Nucleoside transport in guinea pig myocytes. Comparison of the affinities and transport velocities for adenosine and 2-chloroadenosine.
The affinities of adenosine and 2-chloroadenosine for the nucleoside transport system of guinea pig myocytes were evaluated indirectly by studying the inhibition of the binding of [3H]nitrobenzylthioinosine and directly by measuring the influx of [3H]radiolabeled substrates. Maximal transport velocities of the two nucleosides were also obtained. [3H]Nitrobenzylthioinosine bound to a single class of high-affinity sites (KD of 0.8 nM) which possessed a maximal binding capacity (Bmax) of 870,000 sites/cell. Adenosine, 2-chloroadenosine or the nucleoside transport inhibitor, dipyridamole, competitively inhibited the site-specific binding of [3H]nitrobenzylthioinosine with Ki values of 318 microM, 22 microM and 75 nM respectively. Both [3H]adenosine and [3H]2-chloroadenosine entered myocytes in a saturable and inhibitible manner. Observed transport kinetic constants (Km and Vmax) were 146 microM and 24.2 pmoles/10(6) cells/sec, respectively, for adenosine and 36 microM and 11.7 pmoles/10(6) cells/sec, respectively for 2-chloroadenosine. Affinities of adenosine, 2-chloroadenosine, nitrobenzylthioinosine and dipyridamole for the nucleoside transport system derived from binding and influx methodologies were equivalent which confirms that [3H]nitrobenzylthioinosine binding sites are closely associated with the nucleoside transporter.
D015762:2-Chloroadenosine; D000241:Adenosine / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D001692:Biological Transport; D006168:Guinea Pigs; D066298:In Vitro Techniques; D007700:Kinetics; D008297:Male; D009206:Myocardium / Q000378:metabolism*; D013868:Thioinosine / Q000031:analogs & derivatives / Q000378:metabolism; D014316:Tritium
D014316:Tritium; D015762:2-Chloroadenosine; D013868:Thioinosine; C001789:4-nitrobenzylthioinosine; D000241:Adenosine