DEOXYCYTIDINE KINASE: REGULATION & MOLECULAR BIOLOGY

新型多底物亲和色谱的研究进展 介质针对脱氧胞苷激酶-和相关的嘌呤特异性 活性-现在可以解析和分离这些酶 在纯净的状态下。 这些活动对于激活 重要的化疗核苷。 然而， 在已报道的几种同工酶中， 磷酸化每种药物，也不知道这种同工酶是否是 不同的基因产物或仅仅是蛋白水解产物。 的能力 获得纯蛋白质提供了获得肽图谱的机会， 部分氨基酸序列，并产生多克隆抗体。 将进行初步的结构比较， 确定交叉反应性，以评估推定的 同工酶 每一种分离的酶都将详细描述其底物 特异性及其物理和动力学特性。 机制 脱氧核苷三磷酸终产物抑制这些酶 从未被定义过，但实验室的新证据表明， 这些核苷酸是多底物抑制剂。 这 现在将对模型进行动力学测试，反应性类似物将 随后用于绘制脱氧核苷酸三磷酸结合位点。 这一阶段的最终目标是克隆DNA 与激酶mRNA（cDNA）互补，并确定它们的 序列;这些将与氨基酸序列数据相关，并将 允许更完整的同工酶结构比较。 的克隆 cDNA将用聚合酶链产生的探针完成 反应，使用反映氨基酸的寡核苷酸作为引物 序列的 cDNA的可用性将为未来的分离开辟道路 基因组序列，研究基因表达的调控， 基于定点突变的结构-功能研究。

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.