能够激活纤维蛋白酶原而溶解纤维蛋白。总之,本研究获得了海蚯蚓纤溶酶的c DNA序列和氨基酸序列,并初步证明其具有纤维蛋白酶原激活活性,为临床新型溶栓药物的开发提供实验基础。     

抗凝血蛋白质分子的研究现状

凝血酶抑制剂,纤溶酶原激活剂（t-Pa,u-Pa,SK和SAK等）,蚯蚓纤溶酶和蛇毒抗凝血蛋白等是血栓类疾病领域的研究重点,纤溶分子突变体的应用,纤维蛋白水解特异性的增加,溶栓效率的提高以及半衰期的延长等是溶栓研究的方向。     

赤爱胜蚯蚓(Eisenia Foetida Sarigny)纤溶酶的研究Ⅱ药理学作用

对蚯蚓纤溶酶进行了哈白兔体内、体外溶栓、抗凝试验,分别以蛇毒抗栓酶和尿激酶不同剂量组作对照试验,均有显著的溶解血栓功能.另外,蚯蚓纤溶酶溶解天然血块,对纤维蛋白原含量及凝血时间影响的药理学试验,结果证明蚯蚓纤溶酶具有溶栓与抗凝作用.     

抗凝血蛋白质分子的研究现状*

凝血酶抑制剂、纤溶酶原激活剂 (t PA、u PA、SK和SAK等 )、蚯蚓纤溶酶和蛇毒抗凝血蛋白等是血栓类疾病领域的研究重点。纤溶分子突变体的应用、纤维蛋白水解特异性的增加、溶栓效率的提高以及半衰期的延长等是溶栓剂研究的方向。     

蚯蚓纤溶酶的成分分析

蚯蚓纤溶酶（ｅａｒｔｈｗｏｒｍｆｉｂｒｉｎｏｌｙｔｉｃｅｎｚｙｍｅｓ,ＥＦＥ）是从蚯蚓体内提取的一类纤维蛋白水解酶,是一种新的溶栓药．利用亲和层析技术,自蚯蚓匀浆液一步提取蚯蚓纤溶酶,并对该酶的成分进行了分析．实验表明,蚯蚓纤溶酶是一组非均一的糖蛋白,含糖量为５％左右,以中性已糖为主;等电点（ｐＩ）在４．０以下;富含Ａｓｐ,而Ｍｅｔ、Ｔｒｐ和Ｌｙｓ含量很少;ｌｍｇ蚯蚓纤溶酶相当于２５０～３００尿激酶单位．     

华广虻肠道溶纤活性蛋白的纯化和性质(英文)

经过 75 %饱和度硫酸铵沉淀、SephadexG 75凝胶过滤层析、Lys Sepharose 4B亲和层析和电泳制备洗脱 ,从华广虻 (TabanusamaenusWalker)腹部组织匀浆液中分离纯化出分子量约为 6 7KD的溶纤活性蛋白TAFP。经纤维蛋白平板测定表明 ,TAFP不仅具有纤溶酶作用 ,还具有激活纤溶酶原的作用 ;通过三肽生色底物测定发现 ,TAFP能分解纤溶酶原激活剂的生色底物—ChromozymUK及S 2 2 88。还能水解胰蛋白酶专一底物Bz Phe Val Arg NA及CBZ Gly Pro Arg NA ,表明TAFP具有类胰蛋白酶活性 ,专一水解精氨酸形成的酰胺键 (或肽键 )。TAFP无胰凝乳蛋白酶活性     

