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Identification of the Adenosine Uptake Sites in Guinea Pig Brain
Author(s) -
Jarvis Simon M.,
Ng Andrew S.
Publication year - 1985
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1111/j.1471-4159.1985.tb07129.x
Subject(s) - adenosine , nucleoside , dithiothreitol , chemistry , nucleoside transporter , guinea pig , biochemistry , uridine , membrane , covalent bond , binding site , inosine , transporter , stereochemistry , biology , enzyme , rna , endocrinology , organic chemistry , gene
Nitrobenzylthioinosine (NBMPR), a potent and specific inhibitor of nucleoside transport, was employed as a photolabile probe of the adenosine transporter in guinea pig brain membranes. Reversible, high‐affinity binding of [ 3 H]NBMPR to a crude preparation of guinea pig brain membranes was demonstrated (apparent K D 0.075 ± 0.012 n M ; B max values of 0.24 ± 0.04 pmol/mg protein). Adenosine, uridine, dipyridamole, and nitrobenzylthioguanosine inhibited high‐affinity binding. Low concentrations of cyclohexoadenosine (10–300 n M ) had no effect on NBMPR binding. These properties of the high‐affinity NBMPR binding sites were consistent with NBMPR binding to the nucleoside transport protein. Exposure of brain membranes in the presence of [ 3 H]NBMPR and dithiothreitol, a free‐radical scavenger, to ultraviolet light resulted in covalent incorporation of 3 H into polypeptides of apparent MW 66,000–45,000, a value similar to that for the human erythrocyte nucleoside transporter. Covalent attachment of [ 3 H]NBMPR was inhibited by adenosine, dipyridamole, and nitrobenzyl‐thioguanosine.