小麦巯基蛋白酶抑制剂的纯化和性质研究

将小麦麦胚和麸皮的浸取液，经加热和利用ｐＨ沉淀，获得疏基蛋白酶抑制剂粗品；再经ＤＥＡＥ－Ｓｅｐｈａｒｏｓｅ，Ｓｅｐｈａｄｅｘ Ｇ－１００分子筛层析，在ＳＤＳ－ＰＡＧＥ和ＨＰＬＣ柱上得到均为单一蛋白带的小麦ＣＰＩ纯品。其纯化度为原料浸液的４２倍。由ＳＤＳ－ＰＡＧＥ测得ＣＰＩ分子为单一肽链     

猪心肌细胞质苹果酸脱氢酶的制备

用匀浆提取，９０％硫酸铵沉淀和ＣＭ－纤维素、ＤＥＡＥ－ＳｅｐｈａｂｅｘＡ－５０、ＳｅｐｈａｄｅｘＧ－１００柱层析的方法从猪心肌中分离纯化了细胞质苹果酸脱氨酶。酶制品达到了ＩＦＣＣ规定的标准。     

杜氏藻中多糖的化学研究（Ⅰ）

用０．２％稀碱溶液萃取杜氏藻提取出β－胡萝卜素后的残渣，经浓缩后用乙醇沉淀，得粗多糖，以Ｓｅｖａｇｅ法除去蛋白质，再经ＤＥＡＴ－纤维素及葡聚糖凝胶Ｇ－１００柱层析，得杜氏藻多糖（简称ＰＤＳ）．多糖经薄层层析、纸层析、气相色谱及经甲基化后的色谱／质谱分析，获知有５种糖，即半乳糖、木糖、鼠李糖、葡萄糖、甘露糖，并测得各种糖的可能连接．     

黑曲霉胞外葡萄糖淀粉酶的纯化及特性

黑曲霉（Ａｓｐｅｒｇｉｌｌｕｓｎｉｇｅｒ７２８）的粗酶液，经硫酸铵沉淀，ＳｅｐｈａｄｅｘＧ－２５柱凝胶过滤脱盐，ＤＥＡＥ－Ｔｏｙｏｐｅａｒｌ离子交换柱层析和ＳｅｐｈａｃｒｙＳ－１００凝胶层析纯化，经ＰＡＧＥ鉴定为一条带ＳＤＳ凝胶电泳测定分子量为７００００．金属离子Ｆｅ３＋对该酶有一定的抑制作用，该酶的等电点为３．７，最适ｐＨ为４，最适温度为６０℃．Ｅ２８０１％为１５．７２，Ｋｍ值为０．１１２×１０－３ｍ．     

饭豆铜锌超氧物歧化酶的纯化及性质

饭豆种子粗提液中有６条Ｃｕ·Ｚｎ—ＳＯＤ活性谱带，经氯仿－乙醇混合液处理，硫酸铵盐析，ＳｅｐｈａｄｅｘＧ－１００凝胶过滤和ＤＥＡＥ－纤维素柱层析被分离，获得了两组ＳＯＤ同工酶组分（ＳＯＤ—１，ＳＯＤ—２）．它们在酶分子结构上可能存在一定差异．ＳＯＤ－１含与粗提液相对的、电泳迁移率较小的活性谱带，它在聚丙烯酰胺凝胶电泳（ＰＡＧＥ）图谱上的活性染色带和蛋白染色带相互对在；在ＳＤＳ－ＰＡＧＥ和等电聚焦电泳图谱上均呈现单一区带，说明该酶已纯化到均一程度．该酶分子量为３１３００，亚基分子量为１６５００，等电点为４．２，在紫外区的吸收值为２６４．０ｎｍ．该必在０—７５℃范围内稳定．纯化的ＳＯＤ—１比活性为１７８９Ｕ／ｍｇ，ＳＯＤ—２比活性为２８４Ｕ／ｍｇ，ＳＯＤ活力回收率为３２％．     

