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Title: A new fluorescent probe for the equilibrative inhibitor-sensitive nucleoside transporter. 5'-S-(2-aminoethyl)-N6-(4-nitrobenzyl)-5'-thioadenosine (SAENTA)-chi 2-fluorescein.
Austin Authors: Wiley, J S;Brocklebank, A M;Snook, M B;Jamieson, Gary P;Sawyer, W H;Craik, J D;Cass, C E;Robins, M J;McAdam, D P;Paterson, A R
Affiliation: Department of Haematology, Austin Hospital, Heidelberg, Melbourne, Victoria, Australia
Issue Date: 1-Feb-1991
Publication information: The Biochemical Journal; 273 ( Pt 3)(): 667-72
Abstract: 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 [3H]nitrobenzylthioinosine ([3H]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 [3H]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 Kd 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.
Gov't Doc #: 1996965
Journal: The Biochemical journal
Type: Journal Article
Subjects: Adenosine.analogs & derivatives.chemical synthesis.pharmacology
Affinity Labels
Carrier Proteins.antagonists & inhibitors
Cell Line
Flow Cytometry
Fluorescent Dyes.chemical synthesis
Membrane Proteins.antagonists & inhibitors
Molecular Structure
Nucleoside Transport Proteins
Spectrometry, Fluorescence
Thioinosine.analogs & derivatives.metabolism
Thionucleosides.chemical synthesis.pharmacology
Appears in Collections:Journal articles

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