INVESTIGATIONS OF MAMMALIAN AMINOPROPYLTRANSFERASES

Project: Research project

Project Details

Description

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.
StatusFinished
Effective start/end date12/31/896/30/99

Funding

  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences
  • National Institute of General Medical Sciences

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