Inhibition of polyamine biosynthesis in Crithidia fasciculata by D,L-alpha-difluoromethylornithine and D,L-alpha-difluoromethylarginine.
Using Crithidia fasciculata as a model organism for Trypanosoma cruzi, we have examined the effects of D,L-alpha-difluoromethylornithine (DFMO) and D,L-alpha-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-1 mg-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-1 mg-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-1 mg-1) and ODC. Thus DFMA appears to inhibit growth of C. fasciculata via conversion to DFMO and subsequent inhibition of ODC.
Hunter
K J
KJ
Department of Medical Parasitology, London School of Hygiene and Tropical Medicine, U.K.
Strobos
C A
CA
Fairlamb
A H
AH
D000376:Agmatine / Q000494:pharmacology; D000579:Amidinotransferases / Q000378:metabolism; D000818:Animals; D001119:Arginase / Q000378:metabolism; D001120:Arginine / Q000031:analogs & derivatives* / Q000494:pharmacology; D002262:Carboxy-Lyases / Q000378:metabolism; D003421:Crithidia / Q000187:drug effects / Q000201:enzymology / Q000378:metabolism*; D000518:Eflornithine / Q000494:pharmacology*; D007700:Kinetics; D009955:Ornithine Decarboxylase / Q000378:metabolism; D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines / Q000378:metabolism*; D011700:Putrescine / Q000494:pharmacology
D065108:Ornithine Decarboxylase Inhibitors; D011073:Polyamines; C030965:alpha-(difluoromethyl)arginine; D000376:Agmatine; D001120:Arginine; D000579:Amidinotransferases; C020681:glycine amidinotransferase; D001119:Arginase; D002262:Carboxy-Lyases; D009955:Ornithine Decarboxylase; C019722:arginine decarboxylase; D011700:Putrescine; D000518:Eflornithine