The objective is to understand the reactions responsible for the synthesis and interconversion of polyamines in mammalian cells, to determine how polyamine levels are regulated in the cell, and to ascertain the function of polyamines. The particular enzymes which are the focus of this study are the two aminopropyltransferases, spermidine synthase and spermine synthase, and the enzymes spermidine/spermine N1-acetyltransferase and polyamine oxidase which together reverse the effects of the synthesis. Measurements are also being made of the content of the two nucleosides, decarboxylated S-adenosylmethionine (AdoMet) and 5'-methylthioadenosine (MTA) which are respectively a substrate and product of the aminopropyltransferase reactions. The experiments proposed are: 1) to continue the study of the regulation of spermine/spermine N1-acetyltransferase activity by hepatotoxins and polyamines. Specific antibodies to this enzyme and an affinity chromatographic procedure for its purification in high yield have been developed and it is planned to obtain a cDNA probe. The acetyltransferase is very rapidly and highly inducible (greater than 200-fold in 6 h) and the mechanism underlying this change will be studied in detail. 2) To study the role of the acetylase/oxidase pathway in maintaining polyamine levels and in cell growth and differentiation. This will be accomplished using the specific inhibitors, N-[2-(S-Coenzyme A)acetyl]-sym-norspermidine amide for the acetylase and N1-methyl, N2-(2,3-butanedienyl)1,4-butane-diamine for the oxidase. 3) To study the role of the aminopropyltransferases in these processes using specific nucleoside inhibitors, S-adenosyl-1,8-diamino-3-thiooctane, S-methyl-MTA and S-adenosyl-1,12-diamino-3-thio-9-aza-dodecane. 4) To measure acetylated polyamine and polyamine levels and the content of MTA and decarboxylated AdoMet in cells treated with the inducers and inhibitors described above. Polyamine levels will be measured using HPLC and U.V. or fluorescence detection of derivatives. MTA and decarboxylated AdoMet will be quantitated using HPLC or cation exchange columns and U.V. detection. The further metabolism of decarboxylated AdoMet and the fate of its acetylated derivative will be studied. 5) The regulation of spermidine synthase activity will be investigated. 8-Azido derivatives of decarboxylated AdoMet and MTA have been synthesized and will be used as photoactivatable probes for spermidine synthase, spermine synthase and MTA phosphorylase.
| Shi, Chenxu; Welsh, Patricia A; Sass-Kuhn, Suzanne et al. (2012) Characterization of transgenic mice with overexpression of spermidine synthase. Amino Acids 42:495-505 |
| Green, Robert; Hanfrey, Colin C; Elliott, Katherine A et al. (2011) Independent evolutionary origins of functional polyamine biosynthetic enzyme fusions catalysing de novo diamine to triamine formation. Mol Microbiol 81:1109-24 |
| Pegg, Anthony E; Casero Jr, Robert A (2011) Current status of the polyamine research field. Methods Mol Biol 720:3-35 |
| Pegg, Anthony E; Wang, Xiaojing; Schwartz, Charles E et al. (2011) Spermine synthase activity affects the content of decarboxylated S-adenosylmethionine. Biochem J 433:139-44 |
| Wang, Xiaojing; Pegg, Anthony E (2011) Use of (Gyro) Gy and spermine synthase transgenic mice to study functions of spermine. Methods Mol Biol 720:159-70 |
| Pegg, Anthony E; Michael, Anthony J (2010) Spermine synthase. Cell Mol Life Sci 67:113-21 |
| Pegg, Anthony E; Wang, Xiaojing (2009) Mouse models to investigate the function of spermine. Commun Integr Biol 2:271-4 |
| Pegg, Anthony E (2009) Mammalian polyamine metabolism and function. IUBMB Life 61:880-94 |
| Wang, Xiaojing; Levic, Snezana; Gratton, Michael Anne et al. (2009) Spermine synthase deficiency leads to deafness and a profound sensitivity to alpha-difluoromethylornithine. J Biol Chem 284:930-7 |
| Pegg, Anthony E (2009) S-Adenosylmethionine decarboxylase. Essays Biochem 46:25-45 |
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