APPLICATION OF PHOTOAFFINITY NUCLEOTIDE ANALOGS

光亲和核苷酸类似物的应用

• 批准号: 3288954
• 负责人: BOYD E HALEY
• 金额: $ 22.02万
• 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 1991-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3288954
• 关键词: G protein; RNA directed DNA polymerase; adenine nucleotides; adenosine triphosphate; adenylate cyclase; affinity chromatography; affinity labeling; autoradiography; binding proteins; cell membrane; chemical synthesis; cholera toxin; cyclic nucleoside monophosphate; electrofocusing; endonuclease; enzyme inhibitors; enzyme mechanism; erythrocyte membrane; gel electrophoresis; guanosine diphosphate; guanosine triphosphate; human tissue; molecular site; nucleotide analog; oncogenes; phosphorylation; photochemistry; protein kinase A; radiotracer; ultraviolet spectrometry

The major, long term objective of this laboratory is to develop active site directed photoaffinity probes for important nucleotides and to expand this to include polynucleotides of known sequence which contain photoactive bases at specific locations. Photoaffinity probes are useful when they interact with enzymes either behaving as biological mimics or inhibitors of the effect produced by the natural compound. In this proposal, the following enzyme systems will be studied using previously synthesized photoprobes as well as new photoprobes not previously reported. The energetics of activation of Adenylate cyclase (AC) will be studied using Alpha32P and Gamma32P labeled 8N-3GTP (mimic of GTP) and various probes of TNP-GTP (a potent fluorescent inhibitor of AC) modified to contain a photoactive group and non-hydrolyzable phosphates. Also, [32P]8N3NAD will be used with cholera toxin and islet activating protein to attempt to 8N-3ADP-ribosylate the stimulatory and inhibitory GTP regulatory proteins of AC. Similar studies will be done on the ras oncogene product, p21. The catalytic subunit of type II cAMP dependent protein kinase (C-II) will be studied with regards to its autophosphorylation and photolabeling by [Gamma32P]8N-3dATP which appears to be more effective at autophosphorylation than [Gamma32P]ATP. Similar studies will be done on the cGMP dependent kinase, 3-PGA, pyruvate and creatine kinases. Newly synthesized 5-N3-dUTP is enzymatically incorporated by PolI into DNA in place of dTTP resulting in photoactive DNA. Similar 5-N-3U probes will be tested as substrates and active site probes of AMV reverse transcriptase and other DNA and RNA polymerases. 5N3-UDPG will be used to study glycogen synthetase. Both chemical and enzymatic synthesis of photoactive polynucleotides sequences will be done. The most obvious health related application of this research is the detection of viral induced proteins, some of which are oncogene protein products. Other uses are the detection of enzymes that are potential markers of neoplasia such as terminal deoxynucleotidyl transferase, adenosine deaminase, thymidlate kinase, etc., using Mug quantities of tissue or cells.

该实验室的主要长期目标是开发主动地点 重要核苷酸的定向光性探针，并扩展 包括已知序列的多核苷酸，其中包含光活性 基地在特定位置。 光性探针在它们时很有用 与表现为生物模仿的酶相互作用或 天然化合物产生的效果。 在此提案中， 将使用先前合成的酶系统研究酶系统 光探测器以及先前未报告的新光探针。 这 将使用腺苷酸环化酶（AC）激活的能量学研究 alpha32p和Gamma32p标记为8N-3GTP（GTP的模仿）和各种探针 TNP-GTP（AC的有效荧光抑制剂）被修改为包含A 光活性基和非氢化磷酸盐。 另外，[32p] 8n3nad Will 与霍乱毒素和胰岛一起激活蛋白质以尝试 8N-3ADP-核酰基刺激和抑制性GTP调节蛋白 AC。 将对RAS癌基产品P21进行类似的研究。 这 II型cAPP依赖性蛋白激酶（C-II）的催化亚基将是 研究了其自动磷酸化和光标记 [gamma32p] 8n-3DATP似乎更有效 自磷酸化比[gamma32p] ATP。 类似的研究将进行 CGMP依赖性激酶，3-PGA，丙酮酸和肌酸激酶。 新 合成的5-N3-DUTP是由Poli酶纳入DNA中的 DTTP的位置导致光活动DNA。 类似的5-N-3U探针将是 作为底物和AMV逆转录酶的活性位点探针测试 以及其他DNA和RNA聚合酶。 5N3-UDPG将用于研究糖原 合成酶。 光活性的化学和酶促合成 将完成多核苷酸序列。 与健康有关的最明显的 这项研究的应用是检测病毒诱导的蛋白质， 其中一些是癌基蛋白产品。 其他用途是检测 是肿瘤的潜在标记的酶，例如末端 脱氧核苷酸转移酶，腺苷脱氨酶，胸酸激酶等，等等 使用杯子量的组织或细胞。