摘要
Equilibrative nucleoside transporters （ENTs）, which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and bio- chemical characterization of human equilibrative nucle- oside transporter 1 （hENT1） in vitro. The HEK293F- derived, recombinant hENT1 is homogenous and func- tionally active in proteoliposome-based counter flow assays, hENT1 transports the substrate adenosine with a Km of 215 ＋ 34 pmol/L and a Vmax of 578 ＋ 23.4 nmol mg-1 min-1. Adenosine uptake by hENT1 is competi- tively inhibited by nitrobenzylmercaptopurine ribonu- cleoside （NBMPR）, nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies.
Equilibrative nucleoside transporters （ENTs）, which facilitate cross-membrane transport of nucleosides and nucleoside-derived drugs, play an important role in the salvage pathways of nucleotide synthesis, cancer chemotherapy, and treatment for virus infections. Functional characterization of ENTs at the molecular level remains technically challenging and hence scant. In this study, we report successful purification and bio- chemical characterization of human equilibrative nucle- oside transporter 1 （hENT1） in vitro. The HEK293F- derived, recombinant hENT1 is homogenous and func- tionally active in proteoliposome-based counter flow assays, hENT1 transports the substrate adenosine with a Km of 215 ＋ 34 pmol/L and a Vmax of 578 ＋ 23.4 nmol mg-1 min-1. Adenosine uptake by hENT1 is competi- tively inhibited by nitrobenzylmercaptopurine ribonu- cleoside （NBMPR）, nucleosides, deoxynucleosides, and nucleoside-derived anti-cancer and anti-viral drugs. Binding of hENT1 to adenosine, deoxyadenosine, and adenine by isothermal titration calorimetry is in general agreement with results of the competitive inhibition assays. These results validate hENT1 as a bona fide target for potential drug target and serve as a useful basis for future biophysical and structural studies.
