NEW PROBES AND REAGENTS FOR BIOLOGICAL STUDIES

用于生物学研究的新探针和试剂

• 批准号: 6018512
• 负责人: JOHN F KEANA
• 金额: $ 18.63万
• 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 2001-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6018512
• 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：主要研究者的中心主题 NIH支持的研究是设计，化学合成，表征， 和新的探针和试剂的合作应用， 生物系统。 靶分子被设计为响应于 研究人员和从业人员目前的需求和局限性 生物化学、分子和细胞生物学以及医学。 项目 涉及与两个杰出的研究小组在澳大的合作： Drs. C.布斯塔曼特和O.H.格里菲斯，化学系和研究所 分子生物学 两个分领域构成了拟议的五年 项目 a）用于生物化学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）相关研究。 PI-PLC酶是一个受体控制的家族， 在细胞信号传导过程的放大中很重要。 此外，细菌PI-PLC催化细胞表面的裂解 通过糖基磷脂酰肌醇附着在膜上的蛋白质 (GPI)锚。 GPI锚切割活性对医学具有重要意义 作为分析蛋白质的诊断工具， 细胞膜表面。 锚定蛋白包括以下的活化抗原： 免疫系统，粘附分子，羊瘙痒症朊病毒蛋白和 癌胚抗原，一种人类肿瘤标志物。 我们的重要性 PI-PLC的工作在于为研究结构提供了强有力的新工具 （抑制剂结合在晶体酶的活性位点）和 作用机制（即，新的显色、荧光或 PI-PLC的发光底物）在其在 细胞信号转导 与癌症和老年痴呆症的联系 也报告了PI-PLC。 B）用于原子力的新型试剂 显微镜（AFM）。 直接观察单个生物分子， 它们在缓冲器中的原生状态是使用相对较新的 强大的AM技术。 生物样本的分辨率极限为 通常为50-100 A，并受到尖端锋利度的限制。 最锋利的 可用的尖端是曲率半径约为100的碳尖端。 一个目标是设计和合成化学定义良好的尖端， 到一个原子，从而接近最终的决议时间。 的 研究人员还提出了AFM第二种应用的局限性， 即官能团成像（FGI）。 FGI提供直接信息 关于表面不同的化学基团， 在配体-受体相互作用等不同领域的分支， 粘附，润滑和分子加工，后者与酶 固定在尖端上。