Ecto-5′-nucleotidase activity in brain membranes of zebrafish (Danio rerio)

Adenosine, a well-known neuromodulator, may be formed intracellularly in the CNS from degradation of AMP and then exit via bidirectional nucleoside transporters, or extracellularly by the metabolism of released nucleotides. This study reports the enzymatic properties of an ecto-5'-nucleotidase activity in brain membranes of zebrafish (Danio rerio). This enzyme was cation-dependent, with a maximal rate for AMP hydrolysis in a pH range of 7.0-7.5 in the presence of Mg2+. The enzyme presented a maximal activity for AMP hydrolysis at 37 degreesC. The apparent K-m and V-max values for Mg2+-AMP were 135.3 +/- 16 muM and 29 +/- 4.2 nmol Pi-min(-1). mg(-1) protein, respectively. The enzyme was able to hydrolyze both purine and pyrimidine monophosphate nucleotides, such as UMP, GMP and CMP. Levamisole and tetramisole (I mM), specific inhibitors of alkaline phosphatases, did not alter the enzymatic activity. However, a significant inhibition of AMP hydrolysis (42%) was observed in the presence of 100 muM alpha,beta-methylene-ADP, a known inhibitor of ecto-5' -nucleotidase. Since 5'-nucleotidase represents the major enzyme responsible for the formation of extracellular adenosine, the enzymatic characterization is important to understand its role in purinergic systems and the involvement of adenosine in the regulation of neurotransmitter release. (C) 2004 Elsevier Inc. All rights reserved.