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(Alpha2M)，一种普遍存在的蛋白酶抑制剂 在脊椎动物的血浆中，被认为是一种 蛋白酶清除剂，从而保护血液和组织蛋白不受 退化。作为一种蛋白水解酶抑制物，它参与调节 纤溶、凝血和补体中的蛋白酶活性 激活。它与细胞因子和生长因子相互作用，它可能 在血栓的降解过程中具有生理重要性。更好的 理解Alpha2M Will的结构与功能关系 其各种络合物的结构测定的结果 通过染色和低温电子显微镜，以及图像处理。这个 在反应过程中被切割的硫代酯位置的重要性 将Alpha2M转变为蛋白水解型(激活的)形式 通过确定该站点所处的结构进行评估 通过定点突变消除。纤溶酶(Mr=80,000)形成 与Alpha2M的二元复合体，而较小的蛋白酶，如Alpha2M- 凝乳酶(Mr=25,000)形成三元络合物。对…的测定 二元络合物的三维结构及其与我们三维结构的比较 三元α2M-糜蛋白酶复合体的结构应揭示 凝血酶在结构中的定位及有助于评估其意义 更大尺寸的纤溶酶与其结合的方式 字母2M。凝血酶(MR=33,500)也可能形成二元络合物 因为它与Alpha2M的反应比那些 形成三元复合体的蛋白酶。一种基因工程 在Alpha2M中，诱饵区域与 将制备凝血酶以确定是否增加 该反应的速率常数导致形成三元体系 很复杂。工程化α2M及其复合体的三维结构 凝血酶将补充这些研究。进一步认识 在激活过程中涉及的复杂转换 α2M将由(1)的三维结构的确定得到 中间形式和(2)N-端和C-端光纤的配置 绑定到自然结构、中间结构和激活结构。 免疫电子显微镜也将被用来确定位置 诱饵和受体结合部位和特定部位的金标记物 将确定从硫醇酯中释放的硫醇的位置 中间结构和活化结构中的位置。更好的 对蛋白酶与α2M结合方式的理解 应由确定的活动位置的方向产生 木瓜酶在建筑群中贴上了金色的标签。亚单位 组成α2M的两个二聚体的组织将通过 具有广泛性的大鼠单体α1I3的形状测定 与大鼠α2M的序列同源性。