ELECTRON MICROSCOPY OF HUMAN ALPHA-2 MACROGLOBULIN

人 ALPHA-2 巨球蛋白的电子显微镜

• 批准号: 3361238
• 负责人: JAMES K STOOPS
• 金额: $ 15.56万
• 依托单位: UNIVERSITY OF TEXAS HLTH SCI CTR HOUSTON
• 依托单位国家: 美国
• 财政年份: 1990
• 资助国家: 美国
• 起止时间: 1990-09-01 至 1997-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3361238
• 关键词: chemical binding; chemical cleavage; chymotrypsin; conformation; cryoscopy; electron microscopy; enzyme complex; gold; image processing; immunoelectron microscopy; macroglobulins; methylamines; papain; plasmin; plasminogen activator; protease inhibitor; protein engineering; protein purification; protein structure function; recombinant proteins; site directed mutagenesis; stainings; thioester; thrombin; transfection

Alpha-Macroglobulin (alpha2M), a general proteinase inhibitor ubiquitous in the plasma of vertebrates, is believed to serve as a general proteinase scavenger thereby protecting blood and tissue proteins from degradation. As a proteinase inhibitor, it is involved in the regulation of proteinase activity in fibrinolysis, coagulation and complement activation. It interacts with cytokines and growth factors and it may be of physiological importance in the degradation of thrombi. A better understanding of the structure-function relationships of alpha2M will result from the determination of the structure of its various complexes by stain and cryo-electron microscopy, and image processing. The importance of the thioester site which is cleaved during the transformation of alpha2M to the proteolyzed (activated) form will be evaluated by determining the structure in which this site has been eliminated by site directed mutagenesis. Plasmin (Mr=80,000) forms a binary complex with alpha2M whereas smaller proteinases such as alpha- chymotrypsin (Mr=25,000) form a ternary complex. The determination of the 3-D structure of the binary complex and its comparison with our 3-D structure of the ternary alpha2M-chymotrypsin complex should reveal the location of thrombin in the structure and help to assess the significance of the larger size of plasmin on the manner in which it is bound to alpha2M. Thrombin (Mr=33,500) also forms a binary complex, possibly because its reaction with alpha2M is considerably slower than those proteinases that form a ternary complex. A genetically engineered alpha2M in which the bait region more closely matches the specificity of thrombin will be prepared in order to determine whether increasing the rate constant for the reaction results in the formation of ternary complex. The 3-D structure of the engineered alpha2M and its complex with thrombin will complement these studies. Further understanding of the complex transformations that are involved in the activation of alpha2M will result from the determination of (1) the 3_D structure of intermediate forms and (2) the disposition of N- and C-terminal Fabs bound to the native, intermediate, and activated structures. Immunoelectron microscopy will also be utilized to determine the position of the bait and receptor binding sites and a site-specific gold label will determine the position of the thiols released from the thiol ester sites in the intermediate and activated structures. A better understanding of the manner in which proteinases are bound to alpha2M should result from determining the orientation of the active site of papain labelled with a gold cluster in the complex. The subunit organization of the two dimers that comprise alpha2M will be assessed by determining the shape of monomeric rat alpha1I3 which has extensive sequence identity with rat alpha2M.

α-巨球蛋白（α2M），一种通用蛋白酶抑制剂无处不在的 在脊椎动物的血浆中，被认为是一般的 蛋白酶清道夫，因此保护血液和组织蛋白免受 降解。 作为蛋白酶抑制剂，它参与了调节 纤维蛋白溶解，凝结和补体中蛋白酶活性 激活。 它与细胞因子和生长因子相互作用，可能 在血栓降解中具有生理重要性。 更好 了解α2M的结构功能关系 由其各种复合物的结构的确定产生 通过染色和冷冻电子显微镜以及图像处理。 这 在硫化遗址的重要性 将α2M转化为蛋白水解（激活）形式将是 通过确定本网站的结构来评估 通过位置定向诱变消除。 纤溶酶（MR = 80,000）形成A 具有α2M的二元复合物，而较小的蛋白酶，例如α- 胰凝乳蛋白酶（MR = 25,000）形成三元络合物。 确定 二进制复合物的3-D结构及其与我们的3-D的比较 三元α2M-chymotrypsin络合物的结构应揭示 凝血酶在结构中的位置并有助于评估重要性 纤毛蛋白较大尺寸的结合方式 alpha2m。 凝血酶（MR = 33,500）也形成了二元复合物，可能 因为它与α2M的反应比那些的反应要慢得多 形成三元复合物的蛋白酶。 经过基因工程的 α2M诱饵区域更紧密地匹配的特异性 将准备凝血酶，以确定是否增加 反应的速率常数导致三元形成 复杂的。 工程α2M及其复合物的3-D结构 与凝血酶一起将补充这些研究。 进一步理解 激活涉及的复杂变换 alpha2m将由（1）的3_D结构的确定产生 中间形式和（2）n-和c末端晶圆厂的处置 与天然，中间和活化的结构结合。 免疫电子显微镜也将用于确定位置 诱饵和受体结合位点以及特定于位点的金标签 将确定从硫醇酯释放的硫醇的位置 中间和激活结构中的位置。 更好 了解蛋白酶与α2M结合的方式 应由于确定活性位点的方向而产生 木瓜木瓜在综合体中标有金簇。 亚基 组成α2M的两个二聚体的组织将由 确定具有广泛的单体大鼠α1I3的形状 与大鼠α2M的序列同一性。