Comparing amino acid identities between 11 TcAAAP analysed, TcAAA

Comparing amino acid identities between 11 TcAAAP analysed, TcAAAP411 is located close to TcAAAP545 in the identity-based phenogram (Fig. 2b). These data correlate perfectly with in vitro results where both genes were capable of reversing canavanine resistance in yeasts. However, the Leishmania donovani arginine permease LdAAP3 (Shaked-Mishan et al., 2006) is located in a branch distant from TcAAAP411. In silico topological analysis of TcAAAP411 using TMpred ( predicted 10 transmembrane helices and the variable N-terminal domain outside the cell. Two copies of TcAAAP411 were found in the T. cruzi genome database

(GeneDB,, one characterized

herein, and the other haplotype with three different amino acid positions (GeneDB systematic ID: see more Tc00.1047053506053.10). To define the substrate specificity of the permease, competitive transport studies were undertaken. The initial rate of arginine uptake was measured in the presence of 20 μM arginine and 20-fold excess of unlabelled competing molecule. INCB024360 in vitro Considering the participation of other endogenous yeast amino acid permeases, control experiments were also performed using pDR196 yeasts. None of the tested compounds produced a significant decrease on arginine uptake except unlabelled arginine, as expected (Fig. 2c). To test whether canavanine can enter the cells through TcAAAP411, as occurs in the selection yeast media, the same assay Smoothened was repeated using a 50-fold excess of canavanine. The inset in Fig. 2c shows that, in these conditions, canavanine produced a significant decrease on arginine uptake of about 50%. Transport of l-arginine by TcAAAP411 yeasts was found to be roughly proportional to an incubation time up to 20 min (Fig. 2d, inset). Data obtained from concentration-dependent arginine influx curves were analysed using Lineweaver–Burk plots and the apparent Michaelis–Menten constant (Km) value was estimated as about 30 μM (Fig. 2d).

Ten years ago, T. cruzi arginine transport, coupled to phosphoarginine synthesis, was identified and biochemically characterized (Pereira et al., 1999). This transport system showed very similar kinetic parameters and substrate specificity to TcAAAP411, suggesting that this permease is at least one component of the previously measured arginine transport system. Recently, a similar arginine transporter (LdAAP3) has been identified in the protozoan parasite L. donovani (Shaked-Mishan et al., 2006). Its regulation depends on the availability of the extracellular substrate, as amino acid starvation produces an increase in arginine transport and LdAAP3 abundance (Darlyuk et al., 2009). Interestingly, this mechanism of regulation was described in T.

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