L‐Arginine Uptake and L‐Phosphoarginine Synthesis In Trypanosoma Cruzi

Abstract

A very specific L-arginine transporter showing high affinity has been characterized in Trypanosoma cruzi epimastigotes. Uptake was found to be dependent on L-arginine concentration and it was saturable. Values for maximum velocity and Km ranged between 48.1-57.5 pmol.min-1 per 3 x 10(7) cells and between 4.2-5.5 microM, respectively. The calculated activation energy and Q10 were 31.1 KJ.mol-1, and 1.7, respectively. Uptake velocity significantly increased when cells were preincubated in the absence of L-arginine. Cells retained the labeled amino acid independently of the presence or absence of exogenous L-arginine. The specificity of L-arginine uptake was demonstrated by competition assays in the presence of 80-fold molar excess of natural amino acids and several L-arginine derivatives. The highest levels of inhibition were caused by L-homoarginine, D-arginine, L-canavanine, L-ornithine, and L-citrulline. L-arginine uptake by T. cruzi epimastigotes was not affected by the presence of potassium or sodium ions in the incubation mixture or by pH changes in the range between 5.5-8.5. The major product of L-arginine uptake was characterized as phosphoarginine. Moreover, arginine kinase activity was detected in soluble extracts from T. cruzi epimastigotes.

Trypanosoma cruzi Arginine Kinase Characterization and Cloning

“…Other compounds such as L-homoarginine, L-glutamate, N-methyl-L-arginine, Lnitroarginine, L-lysine, L-aspartate, agmatine, and ethylguanidine elicited inhibitions of between 20 and 35% (Table II). Inhibition constant (K i ) values for canavanine and homoarginine were calculated because both metabolites produced inhibition of arginine kinase activity as well as arginine transport in epimastigote cells (9). K i values for canavanine and homoarginine were 6.0 mM, and 7.0 mM respectively (results not shown).…”

“…The existence of a high affinity and very specific L-arginine transporter was also demonstrated in T. cruzi epimastigotes. One of the major products of L-arginine uptake was characterized as phosphoarginine (9).…”

The effects of nitric oxide on the immune system during Trypanosoma cruzi infection

“…Interestingly, epimastigote forms of T. cruzi synthesise their own NO through a partially characterized NOS enzyme which displays regulatory and immunochemical properties resembling those of endogenous NOS1 (Pereira et al 1999, Goldstein et al 2000, Piacenza et al 2001.…”

The crystal structure of Trypanosoma cruzi arginine kinase

“…The protein was subsequently concentrated to 20 mg/mL and tested for biochemical activity as described. 8 Crystals of TcAK were obtained by the hanging drop method, mixing 1.5 lL of protein solution and 1.5 lL of the reservoir solution containing 2.5M ammonium sulfate, 0.1M Tris HCl, pH 7.5. Crystals grew within a week at 188C to a maximum size of 0.2 mm 3 0.2 mm 3 0.3 mm.…”