Kinetic characterization and immunodetection of ecto-ATP diphosphohydrolase (EC 3.6.1.5) in cultured hippocampal neurons.
Extracellular ATP and adenosine modulate synaptic transmission in hippocampal neurons. ATP released from neural cells is hydrolyzed to adenosine by a chain of ecto-nucleotidases. ATP diphosphohydrolase hydrolyses ATP and ADP nucleotides to AMP and 5'-nucleotidase hydrolyses AMP to adenosine. In this work, we investigated the ATPase and ADPase activities of ATP diphosphohydrolase in cultured hippocampal neurons. The apparent Michaelis-Menten constant (K(m)) was 233.9 +/- 14.6 and 221.8 +/- 63.6 microM, with a calculated maximal velocity (V(max), approximately) of 49.2 +/- 10.7 and 10.9 +/- 5.2 nmol Pi/mg protein/min for ATP and ADP, respectively. The horizontal straight line obtained in the competition plot indicated that only one active site is able to hydrolyze both substrates. Furthermore, we detected the presence of this enzyme using anti-CD39 antibody, which strongly stained the soma of pyramidal and bipolar neurons, but the neurites connecting the cell clusters were also immunopositive. This antibody recognized three bands with a molecular mass close to 95, 80 and 60kDa in immunoblotting analysis. The present results show, for the first time, the kinetic and immunocytochemical characterization of an ATP diphosphohydrolase in cultured hippocampal neurons. Probably, the widespread distribution of this enzyme on the surface of neurons in culture could reflect its functional importance in studies of synaptic plasticity hippocampal.
Boeck
Carina Rodrigues
CR
Departamento de Bioquímica, Instituto de Ciências Básicas da Saúde, Universidade Federal do Rio Grande do Sul, Rua Ramiro Barcelos, 2600 (anexo), 90035-003, Porto Alegre, RS, Brazil.
Sarkis
João José Freitas
JJ
Vendite
Deusa
D
D000244:Adenosine Diphosphate / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D015703:Antigens, CD; D001081:Apyrase / Q000378:metabolism*; D002478:Cells, Cultured; D006624:Hippocampus / Q000166:cytology / Q000378:metabolism*; D006868:Hydrolysis; D015151:Immunoblotting; D007150:Immunohistochemistry; D007700:Kinetics; D009474:Neurons / Q000378:metabolism*; D051381:Rats; D017208:Rats, Wistar
D015703:Antigens, CD; D000244:Adenosine Diphosphate; D000255:Adenosine Triphosphate; D001081:Apyrase; C067869:CD39 antigen