POLYAMINE BIOSYNTHESIS AND FUNCTION

多胺、腐胺、亚精胺和精胺是所有活细胞中的主要多碱化合物。这些胺已被证明对许多与生长和分化有关的系统都很重要。我们一直感兴趣的是这些多胺是如何合成的，它们的生物合成和降解是如何调节的，它们的生理功能，它们在体内的作用，以及各种生物合成酶的结构特征。为此，我们在大肠杆菌和酿酒酵母的每个生物合成步骤中都构建了零突变体，并准备了生物合成酶的超表达系统。我们目前的研究旨在扩大我们对这些胺的生物化学、调控和遗传学以及酿酒酵母生物合成酶的研究。在这一年中，我们继续研究鸟氨酸脱羧酶的调节，它是多胺形成的关键酶，对多胺的生理功能研究特别感兴趣。在培养基中加入亚精胺后，鸟氨酸脱羧酶水平显著降低，这既是由于抑制了新的合成，也是由于现有酶的降解。我们发现，与以前的文献报道相反，这种降解的一个重要部分涉及蛋白水解酶活性的诱导。如果蛋白质合成受到抑制，这种诱导就不会发生。利用在大肠杆菌中的高效表达系统，我们建立了大量纯化的酵母鸟氨酸脱羧酶、S腺苷甲硫氨酸脱羧酶、精胺合成酶和精胺合成酶的分离方法，用于结晶学和其他结构研究。S-腺苷甲硫氨酸脱羧酶的结构特别令人感兴趣，因为它是以酶原的形式形成的，翻译后从丙酮酰基团裂解而来，丙酮酰基是酶活性所必需的。我们还克隆了(用聚合酶链式反应技术)大肠杆菌的谷胱甘肽亚精胺合成酶基因，作为研究该化合物在大肠杆菌中功能的初始阶段。-腐胺、亚精胺、精胺、酵母、多胺。鸟氨酸脱羧酶、S腺苷蛋氨酸脱羧酶

The polyamines, putrescine, spermidine, and spermine, are major polybasic compounds in all living cells. These amines have been shown to be important for many systems related to growth and differentiation. We have been interested in how these polyamines are synthesized, how their biosynthesis and degradation are regulated, their physiologic functions, how they act in vivo, and in characterizing the structure of the various biosynthetic enzymes. For this purpose we have constructed null mutants in each of the biosynthetic steps in both Escherichia coli and in Saccharomyces cerevisiae, and have prepared overexpression systems for the biosynthetic enzymes. Our present studies are directed at extending our studies on the biochemistry, regulation and genetics of these amines and of the biosynthetic enzymes in S. cerevisiae. During the current year we have continued our studies on the regulation of the enzyme ornithine decarboxylase which is a key enzyme in the formation of the polyamines, and is of special interest in studies on the physiological function of the polyamines. The ornithine decarboxylase levels are markedly lowered after the addition of spermidine to the medium, both by repression of new synthesis and by degradation of the existing enzyme. We have found that, contrary to previous reports in the literature, an important part of this degradation involves the induction of a proteolytic activity. This induction does not occur if protein synthesis is inhibited. Using overexpression systems in E. coli, we have developed methods for the isolation of large quantities of purified yeast ornithine decarboxylase, S-adenosylmethionine decarboxylase, spermidine synthase and spermine synthase for crystallographic and other structural studies. The structure of S-adenosylmethionine decarboxylase is of particular interest since it is formed as a proenzyme and is post- translationally cleaved to from a pyruvoyl group that is essential for enzymatic activity. We have also cloned (with PCR technology) the E. coli gene for glutathionylspermidine synthase as the initial phase of a study of the function of this compound in E. coli. - putrescine, spermidine, spermine, yeast, polyamines. ornithine decarboxylase, S- adenosylmethionine decarboxylase