APPLICATION OF PHOTOAFFINITY NUCLEOTIDE ANALOGS

光亲和核苷酸类似物的应用

• 批准号: 3288957
• 负责人: BOYD E HALEY
• 金额: $ 15.62万
• 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 1995-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288957
• 关键词: Alzheimer's disease; active sites; adenine nucleotides; adenosine triphosphate; affinity chromatography; affinity labeling; binding proteins; biological response modifiers; cerebrospinal fluid; chemical binding; chemical synthesis; cytokine; fluorescent dye /probe; glutamate dehydrogenase; guanosine diphosphate; guanosine triphosphate; human tissue; hydroxyl group; interferons; ion exchange chromatography; molecular site; nucleic acid probes; nucleotide analog; oncogenes; peptide hormone; phosphoglycerate kinase; photochemistry; protein sequence; radiotracer; second messengers; Alzheimer's disease; active sites; adenine nucleotides; adenosine triphosphate; affinity chromatography; affinity labeling; binding proteins; biological response modifiers; cerebrospinal fluid; chemical binding; chemical synthesis; cytokine; fluorescent dye /probe; glutamate dehydrogenase; guanosine diphosphate; guanosine triphosphate; human tissue; hydroxyl group; interferons; ion exchange chromatography; molecular site; nucleic acid probes; nucleotide analog; oncogenes; peptide hormone; phosphoglycerate kinase; photochemistry; protein sequence; radiotracer; second messengers

The major objective is to synthesize nucleotide photoaffinity and to validate their selective interaction at the active site of enzymes and regulatory proteins. All of the prominent mononucleotides have been modified to make effective photoprobes of cAMP, cGMP, ATP, GTP, UTP, TTP, NAD+, CoA, UDPG, etc. These probes have proven very effective for the identification of specific nucleotide binding proteins, even in crude homogenates. They have been relatively ineffective in detecting active side peptides due to the sometimes low efficiency of insertion and the lability of the photoinserted label to all forms of HPLC. We have resolved this latter problem by taking advantage of the properties of the photoinserted nucleotide to selectively isolate the photolabeled peptide without the use of HPLC except to verify the purity of the final material. Using these procedures we propose to identify the tryptic peptides of various nucleotide binding proteins. Proteins that we will study include the various "biological response modifiers" such as tumor necrosis factor, interferon-alphaA, glucagon, IL-1 alpha & beta, prolactin, certain commercially available key enzymes such as glutamate dehydrogenase, phosphoglycerate & pyruvate kinase & unique proteins found in the cerebral spinal fluid (CSF) if Alzheimer's (AD) & ALS diseased individuals. All of these are known to have specific nucleotide binding sites. We also propose the synthesis of new nucleotide photoprobes designed to: (i) remove inherent weaknesses of previous probes (e.g., [alpha32P]8N3GTPdeltaS to prevent hydrolysis during long preincubations) (ii) make isolation of the photolabeled peptides easier (e.g. probes with a cis hydroxyl group attached through the alpha-phosphate increase the selective retention by boronate columns) and (iii) be new probes that will be selective for uncommon nucleotide sites (e.g. cyclic-ADPribose, cyclic-diGMP, 7-Me- Guanosine) or are small nucleic acid probes for proteins that selectively bind known small sequences (e.g., [32P]8N3ApUp which binds to diphtheria toxin). The most obvious health related application of this research is the determination of the nucleotide binding properties of various polypeptide hormones which may help explain their mechanism of action, the detection of certain oncogene protein products viral induced proteins, CSF proteins unique to AD, ALS, etc. and the study of several enzymes that are important in metabolism and certain enzymes that may be potential indicators of neoplasia.

主要目的是合成核苷酸光附属物和到 在酶的活动部位验证其选择性相互作用，并 调节蛋白。 所有突出的单核苷酸都已 经过修改以制造有效的CAMP，CGMP，ATP，GTP，UTP，TTP， NAD+，COA，UDPG等。这些探针已证明对 鉴定特定核苷酸结合蛋白，即使在粗糙 匀浆。 他们在检测活动方面相对无效 由于插入效率较低而引起的侧肽和 摄影标签的不利于所有形式的HPLC。 我们已经解决了 后一个问题通过利用 光毒的核苷酸可有选择地隔离光标记肽 不使用HPLC，除了验证最终材料的纯度外。 我们建议使用这些过程来识别 各种核苷酸结合蛋白。 我们将研究的蛋白质包括 各种“生物反应修饰符”，例如肿瘤坏死因子， 干扰素-alphaa，胰高血糖素，IL-1α和β，催乳素，某些 市售的关键酶，例如谷氨酸脱氢酶， 在大脑中发现的磷酸甘油酸和丙酮酸激酶和独特的蛋白质 如果阿尔茨海默氏病（AD）和ALS患者患者，脊柱液（CSF）。 所有人 已知这些具有特定的核苷酸结合位点。 我们也建议 新型核苷酸光四剂杆的合成：（i）去除 先前探针的固有弱点（例如[alpha32p] 8n3gtpdeltas to 在长期预孵育期间防止水解）（ii）进行分离 光标记的肽更容易（例如用顺式羟基探针 通过α-磷酸盐附着 硼酸盐列）和（iii）是新探针，将是选择性的 不常见的核苷酸位点（例如环状辅助，环状二孔，7-me- 鸟苷）或小核酸探针的蛋白质探针有选择地 结合已知的小序列（例如[32p] 8n3apup，与白喉结合 毒素）。 这项研究最明显的与健康相关的应用是 确定各种核苷酸结合特性 多肽激素可能有助于解释其作用机理， 检测某些癌基因蛋白产品病毒诱导蛋白CSF AD，ALS等独有的蛋白质以及对几种酶的研究 在新陈代谢和某些可能是潜在的酶中很重要 肿瘤的指标。