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 bi-directional 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 Mg(2+). The enzyme presented a maximal activity for AMP hydrolysis at 37 degrees C. The apparent K(m) and V(max) values for Mg(2+)-AMP were 135.3+/-16 microM 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 (1 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 microM 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.
Departamento de Ciências Fisiológicas, Laboratório de Pesquisa Bioquímica, Faculdade de Biociências, Pontifícia Universidade Católica do Rio Grande do Sul, Avenida Ipiranga 6681, 90619-900, Porto Alegre, RS, Brazil.
Mario Roberto
MR
Senger
Eduardo Pacheco
EP
Rico
Renato Dutra
RD
Dias
Maurício Reis
MR
Bogo
Carla Denise
CD
Bonan
D015720:5'-Nucleotidase / Q000378:metabolism*; D000249:Adenosine Monophosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology*; D002413:Cations, Divalent / Q000494:pharmacology; D002462:Cell Membrane / Q000201:enzymology; D004791:Enzyme Inhibitors / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008274:Magnesium / Q000494:pharmacology; D015027:Zebrafish
D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D000249:Adenosine Monophosphate; D015720:5'-Nucleotidase; D008274:Magnesium