花椰菜凝集素的纯化及部分性质的研究

花椰菜凝集素由花蕊组织经生理盐水抽提、硫酸铵沉淀和ＳｅｐｈａｄｅｘＧ－１００、Ｓｅｐｈａｒｏｓｅ－４Ｂ凝胶层析纯化获得．ＰＡＧＥ鉴定和高碘酸－Ｓｃｈｉｆｆ试剂反应均显单一条带，说明该凝集素为纯化的糖蛋白．经ＳｅｐｈａｄｅｘＧ－２００柱过滤分析，其相对分子质量约为９４０００．ＳＤＳ－ＰＡＧＥ分析表明该凝集素含有４个亚基，其相对分子质量分别约为２８０００、２５０００、２２０００和２００００．花椰菜凝集素经５０℃处理５分钟仍保持高的凝集活性，该凝集素对酸处理较碱处理稳定．     

柱层析法纯化且人肿瘤坏死因子α及其抑瘤作用

半合成人肿瘤坏死因子ＴＮＦ－α在大肠杆菌中高表达，发酵后收集菌体经超声破碎，然后在１２０００ｒ／ｍｉｎ４℃离心１０ｍｉｎ，上清经６０℃热处理３０ｍｉｎ，离心上清用６０％饱和度的硫酸铵沉淀，再依次经过ＳｅｐｈａｄｅｘＧ－２５，ＤＥＡＥ－５２纤维素和Ｓｅｐｈａｄｅｘ－Ｇ－７５柱层析，得到的纯化ｒｈＴＮｆ－ａ比活性为２×１０＾７ｕ／ｍｇ，回收率为２４％，经过ＳＤＳ－ＰＡＧＥ电泳和ＨＰＬＣ鉴定，纯度达９５     

人尿胰蛋白酶抑制剂的分离纯化

人尿经吸附剂处理，硫酸铵沉淀后，得到胰蛋白酶抑制剂粗品。粗品经ＤＥＡＥ－ＳｅｐｈａｄｅｘＡ－５０层析及ｔｒｙｐｓｉｎ－Ｓｅｐｈａｒｏｓｅ亲和层析，得到两种ＨＵＴＩ：ＨＵＴＩ１与ＨＵＴＩ２。ＨＵＴＩ１的比活为３５５６ｕｎｉｔｓ／ｍｇ，活性回收率为３５％，含分子量６７０００与２５０００两种组粉；ＨＵＴＩ２的比活为３３８５ｕｎｉｓ／ｍｓ，活性回收率为３１％，分子量为６７０００。经二步层析纯化，纯化因子达     

短裙竹荪（Dictyophor a duplicata）多糖的研究（I）：Dd多糖的分离…

竹荪子实体经热水提取，乙醇沉淀，蛋白酶法和Ｓｅｖａｇ法相结合去蛋白，再通过ＤＥＡＥ－Ｓｅｐｈａｄｅｘ Ａ－２５和Ｓｅｐｈａｄｅｘ Ｇ－２００柱层析纯化，得到竹荪多糖（简称Ｄｄ）纯品，经凝胶过滤法和聚丙烯酰胺凝胶电泳法测定表明，Ｄｄ多糖为均一的物质，Ｄｄ经紫外扫描，无核酸和蛋白质的特征吸收峰，ＩＲ－４００扫描表明典型多糖吸收峰，Ｄｄ的相对分子质量约为１９６０００。     

海洋细菌11211的生长条件及其几丁质酶研究

从南海海水中分离到产几丁质酶的弧菌（Ｖｉｂｒｉｏ ｓｐ．）,研究了菌株１１２１１的生长条件和产酶条件。１１２１１菌生长需要Ｎａ＾＋,海水的其他成分对菌生长也有影响。胶体几丁质可诱导几丁质酶产生。在几丁质培养基中１１２１１菌的产酶高峰为生长的第７天。经硫酸铵沉淀,Ｓｅｐｈａｄｅｘ Ｇ－１００、ＤＥＡＥ－纤维素及几丁质柱层析,酶被纯化了１８倍。经ＳＤＳ－ＰＡＧＥ分析,酶的相对分子质量约为３００００。该     

Domains specifying thrombin-receptor interaction.

