A NEW FLUORESCENT-PROBE FOR THE EQUILIBRATIVE INHIBITOR-SENSITIVE NUCLEOSIDE TRANSPORTER - 5'-S-(2-AMINOETHYL)-N6-(4-NITROBENZYL)-5'-THIOADENOSINE (SAENTA)-X2-FLUORESCEIN

The N6-(4-nitrobenzyl) derivative of adenosine is a tight-binding inhibitor of the equilibrative inhibitor-sensitive nucleoside transporter of mammalian cells. A fluorescent ligand for this transporter has been synthesized by allowing an adenosine analogue, 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA), to react with fluorescein isothiocyanate. The purified adduct had a SAENTA/fluorescein molar ratio of 0.92:1 calculated from its absorption spectrum. The intensity of fluorescent emission from the SAENTA-chi-2-fluorescein adduct was 30% that of fluorescein isothiocyanate (chi-2 is the number of atoms in the linkage between fluorescein and SAENTA). SAENTA-chi-2-fluorescein inhibited the influx of nucleosides into cultured leukaemic cells with an IC50 (total concentration of inhibitor producing 50% inhibition) of 40 nM. The adduct inhibited the binding of [H-3]nitrobenzylthioinosine ([H-3]NBMPR) with half-maximal inhibition at 50-100 nM. Mass Law analysis of the competitive-binding data suggested the presence of two classes of sites for [H-3]NBMPR binding, only one of which was accessible to SAENTA-chi-2-fluorescein. Flow cytometry was used to analyse equilibrium binding of SAENTA-chi-2-fluorescein to leukaemic cells, and a K(d) of 6 nM was obtained. SAENTA-chi-2-fluorescein is a high-affinity ligand for the equilibrative inhibitor-sensitive nucleoside transporter which allows rapid assessment of transport capacity by flow cytometry.