AMINOPROPYLTRANSFERASES

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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.