INVESTIGATIONS OF MAMMALIAN AMINOPROPYLTRANSFERASES

研究的总体目标是：(A)提供详细的 了解对合成负责的反应， 哺乳动物细胞中多胺的相互转化和去除；(B) 确定多胺的功能；和(C)利用这一信息 设计和测试具有以下特性的多胺类抑制剂和拮抗剂 有用的治疗属性。 这些研究将集中在三种酶上：亚精胺合成酶和 精胺合成酶，分别是氨丙基转移酶 负责亚精胺和精胺的合成，以及 亚精胺/精胺-N1-乙酰转移酶(SAT)是一种高度敏感的 可诱导的速度控制酶在降解和排泄中的作用 这些多胺。建议的实验是：调查 SAT诱导机制及对SAT调控作用的评价 多胺的含量和外流；研究多胺外流的机制 以及腐胺在保护细胞免受感染方面的可能作用 低张休克；研究精胺的功能；并比较 亚精胺合成酶的结构、特异性和表达 精胺合成酶。 在这些研究中使用的具体工具，这些工具已经 在这个项目的工作中开发的，包括所有抗体 三种酶，精胺合成酶和SAT的cDNA克隆，特异性 氨丙基转移酶的抑制剂，多胺类似物 不同程度的能力来替代天然多胺和 诱导SAT，以及分析多胺和 相关核苷如脱羧基S-腺苷甲硫氨酸和 5‘-甲硫腺苷。SAT的抑制剂，可用于 对其功能的研究正在开发中。它的目的是 获得精胺合成酶的cdna，这些酶的基因组克隆 并在大肠杆菌中表达哺乳动物酶以产生 足够进行研究的蛋白质。它还计划使用 带有可诱导启动子的适宜表达SAT的质粒载体 转基因哺乳动物细胞。 已知多胺对哺乳动物细胞的生长和 多胺拮抗剂已被证明具有潜在的 抗肿瘤药物，但这些作用被严格的 代偿对细胞内多胺水平的调节 它们的合成、降解、吸收和吸收速度的调节 排泄物。这些研究将确定化合物和方案，以 最大限度地提高抗肿瘤策略的有效性 细胞型多胺含量。

The overall objectives of the research are: (a) to provide a detailed understanding of the reactions responsible for the synthesis, interconversion and removal of polyamines in mammalian cells; (b) to determine the functions of polyamines; and (c) to use this information to design and test inhibitors and antagonists of polyamines which have useful therapeutic properties. The studies will be focussed on three enzymes; spermidine synthase and spermine synthase, which are aminopropyltransferases respectively responsible for the synthesis of spermidine and spermine, and spermidine/spermine- N1-acetyltransferase (SAT) which is a highly inducible, rate controlling enzyme in the degradation and excretion of these polyamines. The experiments proposed are: to investigate the mechanism of SAT induction and evaluate the role of SAT in regulating polyamine content and efflux; to study the mechanism of polyamine efflux and the possible role of putrescine in protecting cells against hypotonic shock; to investigate the function of spermine; and to compare the structure, specificity and expression of spermidine synthase and spermine synthase. Specific tools to be used in these studies, which have already been developed during the work on this project, include antibodies to all three enzymes, cDNA clones for spermidine synthase and SAT, specific inhibitors for the aminopropyltransferases, polyamine analogs which have varying degrees of ability to substitute for the natural polyamines and to induce SAT, and methods for the analysis of the polyamines and related nucleosides such as decarboxylated S-adenosylmethionine and 5'-methylthioadenosine. Inhibitors of SAT that can be used to investigate its function are under development. It is intended to obtain a cDNA for spermine synthase, genomic clones for these enzymes and to express the mammalian enzymes in E. coli in order to generate sufficient protein for the studies. It is also planned to use appropriate plasmid vectors with inducible promoters to express SAT in transfected mammalian cells. Polyamines are known to be essential for mammalian cell growth and polyamine antagonists have been demonstrated to have potential as antineoplastic agents but these effects are counteracted by the strict regulation of cellular polyamine levels brought about by compensatory adjustments of their rate of synthesis, degradation, uptake and excretion. These studies will identify compounds and protocols to maximize the effectiveness of antitumor strategies which perturb cellular polyamine content.