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• 批准号: 6120981
• 负责人: JOHN LUTE MARKLEY
• 金额: $ 22.64万
• 依托单位: UNIVERSITY OF WISCONSIN-MADISON
• 依托单位国家: 美国
• 财政年份: 1999
• 资助国家: 美国
• 起止时间: 1999-03-01 至 2000-02-29
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6120981
• 关键词: biological products; biomaterials; biomedical resource; enzymes; nuclear magnetic resonance spectroscopy; proteins

Glycolipids and glycoproteins are involved in a variety of molecular interactions which are crucial to eukaryotic cellular function. The enzymatic mechinery required for the initial assembly of the majority of cell surface carbohydrates is located in the membrane of the endoplasmic reticulum (ER). This glycan-assembly apparatus consists of a wide spectrum of proteins (mainly glycosyltransferases (GTs)) involved in the construction of N-glycans and glycosylphosphatidylinositol (GPI) anchors. These ER proteins are typically membrane-bound, highly labile and, unlike their counterparts in the Golgi, not generally amenable to conventional protein purification; with very few exceptions only partial purifications have been achieved. Photoaffinity labeling provides an alternative to conventional chromatographic methods in which enzymatic activity must be retained. In photoaffinity labeling, a radiolabeled substrate analog which contains a functional group that can be photoactivated to a highly reactive species upon irradiation with ultraviolet light and thus form covalent bonds with nearby molecules (i.e., proteins) is used. Therefore, one needs enzymatic activity only once to form the non-covalently bound analog-enzyme complex, and following UV-irradiation, the radiolabel is covalently incorporated into the protein. Purification of the protein using conventional chromatographic techniques can then be perfoirmed using the photo-incorporated radiolabel as a marker for the protein. We have developed synthesis schemes for two classes of photoaffinity probes. The structures of the probes are based on structural motifs present in glycosyltransferase substrates which are essential for enzymatic recognition of substrate. In the case of nucleotide-sugar utilizing GTs, the nucleotide itself acts as a competitive inhibitor to enzymatic acitivity, whereas the sugar or sugar-phosphates do not. This demonstrates specific recognition of the nucleotide by the enzyme. Therefore, we have initially based the structure of our probes on uridine-nucleotides. The two classes of probes are 1) 5-azido-UTP and 5-azido-UMP and 2) P3-(4-azidoanilido)-uridine 5?-triphosphate. The synthesis scheme for the first class of probes is an alternative but improved method to existing procedures for synthesis of this class. The advantage of this procedure is ease of separation of reaction products (using chromatographic methods, silica gel and anion exchange) due to the hydrophobicity of the benzoyl-blocking group of the starting material (2?, 3?-dibenzoyluridine). The synthesis of P3-(4-azidoanilido)-uridine 5?-triphosphate is based on analogy to an existing procedure for the guanosine derivative. The uridine derivative synthesis has not been reported previously in the literature. Our motive for utilizing the NMRFAM is to provide routine 1D spectra (1H, 13C, 15N and 31P) for structural characterization of synthetic intermediates and products of the photoaffinity probes described above. The samples would be submitted for service spectroscopy on the DMX 400 MHz spectrometer.

糖脂和糖蛋白参与多种 分子间的相互作用对真核细胞 功能 初始组装所需的酶促机制 大多数细胞表面碳水化合物位于 内质网（ER）膜。 这种聚糖组装 一种由多种蛋白质组成的装置（主要是 糖基转移酶（GT））参与N-聚糖的构建 和糖基磷脂酰肌醇（GPI）锚。 这些ER蛋白是 典型的膜结合，高度不稳定， 在高尔基体中，一般不适合常规蛋白质 纯化;除了极少数例外，只有部分纯化 办妥了一批多年想 光亲和标记提供了一种替代方法， 常规的色谱方法，其中酶活性必须 保留。 在光亲和标记中，放射性标记的底物 类似物，其含有可被光活化以 在紫外光照射下的高反应性物质， 从而与附近的分子形成共价键（即，蛋白质）是 采用 因此，人们只需要一次酶活性就可以形成 非共价结合的类似物-酶复合物，以及 在UV-照射下，放射性标记共价结合到 蛋白 使用常规方法纯化蛋白质 色谱技术可以使用 光掺入放射性标记作为蛋白质的标记。 我们有 开发了两类光亲和探针的合成方案。 探针的结构基于存在于DNA中的结构基序。 糖基转移酶底物是酶促反应所必需的， 识别底物。 在利用核苷酸-糖的情况下， GT，核苷酸本身作为竞争性抑制剂， 酶活性，而糖或糖磷酸盐则没有。 这证明了核苷酸的特异性识别， 酵素 因此，我们已经初步建立了我们的结构， 尿苷核苷酸探针。 这两类探测器是1） 5-叠氮基-UTP和5-叠氮基-UMP和2）P3-（4-叠氮基苯胺基）-尿苷 五个？三磷酸盐 第一类探针的合成方案 是现有程序的替代但改进的方法， 综合这一课。 该程序的优点是易于 反应产物的分离（使用色谱法，二氧化硅 凝胶和阴离子交换），这是由于 起始材料的苯甲酰基封端基团（2 R， 三个？二苯甲酰尿苷）。 P3-（4-叠氮苯胺基）-尿苷的合成 五个？三磷酸盐是基于对现有程序的类比， 鸟苷衍生物 尿苷衍生物的合成还没有完成。 以前在文献中报道过。 我们利用 NMRFAM将提供常规1D光谱（1H、13 C、15 N和31 P）， 合成中间体和产品的结构表征 上述光亲和探针。 样本将是 在DMX 400 MHz光谱仪上提交服务光谱。