Platelet activation by the coagulation protease thrombin is central to arterial thrombosis, a major cause of morbidity and mortality. We recently isolated a complementary DNA encoding the platelet thrombin receptor. The extracellular amino-terminal extension of this seven transmembrane domain receptor contains the putative thrombin cleavage site LDPR/S which is critical for receptor activation. By replacing this cleavage site with the cleavage site for enterokinase, we have created a functional enterokinase receptor. This result demonstrates that all information necessary for receptor activation is provided by receptor proteolysis. Nanomolar enterokinase concentrations are required to activate this new receptor, in contrast to the picomolar thrombin concentrations that activate wild-type thrombin receptor. We identified a receptor domain critical for thrombin's remarkable potency at its receptor. This domain resembles the carboxyl tail of the leech anticoagulant hirudin and functions by binding to thrombin's anion-binding exosite. Our studies thus define a model for thrombin-receptor interaction. The utility of this model was demonstrated by the design of novel thrombin inhibitors based on receptor peptides.     

PAR3 is a cofactor for PAR4 activation by thrombin

Identification of the mechanisms by which the coagulation protease thrombin activates platelets is critical for understanding haemostasis and thrombosis. Thrombin activates cells at least in part by cleaving protease-activated G-protein-coupled receptors (PARs). PAR3 and PAR4 are thrombin receptors expressed in mouse platelets. Inhibition of thrombin binding to mPAR3 (ref. 4) and knockout of the mPAR3 gene inhibited mouse platelet activation at low but not high concentrations of thrombin. Thus PAR3 is important for thrombin signalling in mouse platelets. Expression of human PAR3 in heterologous expression systems reliably resulted in responsiveness to thrombin. Curiously, despite its importance for the activation of mouse platelets by thrombin, mouse PAR3 (mPAR3) did not lead to thrombin signalling even when overexpressed. We now report that mPAR3 and mPAR4 interact in a novel way: mPAR3 does not itself mediate transmembrane signalling but instead functions as a cofactor for the cleavage and activation of mPAR4 by thrombin. This establishes a paradigm for cofactor-assisted PAR activation and for a G-protein-coupled receptor's acting as an accessory molecule to present ligand to another receptor.     

Role of thrombin signalling in platelets in haemostasis and thrombosis.

Platelets are critical in haemostasis and in arterial thrombosis, which causes heart attacks and other events triggered by abnormal clotting. The coagulation protease thrombin is a potent activator of platelets ex vivo. However, because thrombin also mediates fibrin deposition and because multiple agonists can trigger platelet activation, the relative importance of platelet activation by thrombin in haemostasis and thrombosis is unknown. Thrombin triggers cellular responses at least in part through protease-activated receptors (PARs). Mouse platelets express PAR3 and PAR4 (ref. 9). Here we show that platelets from PAR4-deficient mice failed to change shape, mobilize calcium, secrete ATP or aggregate in response to thrombin. This result demonstrates that PAR signalling is necessary for mouse platelet activation by thrombin and supports the model that mouse PAR3 (mPAR3) does not by itself mediate transmembrane signalling but instead acts as a cofactor for thrombin cleavage and activation of mPAR4 (ref. 10). Importantly, PAR4-deficient mice had markedly prolonged bleeding times and were protected in a model of arteriolar thrombosis. Thus platelet activation by thrombin is necessary for normal haemostasis and may be an important target in the treatment of thrombosis.     

分光光度法测定盐源山蛭素对凝血酶的抑制活性

报告了一实用的凝血酶抑制剂抑制活性的测定方法——发色底物分光光度法；以发色底物S—2238与标准凝血酶反应，测定未抑制的吸光度Ao，同时以发色底物S—2238与凝血酶抑制剂盐源山蛭素和标准凝血酶反应测定抑制吸光度As，然后以As／Ao计算盐源山蛭素的抑制活性a1．实验证明，这种对盐源山蛭素抑制活性的测定方法简便易行、重复性高、偶然误差小、可信度高．     