竹叶青Trimeresurus stejnegeri蛇毒凝血酶样酶的酶学性质研究

本文报道从竹叶青蛇毒中纯化的两个凝血酶样酶(组分Ⅰ和组分Ⅱ)的酶学性质研究。结果表明二者在体外都能使人纤维蛋白原转变为纤维蛋白,具有精氨酸酯酶活性,都能水解凝血酶专一性底物。纤维蛋白平板法证明组分Ⅰ具有激活纤溶作用;组分Ⅱ同时具有激活纤溶和直接纤溶作用;组分Ⅰ和组分Ⅱ能缓慢降解纤维蛋白原的α链,随着作用时间延长,组分Ⅰ还能进一步部分降解纤维蛋白原的β链,活性中心探讨表明,组分Ⅰ的活性中心由丝氨酸蛋白酶部位和糖基部位组成。组分Ⅰ水解BAEE的最适温度为50℃,最适pH为8.6。     

竹叶青Trimeresurus stejeegeri蛇毒凝血酶样酶的酶学性质研究

本文报道从竹叶青蛇毒中纯化的两个凝血酶样酶（组分Ⅰ和组分Ⅱ）的酶学性质研究。结果表明二者在体外都能使人纤维蛋白原转变为纤维蛋白,具有精氨酸酯酶活性,都能水解凝血酶专一性底物。纤维蛋白平板法证明组分Ⅰ具有激法纤溶作用;组分Ⅱ同时具有激活纤溶和直接纤溶作用;组分Ⅰ和组分Ⅱ能缓慢降解纤维蛋白原的α链,随着作用时间延长,组分Ⅰ还能进一步部分降解纤维蛋白原的β链,活性中心探讨表明,组分Ⅰ的活性中心由丝氨酸蛋白酶部位和糖基部位组成。组分Ⅰ水解BAEE的最适温度为50 ℃,最适pH为8.6。     

地鳖纤溶活性蛋白的纯化及性质研究

通过硫酸铵分段沉淀、DEAE-纤维素柱和SephadexG-75柱层析从雌地鳖(Eupolyphagesinensiswalker)体内分离纯化到一种相对分子质量约为41.3kD的纤溶活性蛋白,纤维蛋白平板测定表明,该蛋白具有纤溶作用,经SDS-PAGE电泳显示为一条带,含糖量为10.5%。其水解纤维蛋白的比活力为547.86u/mg。该成分受蛋白抑制剂和丝氨酸蛋白酶抑制剂PMSF的抑制,但EDTA对其影响不大,提示该成分属于丝氨酸蛋白酶类。该成分在40℃下基本稳定,最适温度40℃,最适pH为8.0,其激活纤维蛋白溶解酶(PLG)的机制与尿激酶(UK)有一定区别。推测其可能是一种新的地鳖纤溶酶组分。     

The Fibrinogen Concentration in Blood of Dairy Cows and its Influence on the Interpretation of the Glutaraldehyde and Formol-Gel Test Reactions

It has earlier been shown that the formol-gel test on serum and glutaraldehyde test on whole blood are simple and rapid methods for evaluation or the immunoglobulin status in the cow. Both tests function as coagulation tests in which aldehyde groups oross-link basic blood globulins at their NH2-groups, forming polymerisates. The glutaraldehyde has in whole blood the capacity to polymerize not only immunoglobulins but also fibrinogen. This investigation was made in order to study whether the fibrinogen level may influence the result of the glutaraldehyde test, so revealing any differences between the results of that and the formol-gel test carried out on serum. In 92 cows with a variety of clinical disorders (most of them with inflammatory processes) the total protein, albumin, total globulin concentration and albumin/globulin ratio in serum and fibrinogen concentration in plasma were recorded. The material was grouped according to glutaraldehyde and formol-gel test reactions. It is shown that increases in the fibrinogen level have an effect on the results of the glutaraldehyde test. A positive glutaraldehyde test in more acute processes is ascribed to a heavy rise of plasma fibrinogen in its capacity of acute-phase protein. A positive glutaraldehyde test in chronic diseases may be viewed as a result of interaction between high immunoglobulin concentrations and elevated fibrinogen concentration. In conclusion the fibrinogen and immunoglobulin status of blood is important to assess in many diseases of cattle. The semiquantitative tests described for field use can separately, or especially in parallel use, provide valuable information about the character and development of a disease and may be regarded as good substitutes for the sedimentation rate (SR), which is not demonstrable in cattle. kw|Keywords|k]bovine fibrinogen; k]bovine serum proteins; k]formol-gel reaction; k]glutaraldehyde test; k]acute and chronic inflammations     

