Inhibition of polyamine biosynthesis in Crithidia fasciculata by d,l-α-difluoromethylornithine and d,l-α-difluoromethylarginine

  • Hunter K
  • Strobos C
  • Fairlamb A
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Abstract

Using Crithidia fasciculata as a model organism for Trypanosoma cruzi, we have examined the effects of d,l-α-difluoromethylornithine (DFMO) and d,l-α-difluoromethylarginine (DFMA) on growth and polyamine synthesis. In a defined, polyamine-free medium growth was markedly inhibited by DFMO (94% at 50 mM; IC50= 37 mM) and to a lesser extent by DFMA (65% at 50 mM). Addition of putrescine, but not agmatine, reverses inhibition of growth, suggesting that the site of inhibition is ornithine decarboxylase (ODC). Consistent with this conclusion, DFMO or DFMA results in a complete loss of putrescine and significant reductions in intracellular spermidine, glutathionylspermidine and N1,N8-bis(glutathionyl)spermidine (trypanothione). In addition, significant concentrations of DFMO (0.8 mM) were present in DFMA-treated cells. However, in contrast to other organisms, conversion of DFMA to DFMO is probably not catalysed by arginase. Substantial ornithine decarboxylase activity (63.1 pmol min-1mg-1; ODC) was observed in control cells, sufficient to account for polyamine synthesis during growth. In addition, a trace arginine decarboxylase (ADC) activity (1.19 pmol min-1mg-1) was found. Evidence is presented showing that the apparent ADC activity is actually due to the concerted action of arginase (1.5 nmol min-1mg-1) and ODC. Thus DFMA appears to inhibit growth of C. fasciculata via conversion to DFMO and subsequent inhibition of ODC. © 1991.

Author-supplied keywords

  • Polyamine
  • Trypanosomatid
  • Trypanothione
  • α-Difluoromethylarginine
  • α-Difluoromethylornithine

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