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Journal of Biochemistry 2005 137(3):323-329; doi:10.1093/jb/mvi035
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© 2005 The Japanese Biochemical Society

Regular Paper

Identification of Specific [3H]Adenine-Binding Sites in Rat Brain Membranes

Shun Watanabe1,2, Masahiko Ikekita2,3 and Hiroyasu Nakata1,*

1 Department Molecular Cell Signaling, Tokyo Metropolitan Institute for Neuroscience, 2-6 Musashidai, Fuchu, Tokyo 183-8526; and 2 Department of Applied Biological Science, Faculty of Science and Technology, and 3 Genome and Drug Research Center, Tokyo University of Science, Chiba

* To whom correspondence should be addressed. Tel: +81-423-253881, Fax: +81-423-218678, E-mail: nakata{at}tmin.ac.jp

ABSTRACT

We recently reported that adenine acts as a neurotrophic factor independent of adenosine or P2 receptors in cultured Purkinje cells [Watanabe S. et al. (2003) J. Neurosci. Res. 74, 754–759], suggesting the presence of specific receptors for adenine in the brain. In this study, the characterization of adenine-binding activity in the rat brain was performed to further characterize the receptor-like adenine-binding sites. Specific binding sites for [3H]adenine were detected in membrane fractions prepared from rat brains. The kinetics of [3H]adenine binding to membranes was described by the association and dissociation rate constants, 8.6 x 105 M–1 min–1 and 0.118 ± 0.045 min–1, respectively. A single binding site for [3H]adenine with a K D of 157.1 ± 20.8 nM and a B max of 16.3 ± 1.1 pmol/mg protein (n = 6) was demonstrated in saturation experiments. A displacement study involving various related compounds showed that the [3H]adenine binding was highly specific for adenine. It was also found that [3H]adenine-binding activity was inhibited by adenosine, although other adenosine receptor ligands were ineffective as to [3H]adenine binding. The brain, especially the cerebellum and spinal cord, showed the highest [3H]adenine-binding activity of the tissues examined. These results are consistent with the presence of a novel adenine receptor in rat brain membranes.


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