Erythrocyte oxidative damage by myeloperoxidase. The protective action of serum proteins

Influence of main serum proteins (albumin, immunoglobulin G) and proteins-antioxidants (ceruloplasmin, transferrin, superoxide dismutase) on the oxidative damage of erythrocytes by myeloperoxidase and hypochlorite was investigated. The proteins were determined to act as protectors and decrease the degree of hemoglobin oxidation, ceruloplasmin and albumin possessing the highest antioxidant activity.     

以聚乙二醇为层析伴侣同时制备SOD、过氧化氢酶和血红蛋白

发展了一条从红细胞裂解液中同时制备超氧化物歧化酶(SOD)、过氧化氢酶和血红蛋白的新工艺。采用0 75 %的聚乙二醇600作为层析伴侣,使血红蛋白直接流过阴离子交换层析柱,同时吸附SOD和过氧化氢酶。经过梯度洗脱获得SOD和过氧化氢酶组分,再经过疏水性相互作用层析与凝胶过滤层析相串联,使SOD和过氧化氢酶得到纯化。纯化后的SOD和过氧化氢酶的比活力分别达到15932u/mg和65918u/mg ,血红蛋白的纯度达到99.9%以上。总回收率为:SOD ,47.4% ;过氧化氢酶,29.6% ;血红蛋白,88.7%。     

Serum protein degradation by hypochlorite

The structural integrity of serum proteins: albumin, immunoglobulin G, transferrin, ceruloplasmin and superoxide dismutase, and the functional activity of the latter two enzymes after their interaction with hypochlorite were studied. It was shown that the interaction between the proteins and hypochlorite resulted in protein injury and degradation of their native structure. In the case of ceruloplasmin and transferrin, a practically complete protein "dissipation" occurred, the albumin and superoxide dismutase structures being injured in a lesser degree. The inactivation of ceruloplasmin was slower than that of superoxide dismutase. The protein degradation by hypochlorite seems to be the main factor restricting the ability of the proteins to act as antiinflammatory drugs.     

The adsorption of bovine blood proteins onto the surface of O-(carboxymethyl)chitin

Chitin was found to interact with bovine blood proteins and the affinities of these proteins for chitin tended to be decreased by the introduction of O-carboxymethyl (CM) groups onto the chitin surface, especially with fibrinogen. As the adsorption of blood proteins to the CM-chitin (d.s. 0.35) was assumed to follow an isothermal adsorption-curve, the adsorption coefficients were estimated by applying the Langmuir equation. Bovine serum albumin showed the highest affinity among the proteins applied in this experiment [KBSA (bovine serum albumin); 20.0, KB gamma G (bovine gamma globulin); 1.96, KBF (bovine fibrinogen); 1.20]. The binding site of BSA for CM-chitin was assumed to be regulated not only by the cationic groups of BSA but also by other factors such as the recognition capacity of BSA to bind to GlcNAc residues in CM-chitin.     

Antioxidative properties and degradation of serum proteins by active oxygen forms (O2-., OCl-) generated by stimulated neutrophils

The ability of major serum proteins (albumin, immunoglobulin G) and free radical scavenger proteins (ceruloplasmin, superoxide dismutase, transferrin) to interact with O2-. and OCl- was studied. The interaction between serum proteins and OCl- was shown to be nonspecific and cause protein degradation. During SDS polyacrylamide gel electrophoresis ceruloplasmin and transferrin were degraded in the highest degree. Protein damage was also recorded by fluorescence changes. It is suggested that the damaging influence of active oxygen species secreted by stimulated neutrophils into the extracellular space can be abolished only by ceruloplasmin.     

