NEW PROBES AND REAGENTS FOR BIOLOGICAL STUDIES

用于生物学研究的新探针和试剂

• 批准号: 2397610
• 负责人: JOHN F KEANA
• 金额: $ 17.56万
• 依托单位: UNIVERSITY OF OREGON
• 依托单位国家: 美国
• 财政年份: 1997
• 资助国家: 美国
• 起止时间: 1997-09-01 至 2000-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2397610
• 关键词: atomic force microscopy; biological signal transduction; chemical synthesis; contrast media; crosslink; image enhancement; immobilized enzymes; membrane proteins; molecular site; phosphatidylinositols; phospholipase C; phospholipase inhibitor; reagent /indicator; receptor binding

DESCRIPTION: The central theme of the principal investigator's NIH-supported research is the design, chemical synthesis, characterization, and collaborative application of new probes and reagents for studying biological systems. The target molecules are designed in response to the current needs and limitations of researchers and practitioners in the fields of biochemistry, molecular and cell biology, and medicine. The project involves a collaboration with two outstanding research groups at the U of O: Drs. C. Bustamante and O.H. Griffith, Chemistry department and Institute of Molecular Biology. Two sub-areas form the focus of this proposed five-year project. a) Novel substrates and inhibitors for biochemical and biophysical studies related to phosphatidylinositol-specific phospholipase C (PI-PLC). The PI-PLC enzymes are a receptor-controlled family that are centrally important in the amplification of cellular signaling processes. Additionally, bacterial PI-PLC catalyzes the cleavages of cell surface proteins attached to the membrane by way of a glycosylphosphatidylinsoitol (GPI) anchor. The GPI anchor cleaving activity is of importance to medicine as diagnostic tools for the analysis of proteins presented on the outer surface of cell membranes. Anchored proteins include activation antigens of the immune system, adhesion molecules, scrapie prion proteins and the carcinoembryonic antigen, a human tumor marker. The significance of our PI-PLC work lies in providing powerful new tools for studying the structure (inhibitors bound at the active site of the crystalline enzymes) and mechanism of action (i.e., novel chromogenic, fluorogenic or chemiluminescent substrates) of the PI-PLCs in their central role in cellular signal transduction. Links to cancer and Alzheimer's disease have also been reported for the PI-PLCs. b) Novel reagents for atomic force microscopy (AFM). The direct visualization of individual biomolecules in their native state in buffer is being achieved using the relatively new and powerful technique of AM. The resolution limit with biological specimens is typically 50-100 A and is limited by the sharpness of the tip. The sharpest tips available are carbon tips with a radius of curvature of about 100 A. One aim is to design and synthesize chemically well-defined tips that taper to a single atom, thus approaching the ultimate time of resolution. The investigators also address limitations in a second application of AFM, namely functional group imaging (FGI). FGI provides direct information about the different chemical groups at the surface and has important ramifications in fields as diverse as ligand-receptor interactions, adhesion, lubrication, and molecular machining, the latter with enzymes immobilized on the tip.

描述：首席调查员的中心主题是 NIH支持的研究包括设计、化学合成、表征、 和合作应用新的探针和试剂进行研究 生物系统。靶分子的设计是为了响应 研究人员和从业者在该领域的当前需求和限制 生物化学，分子和细胞生物学，以及医学。该项目 涉及与密歇根大学两个杰出的研究小组的合作： 首页--期刊主要分类--期刊细介绍--期刊题录与文摘--期刊详细文摘内容 分子生物学。两个分领域构成了这一拟议五年计划的重点 项目。A)用于生化和生物物理的新型底物和抑制剂 磷脂酰肌醇特异性磷脂酶C(PI-PLC)的相关研究。 PI-PLC酶是一个由受体控制的家族，它们集中在 在放大细胞信号过程中起着重要的作用。 此外，细菌PI-PLC还催化细胞表面的裂解 以糖基磷脂酰肌醇的形式附着在膜上的蛋白质 (GPI)锚。GPI锚点切割活动在医学上具有重要意义 作为诊断工具，用于分析外部呈现的蛋白质 细胞膜的表面。锚定蛋白包括活化抗原 免疫系统、黏附分子、瘙痒病毒蛋白和 癌胚抗原，人类肿瘤标记物。我们的重要意义在于 PI-PLC的工作在于为研究结构提供了强有力的新工具 (结合在结晶酶活性部位的抑制剂)和 作用机理(即新的显色、荧光或 化学发光底物)的PI-PLC在其核心作用 细胞信号转导。与癌症和阿尔茨海默氏症的联系 也有关于PI-PLC的报告。B)原子力的新试剂 显微镜(AFM)。单个生物分子的直接可视化 它们在缓冲区中的本机状态是使用相对较新的和 强大的AM技术对生物样品的分辨极限为 通常为50-100安，受限于尖端的锐度。最犀利的 可用的尖端是曲率半径约为100A的碳尖。 其中一个目标是设计和合成化学上定义明确的尖端，使其逐渐变细 到单个原子，从而接近分解的最终时间。这个 研究人员还解决了原子力显微镜第二次应用的局限性， 即功能基团成像(FGI)。FGI提供直接信息 关于表面的不同化学基团，并具有重要的 在各种领域的分支，如配体-受体相互作用， 粘合、润滑和分子加工，后者使用酶 固定在顶端。