INVESTIGATIONS OF MAMMALIAN AMINOPROPYLTRANSFERASES

研究的总体目标是：（a）提供一个详细的 了解合成反应的原理， 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）使用这些信息 设计和测试多胺的抑制剂和拮抗剂， 有用的治疗性质。 这些研究将集中在三种酶上：亚精胺合酶和 精胺合酶，它们分别是氨丙基转移酶 负责亚精胺和精胺的合成， 亚精胺/精胺- N1-乙酰转移酶（SAT）是一种高度 诱导，速率控制酶的降解和排泄 这些多胺。 提出的实验是：调查 SAT的诱导机制，并评估SAT在调节 多胺含量与外排的关系，探讨多胺外排的机制 以及腐胺在保护细胞免受 目的：探讨精胺在低渗休克中的作用， 亚精胺合酶的结构、特异性和表达， 精胺合酶 在这些研究中使用的具体工具， 在这个项目的工作期间开发的，包括抗体， 三种酶，精脒合酶和SAT的cDNA克隆，特异性 氨丙基转移酶的抑制剂，具有 不同程度的取代天然多胺的能力， 诱导SAT的方法，以及分析多胺和 相关核苷如脱羧S-腺苷甲硫氨酸和 5 '-甲硫腺苷。 SAT抑制剂，可用于 正在研究其功能。 其旨在 获得精胺合酶的cDNA，这些酶的基因组克隆 并在E.大肠杆菌， 为研究提供充足的蛋白质。 还计划利用 具有诱导型启动子的适当质粒载体，以在大肠杆菌中表达SAT。 转染的哺乳动物细胞。 已知多胺对于哺乳动物细胞生长是必需的， 多胺拮抗剂已被证明具有作为 但这些影响被严格的 调节细胞多胺水平所带来的补偿 调整其合成、降解、吸收和 排泄 这些研究将确定化合物和方案， 最大限度地提高抗肿瘤策略的有效性， 细胞多胺含量

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.