Luminol assay for microdetermination of superoxide dismutase activity: its application to human fetal blood

A microtechnique for determining the superoxide dismutase activity in erythrocytes is described. This technique involves the inhibition of luminol-enhanced chemiluminescence of superoxide anion generated by xanthine-xanthine oxidase. Measurements required a steady-state chemiluminescence whether superoxide dismutase was present or absent; the level of luminescence was correlated to enzyme activity. Superoxide dismutase activity measured by this technique was 836 +/- 112 micrograms/g of hemoglobin for whole blood and 834 +/- 109 micrograms/g of hemoglobin for erythrocytes. When the reference technique was applied to larger amounts of blood, the results were 862 +/- 58 and 858 +/- 116 micrograms/g of hemoglobin for whole blood and washed erythrocytes, respectively. The enzymatic activity of superoxide dismutase from fetal blood (obtained by venipuncture in utero and of 19-26 weeks gestational age) was similar to that of adult blood, when measured by the new technique.     

Stability of helix-rich proteins at high concentrations

A number of techniques, including circular dichroism, FTIR, front face fluorescence, and UV absorption spectrophotometries, dynamic light scattering, and DSC, were used to directly measure the colloidal and conformational stability of proteins in highly concentrated solutions. Using bovine serum albumin (BSA), chicken egg white lysozyme, human hemoglobin A0, and bovine fibrinogen as model proteins, the thermal transition temperatures of proteins in dilute and concentrated solutions were compared. At 10 degrees C, no significant differences in both secondary and tertiary structures were detected for proteins at different concentrations. When temperature was introduced as a variable, however, hemoglobin and fibrinogen demonstrated higher transition midpoints (T(m)s) in concentrated rather than in dilute solutions (deltaT(m) approximately 2-10 degrees C). In contrast, lysozyme and BSA in concentrated solutions exhibit a lower T(m) than in dilute solutions (deltaT(m) approximately 2-20 degrees C). From these studies, it appears that a variety of factors determine the effect of high concentrations on the colloidal and conformational stability of a particular protein. While the prediction of excluded volume theory is that high concentrations should conformationally stabilize proteins, other factors such as pH, kinetics, protein dynamics, and intermolecular charge-charge effects may affect the overall stability of proteins at high concentrations under certain conditions.     

A comparative study of serum proteins ability to scavenge active oxygen species: O2-. and OCl-

The ability of serum proteins (albumin, immunoglobulin G) and protein antioxidants (ceruloplasmin, superoxide dismutase and transferrin) to react with O2-. and OCl-, was studied. The interaction between serum proteins and OCl- was shown to be non-specific. Ceruloplasmin is the most effective OCl- trapping protein, and it reacts with O2-. with a considerable efficiency. Therefore, ceruloplasmin is supposed to be the main scavenger of toxic oxygen species generated by stimulated neutrophils.     

Purification and characterization of cathepsin B from monkey skeletal muscle

Cathepsin B was purified about 11,000-fold from monkey skeletal muscle by ammonium sulfate fractionation and sequential column chromatographies monitored by assaying of Z-Phe-Arg-MCA hydrolase activity. The purified enzyme gave a single protein band on SDS-polyacrylamide gel electrophoresis, and its molecular weight was estimated to be 24,000 by gel filtration. It had a pH optimum of 6.5, required a thiol reducing agent for activation, and was inhibited by various thiol protease inhibitors. These properties were similar to those reported for cathepsins B from other sources. Although the enzyme scarcely hydrolyzed ordinary proteins, such as casein, hemoglobin, and bovine serum albumin, it degraded myosin and actin among various myofibrillar proteins. These results strongly suggested that skeletal muscle cathepsin B may participate in the degradation of muscle proteins in vivo. In addition, cathepsin B was shown to hydrolyze various neuropeptides such as Leu-enkephalin, beta-neoendorphin, alpha-neoendorphin, dynorphin(1-13), and substance P. It appeared to act on these peptides mainly as a dipeptidyl carboxypeptidase, although not so rigorously, presumably due to its endopeptidase activity.