Kinetics and molecular characteristics of arginine transport by Leishmania donovani promastigotes.

Characteristics of transport of L-arginine were studied in Leishmania donovani promastigotes grown in vitro in a defined medium. The promastigotes exhibited a time-dependent, temperature-sensitive, pH-dependent and saturable uptake of arginine. Metabolic inhibitors caused 81-92% inhibition, indicating that arginine influx in promastigotes is an energy requiring process. The presence of Na+ ions was necessary for full activity. Considerable inhibition was also noticed with valinomycin, gramicidin and amiloride. The transporter seems to involve an -SH group at the active site. The most distinctive feature of the leishmanial transporter was that lysine and ornithine did not show significant competition with arginine transport. Other neutral and acidic amino acids, as well as polyamines were also ineffective. The arginine analogues, viz., nitro-L-arginine methyl ester, N-nitro-L-arginine, aminoguanidine, agmatine and D-arginine were not recognised by the transporter, while N-methyl-L-arginine acetate and phospho-L-arginine showed competition, indicating stereo-specificity of the transporter and recognition of both the guanidino group, as well as the arginine side chain by the transporter. No exchange of intracellular [14C]arginine taken up by the promastigotes was noticed during incubation with 2 or 5 mM arginine in the extracellular medium. Eighty percent of the arginine taken up remained in the trichloroacetic acid-soluble fraction. Pentamidine caused competitive inhibition of arginine transport, exhibiting an IC50 value of 40 microM. Results indicate the presence of a novel distinct arginine transporter in Leishmania promastigotes.

Fluorinated analogues of L-ornithine are powerful inhibitors of ornithine decarboxylase and cell growth of Leishmania infantum promastigotes.

Fluorinated analogues of L-ornithine have been tested on growth and ornithine decarboxylase arising from L.infantum cytosolic extracts. EC50 values estimated from dose/response curves were 38 microM, 2.62 microM and 4.64 microM for alpha-DFMO, delta-MFMO and delta-MFMOme respectively. Also the inhibition produced by all three compounds was effectively reverted by exogenous putrescine, pointing towards the inhibition of L.infantum ODC. ODC from logarithmic phase cytosolic extracts was physicochemically and kinetically characterized, showing a long half-life (more than 24 h) and a km value for L-ornithine of 98 microM. Finally, the inhibitory effect of fluorinated analogues of L-ornithine was analysed on L.infantum ODC showing a time-dependent irreversible behavior, with Ki values estimated on 125 microM, T1/2 3.5 min for alpha-DFMO; 13.3 microM, T1/2 1.8 min for delta-MFMO and 4.3 microM, T1/2 4 min for delta-MFMOme.

Selective inhibition of mammalian diamine oxidase by aquatic biotoxins.

The inhibitory activity of saxitoxin and tetrodotoxin on diamine oxidase and S-adenosyl-l-methionine decarboxylase from mammalian sources have been analysed. Unlike tetrodotoxin, saxitoxin was a reversible non-competitive inhibitor of pig kidney diamine oxidase with an estimated K(i) of 140 mum. S-Adenosyl-l-methionine decarboxylase from a highly purified source was not affected by the concentrations tested of either biotoxin. A possible analytical application of this finding is discussed.