DEOXYCYTIDINE KINASE: REGULATION & MOLECULAR BIOLOGY

脱氧胞苷激酶：调节

• 批准号: 3191614
• 负责人: DAVID H IVES
• 金额: $ 13.92万
• 依托单位: OHIO STATE UNIVERSITY
• 依托单位国家: 美国
• 财政年份: 1989
• 资助国家: 美国
• 起止时间: 1989-05-01 至 1993-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/3191614
• 关键词: antibody; chemical binding; complementary DNA; enzyme inhibitors; enzyme mechanism; enzyme structure; enzyme substrate; high performance liquid chromatography; isozymes; molecular cloning; molecular genetics; natural gene amplification; nucleic acid probes; nucleoside triphosphate; protein purification; protein sequence

The development of new classes of multisubstrate affinity chromatography media directed toward deoxycytidine kinase--and related purine-specific activities--now makes it possible to resolve and isolate these enzymes in a pure state. These activities are essential for activating important chemotherapeutic nucleosides. However, it has not been clear which protein, of several isoenzymes reported, is responsible for phosphorylating each drug, nor is it known whether such isoenzymes are distinct gene-products or merely proteolytic artifacts. The ability to obtain pure proteins provides the opportunity to obtain peptide maps and partial amino acid sequences to each, and raise polyclonal antibodies. Preliminary structural comparisons will be made, and immunological cross-reactivity determined, to assess the relatedness of putative isoenzymes. Each enzyme isolated will be characterized in detail as to its substrate specificities, and its physical and kinetic properties. The mechanism of deoxynucleoside triphosphate end-product inhibition of these enzymes has never been defined, but new evidence from this laboratory suggests that these nucleotides are acting as multisubstrate inhibitors. This model will be tested kinetically now, and reactive analogs will be employed later to map the deoxynucleotide triphosphate binding sites. The final objective during this period is to clone the DNAs complementary to the kinase mRNAs (cDNAs) and to determine their sequences; these will be related to amino acid sequence data and will permit more complete structural comparison of isoenzymes. Cloning of cDNAs will be accomplished with probes generated by the Polymerase Chain Reaction, using as primers oligonucleotides reflecting amino acid sequences. Availability of cDNAs will open the way to future isolation of genomic sequences, study of the regulation of gene expression and to structure-function studies based on site-directed mutagenesis.

新型多底物亲和层析的研究进展 针对脱氧胞苷激酶和相关的嘌呤专一性的介质 活性--现在可以分解和分离这些酶 处于一种纯净的状态。这些活动对于激活 重要的化疗核苷。然而，目前还不清楚 在已报道的几种同工酶中，哪种蛋白质负责 使每种药物磷酸化，也不知道这种同工酶是否 不同的基因产物或仅仅是蛋白质分解产物。有能力 获得纯净的蛋白质提供了获得肽图和 对每个氨基酸序列进行部分编码，并产生多克隆抗体。 将进行初步的结构比较，并进行免疫学 确定交叉反应，以评估推定的 同工酶。 每一种分离的酶都将对其底物进行详细的表征 特性，以及其物理和动力学特性。这一机制 脱氧核苷三磷酸对这些酶的最终产物的抑制作用 从未被定义过，但来自该实验室的新证据表明 这些核苷酸起着多底物抑制剂的作用。这 现在将对模型进行运动学测试，并将进行反应性模拟 随后用于绘制脱氧核苷酸三磷酸结合位点图。 在此期间的最终目标是克隆DNA 与激酶mRNAs(CDNAs)互补并确定其 序列；这些序列将与氨基酸序列数据相关，并将 允许对同工酶进行更完整的结构比较。克隆 CDNA将通过聚合酶链产生的探针来完成 反应，以反映氨基酸的寡核苷酸为引子 序列。CDNA的可用性将为未来的隔离开辟道路 基因组序列，研究基因表达的调节和 基于定点突变的结构功能研究。