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.
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.
authors.affiliation: Department of Haematology, Austin Hospital, Heidelberg, Melbourne, Victoria, Australia.
authors.forename: J S
authors.initials: JS
authors.lastname: Wiley
authors.forename: A M
authors.initials: AM
authors.lastname: Brocklebank
authors.forename: M B
authors.initials: MB
authors.lastname: Snook
authors.forename: G P
authors.initials: GP
authors.lastname: Jamieson
authors.forename: W H
authors.initials: WH
authors.lastname: Sawyer
authors.forename: J D
authors.initials: JD
authors.lastname: Craik
authors.forename: C E
authors.initials: CE
authors.lastname: Cass
authors.forename: M J
authors.initials: MJ
authors.lastname: Robins
authors.forename: D P
authors.initials: DP
authors.lastname: McAdam
authors.forename: A R
authors.initials: AR
authors.lastname: Paterson
mesh_terms: D000241:Adenosine / Q000031:analogs & derivatives* / Q000138:chemical synthesis / Q000494:pharmacology; D000345:Affinity Labels; D002352:Carrier Proteins / Q000037:antagonists & inhibitors*; D002460:Cell Line; D003561:Cytarabine / Q000378:metabolism*; D005434:Flow Cytometry; D005456:Fluorescent Dyes / Q000138:chemical synthesis*; D006801:Humans; D007700:Kinetics; D008565:Membrane Proteins / Q000037:antagonists & inhibitors*; D015394:Molecular Structure; D033703:Nucleoside Transport Proteins; D013050:Spectrometry, Fluorescence; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D013872:Thionucleosides / Q000138:chemical synthesis* / Q000494:pharmacology
chemical_list: D000345:Affinity Labels; D002352:Carrier Proteins; D005456:Fluorescent Dyes; D008565:Membrane Proteins; D033703:Nucleoside Transport Proteins; D013872:Thionucleosides; D003561:Cytarabine; C066257:5'-S-(2-aminoethyl)-N(6)-(4-nitrobenzyl)-5'-thioadenosine; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine