HEXOSE UPTAKE IN TRYPANOSOMA CRUZI - STRUCTURE-ACTIVITY RELATIONSHIP BETWEEN SUBSTRATE AND TRANSPORTER

摘要

The gene encoding a hexose transporter, TcrHT1, from Trypanosoma cruzi has been functionally expressed in mammalian Chinese hamster ovary cells. Kinetic parameters of the heterologously expressed protein are very similar to those of the transporter identified in T. cruzi epimastigotes, confirming that TcrHT1 is the major transporter functioning in these para sites. A detailed analysis of substrate recognition using analogues of D-glucose substituted at each carbon position has been performed. The glucose transporter of T. cruzi does not recognize C-3 or C-6 analogues of D-glucose, whereas these analogues were recognized by the glucose transporter of bloodstream-form T. brucei. As for other kinetoplastid transporters, but in stark contrast to the mammalian GLUT family, TcrHT1 can also transport D-fructose, with relatively high affinity (K-m = 0.682+/-0.003 mM). Amino acid side-chain-modifying reagents were also used to identify residues of the transporter present at the substrate-binding site. While specific modifiers of cysteine, histidine and arginine all inhibited catalytic activity, protection using substrate was only observed using the arginine-specific reagent, phenylglyoxal. Reagents which modify lysine residues had no effect on transport.