Ontogenetic profile of ecto-5′-nucleotidase in rat brain synaptic plasma membranes

Laboratory for Molecular Biology and Endocrinology, Institute of Nuclear Sciences Vinca, University of Belgrade, Mike Petrovica 12-14, 11000 Belgrade, Serbia.
International journal of developmental neuroscience: the official journal of the International Society for Developmental Neuroscience (Impact Factor: 2.58). 03/2011; 29(4):397-403. DOI: 10.1016/j.ijdevneu.2011.03.003
Source: PubMed


Ecto-5'-nucleotidase (CD73; EC, e-5NT) is regarded as the key enzyme in the extracellular formation of adenosine, which acts as a neuromodulator and important trophic and homeostatic factor in the brain. In the present study, we have investigated e-5NT activity, kinetic properties concerning AMP hydrolysis and the enzyme protein abundance in the purified synaptic plasma membrane (SPM) preparations isolated from whole female rat brain at different ages. We observed pronounced increase in AMP hydrolyzing activity in SPM during maturation, with greatest increment between juvenile (15-day-old) and pre-pubertal (30-day-old) rats. Immunodetection of e-5NT protein in the SPM displayed the reverse pattern of expression, with the maximum relative abundance at juvenile and minimum relative abundance in the adult stage. Negative correlation between the enzyme activity and the enzyme protein abundance in the SPM indicates that e-5NT has additional roles in the synaptic compartment during postnatal brain development, other than those related to AMP hydrolysis. Determination of kinetic parameters, K(m) and V(max), suggested that the increase in the enzyme activity with maturation was entirely due to the increase in the enzyme catalytic efficiency (V(max)/K(m)). Finally, double immunofluorescence staining against e-5NT and presynaptic membrane marker syntaxin provided first direct evidence for the existence of this ecto-enzyme in the presynaptic compartment. The results of the study suggest that e-5NT may be a part of general scheme of brain development and synapse maturation and provide rationale for the previously reported inconsistencies between enzyme immunohistochemical and biochemical studies concerning localization of e-5NT in the brain.

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Available from: Ivana Grkovic, Jun 09, 2014
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    • "Ecto-5′-Nucleotidase Assay Ecto-5′-nucleotidase assay was performed in the reaction medium containing the following: 5 mM MgCl 2 and 50 mM Tris- HCl buffer, pH 7.4 and 20 μg of SPM proteins (Stanojevic et al. 2011). The reaction was initiated by the addition of 1.0 mM AMP (Sigma-Aldrich, Germany). "
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