Effect of thrombin on the apoptosis of hippocampal neurons in vitro

Hippocampal neurons were treated by thrombin and thrombin receptor activating peptides (TRAP). Cell survival rate was decreased in a dose-dependent manner by MTT assay. The numbers of apoptotic cell and apoptotic rate of hippocampal neurons treated by different concentrations of thrombin were increased in a dose-dependent manner by terminal deoxynucleotidyl transferase (TdT) mediated dUTP-biotin nick end-labeling (TUNEL) method and Flow Cytometry. When the concentration of thrombin is 40 U/mL, TUNEL positive cells and apoptotic rate of hippocampal neurons reached peak value, were 27.3±4.0 and (29.333±4.633)%, respectively. Immunocytochemistry assay show that Bcl-2 protein expression was down-regulated and Bax protein expression was up-regulated with the concentration of thrombin increased. TRAP can mimic the effect of thrombin to induce apoptosis on hippocampal neurons. These data demonstrated that thrombin induced hippocampal neuron apoptosis in a dose-dependent manner through activating protease-activated protein-1 (PAR-1). The change in expression of Bcl-2 and Bax was related with the effect of high concentration thrombin induced apoptosis on hippocampal neurons. Foundation item: Supported by the Natural Science Foundation of Hainan Province (N30215) Biography: YANG Wen-qiong (1968-), female, Ph.D. candidate, research direction: cerebrovascular disease.     

Structural basis for the anticoagulant activity of the thrombin-thrombomodulin complex

The serine proteinase alpha-thrombin causes blood clotting through proteolytic cleavage of fibrinogen and protease-activated receptors and amplifies its own generation by activating the essential clotting factors V and VIII. Thrombomodulin, a transmembrane thrombin receptor with six contiguous epidermal growth factor-like domains (TME1-6), profoundly alters the substrate specificity of thrombin from pro- to anticoagulant by activating protein C. Activated protein C then deactivates the coagulation cascade by degrading activated factors V and VIII. The thrombin-thrombomodulin complex inhibits fibrinolysis by activating the procarboxypeptidase thrombin-activatable fibrinolysis inhibitor. Here we present the 2.3 A crystal structure of human alpha-thrombin bound to the smallest thrombomodulin fragment required for full protein-C co-factor activity, TME456. The Y-shaped thrombomodulin fragment binds to thrombin's anion-binding exosite-I, preventing binding of procoagulant substrates. Thrombomodulin binding does not seem to induce marked allosteric structural rearrangements at the thrombin active site. Rather, docking of a protein C model to thrombin-TME456 indicates that TME45 may bind substrates in such a manner that their zymogen-activation cleavage sites are presented optimally to the unaltered thrombin active site.     

花生蛋白质的主要种类和等电点的研究

花生蛋白等电聚焦显１１条区带，其中６条很明显，说明花生至少有１１类不同等电点的蛋白质。等电点分别为４，３、４．５、５．１、５．４、６．５、６．７的蛋白质是主要的，他们的聚丙烯酰胺凝胶电泳和花生蛋白乳状液的醋酸纤维薄膜电泳的结果都有３条区带，其中两条比较明显，说明花生蛋白有两种主要组分和一种次要组分；这三种组分可有多种等电点，一种等电点可同时有这三种组分。     

发酵液中谷氨酸分离方法的研究

采用谷氨酸发酵液先浓缩后调节等电点以提取谷氨酸。谷氨酸收得率在８５－９０％，比常用的等电点法收得率可提高１０—２０％．     

Thrombin-stimulated cell division involves proteolysis of its cell surface receptor.

A cell-surface component of molecular weight 43,000 is cleaved by thrombin on cells that divide after thrombin treatment, but is not cleaved on cells that are unresponsive to its mitogenic action. Studies with a photoreactive derivative of thrombin showed that its cell surface receptor has a molecular weight of 43,000. This indicates that thrombin must cleave its receptor to stimulate cell division.     

龙血竭总酚活血化瘀活性成分的虚拟筛选及初步活性研究

应用Discovery Studio虚拟筛选软件,以凝血酶为受体,对龙血竭总酚活血化瘀有效成分进行了虚拟筛选. 虚拟筛选确定的受试成分对体外凝血酶活性的影响数据表明,计算机辅助虚拟筛选结果与体外药理实验结果基本符合. 受试成分龙血素A、龙血素B、龙血素C、7-羟基-4'-甲氧基黄烷、4',7-二羟基高异黄烷能抑制体外凝血酶活性,具有活血化瘀的药理作用.