AJP - Heart and Circulatory Physiology, Vol 246, Issue 5 710-H715, Copyright © 1984 by American Physiological Society
Photoaffinity labeling of adenosine transporter in cardiac membranes with nitrobenzylthioinosine
K. F. Kwan and S. M. Jarvis
The kinetic and molecular properties of the adenosine transporter in guinea
pig cardiac membranes were studied using nitrobenzylthioinosine (NBMPR), a
potent and specific inhibitor of nucleoside transport. [3H]-NBMPR bound
tightly but reversibly to guinea pig cardiac membranes (apparent
dissociation constant 0.24 +/- 0.07 nM; maximum binding capacity 1.24 +/-
0.45 pmol of NBMPR bound/mg protein). Reversible high-affinity [3H]NBMPR
binding was inhibited in an apparent competitive manner by adenosine
(apparent inhibition constant 0.14 mM). L-N-phenylisopropyladenosine
(L-PIA) had no effect on NBMPR binding. Exposure of cardiac membranes in
the presence of [3H]-NBMPR and dithiothreitol, a free-radical scavenger, to
ultraviolet light resulted in covalent incorporation of 3H into
polypeptides of apparent molecular weight 66,000-50,000. Covalent
attachment of [3H]NBMPR under equilibrium binding conditions was inhibited
by adenosine, nitrobenzylthioguanosine , and dipyridamole but was
unaffected by the adenosine receptor agonist L-PIA. These data suggest that
the photolabeled molecular weight protein (apparent mol wt 66,000-50,000)
is involved in adenosine permeation by guinea pig cardiac membranes.
