APPLICATION OF PHOTOAFFINITY NUCLEOTIDE ANALOGS

光亲和核苷酸类似物的应用

• 批准号: 2900617
• 负责人: BOYD E HALEY
• 金额: $ 15.16万
• 依托单位: UNIVERSITY OF KENTUCKY
• 依托单位国家: 美国
• 财政年份: 1985
• 资助国家: 美国
• 起止时间: 1985-07-01 至 2000-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/2900617
• 关键词: ADP ribosylation; affinity labeling; azides; crosslink; guanine nucleotide binding protein; guanosine triphosphate; high performance liquid chromatography; intermolecular interaction; nicotinamide adenine dinucleotide; nucleotide analog; oncoprotein p21; phenone; western blottings; ADP ribosylation; affinity labeling; azides; crosslink; guanine nucleotide binding protein; guanosine triphosphate; high performance liquid chromatography; intermolecular interaction; nicotinamide adenine dinucleotide; nucleotide analog; oncoprotein p21; phenone; western blottings

ADPribosylation factor (ARF) and ras p21 use 8N3GTP as a mimic of GTP. 8N3GTP photoinserts effectively into each may be further modified on the gamma-phosphate with an extendable photoactive benzophenone (BP) group and still compete at the GTP site. ARF is a GTP binding protein that activates some bacterial toxins which use NAD+ to ADPribosylate other GTP binding proteins. Using [32P]8N3GTP and [32P]2N3NAD+ data has been obtained which shows that ARF alone contains both GTP and NAD+ binding domains. Further, the addition of ARF decreases by 50 percent the [32P]2N3NAD+ photoinsertion into interactive toxin with 50 percent of the labeling now appearing on ARF. This indicates a shared NAD+ site between ARF and toxin in the complex and has both structural and mechanistic implications. Therefore, the first specific aim will be to identify the NAD+ and GTP binding domains of ARF using [32P]8N3GTP and [32P]2N3NAD+ and to correlate photoinsertion with modification of biological activity. The second specific aim will be to synthesize and use NAD+ and GTP bifunctional photoprobes (azide plus benzophenone modified) to develop procedures to ~site specifically~ crosslink ARF with interactive toxin(s) through the GTP or NAD+ binding site(s). The ARF-toxin complex is being used as a model system to develop a general procedure which may be used to identify unknown proteins which interact with other G-regulatory proteins. The third specific aim will be to test the bifunctional GTP probes as biological mimics of GTP using ras p21 to develop procedures for identifying p21 ~nearest neighbor~ proteins. While many G-proteins of have been identified, the ~nearest neighbor~ interactive proteins they bind to are usually not known. The first aspect will be to crosslink p21 with known interactive proteins (GAP, Raf, etc) and this will be followed by studies to detect similar proteins in Xenopus homogenates. The major long term objective of this research is to develop a general procedure for the identification of the interactive proteins which bind different G-regulatory proteins, such as p21 and ARF.

Adpribosylation系数（ARF）和RAS P21使用8N3GTP作为模仿 GTP。 8N3GTP Photoinserts有效地进一步 用可扩展的光活性在γ-磷酸盐上修饰 苯甲酮（BP）组，仍在GTP站点竞争。 arf是 一种激活一些使用的细菌毒素的GTP结合蛋白 NAD+依次依次，其他GTP结合蛋白。 使用 [32p] 8n3gtp和[32p] 2N3NAD+数据已获得 表明单独的ARF同时包含GTP和NAD+结合域。 此外，ARF的添加减少了50％ [32p] 2N3NAD+ Photoinsertion成50％的交互式毒素 现在出现在ARF上的标签。 这表明共享的nad+ 在综合体中ARF和毒素之间的位置，既有结构又具有 机械含义。 因此，第一个具体目标是 使用使用ARF的NAD+和GTP结合域使用 [32p] 8n3gtp和[32p] 2n3nad+，并关联摄影 随着生物活性的改变。 第二个特定目标将 合成并使用NAD+和GTP双功能光预探 （叠氮化物加苯酮修饰）开发到〜站点的程序 具体〜与互动毒素通过GTP进行交联ARF 或NAD+结合位点。 ARF毒素复合物被用作 建立一个通用程序的模型系统，该程序可用于 识别未知蛋白质与其他G调控相互作用 蛋白质。 第三个具体目的是测试双功能GTP 使用RAS P21作为GTP的生物模仿探针来发展 识别p21〜最近的邻居〜蛋白质的程序。 尽管 已经确定了许多G蛋白，〜最近的邻居〜 它们与之结合的互动蛋白通常是不知道的。 第一个 方面将是带有已知互动蛋白的交联P21（GAP， RAF等），随后将进行研究以检测类似蛋白 在爪蟾匀浆中。 主要目标的主要目标 研究是为识别的一般程序制定一般程序 互动蛋白结合不同的G调节蛋白，例如 P21和ARF。