ELECTRON MICROSCOPY OF HUMAN ALPHA-2 MACROGLOBULIN

人 ALPHA-2 巨球蛋白的电子显微镜

• 批准号: 2220733
• 负责人: JAMES K STOOPS
• 金额: $ 13.48万
• 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/2220733
• 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（α 2 M），一种普遍存在的蛋白酶抑制剂 在脊椎动物的血浆中，被认为是一种普遍的 蛋白酶清除剂，从而保护血液和组织蛋白， 降解 作为一种蛋白酶抑制剂， 纤维蛋白溶解、凝血和补体中蛋白酶活性的变化 activation. 它与细胞因子和生长因子相互作用， 在血栓的降解中具有生理重要性。 更好的 了解alpha 2 M的结构-功能关系将有助于 结果从确定其各种配合物的结构 通过染色和冷冻电子显微镜和图像处理。 的 硫酯位点的重要性，该位点在 将α 2 M转化为蛋白水解（活化）形式， 通过确定该网站的结构进行评估， 通过定点诱变消除。 纤溶酶（Mr= 80，000）形成 与α 2 M的二元复合物，而较小的蛋白酶如α- 糜蛋白酶（Mr= 25，000）形成三元复合物。 测定 二元复合体的三维结构及其与我们的三维结构的比较 三元α 2 M-胰凝乳蛋白酶复合物的结构应该揭示 凝血酶在结构中的位置，并帮助评估其意义 大尺寸纤溶酶的结合方式 α 2M。 凝血酶（Mr= 33，500）也可能形成二元复合物， 因为它与alpha 2 M的反应比那些 形成三元复合物的蛋白酶。 基因工程 α 2 M，其中诱饵区域更接近地匹配α 2 M的特异性， 凝血酶将被制备，以确定是否增加 反应的速率常数导致形成三元 复杂. 工程改造的alpha 2 M及其复合物的三维结构 将补充这些研究。 进一步理解 参与激活的复杂转化 α 2 M将由（1）α 2 M的3_D结构的确定产生。 中间形式和（2）N-和C-末端Fab的处置 与天然结构、中间结构和活化结构结合。 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位点特异性金标记 将决定从硫羟酸酯释放的硫醇的位置 中间结构和活化结构中的位点。 更好的 了解蛋白酶与α 2 M结合的方式 应该是由确定活性位点的方向而产生的。 木瓜蛋白酶在复合物中标记有金簇。 子单元 组成α 2 M的两个二聚体的结构将通过以下方法进行评估： 确定了单体大鼠α 1 I3的形状， 与大鼠α 2 M的序列同一性。