Biochemical analysis of ecto-nucleotide pyrophosphatase phosphodiesterase activity in brain membranes indicates involvement of NPP1 isoenzyme in extracellular hydrolysis of diadenosine polyphosphates in central nervous system

摘要: Synaptosomes and plasma membranes obtained from rat brain display ectoenzymatic hydrolytic activity responsible for hydrolysis of the neurotransmitter/neuroregulatory nucleotides diadenosine polyphosphates. Intact synaptosomes synaptic isolated by sucrose-gradient ultracentrifugation several regions (hypothalamus, hippocampus, temporal cortex, frontal cortex striatum cerebellum) degraded fluorogenic substrates diethenoadenosine polyphosphates up to ethenoadenosine as by-product. Purified ectoenzyme cleaved always releasing mononucleotide moieties 5'-monophosphate corresponding (n-1) 5'-phosphate. Ectoenzymatic reached maximal at pH 9.0 (pH range 6.5-9.0) was activated Ca(2+) Mg(2+) ions, with effects around 2.0 mM cation. EDTA drastically reduced Zn(2+) required enzyme reactivation. Hydrolysis followed hyperbolic kinetics K(m) values in 3-10 microM range. Diadenosine heparin behaved competitive inhibitors enzymatic AMP, ATP, alpha,beta-methyleneADP, ADPbetaS ATPgammaS, beta,gamma-methyleneATP, suramin diethyl pyrocarbonate were also inhibitors. shared typical characteristics members ecto-nucleotide pyrophosphatase/phosphodiesterase (E-NPP) family inhibition data suggest that NPP1 is involved cleavage extracellular brain. Synaptic cerebellum, hypothalamus hippocampus presented highest activities no differences observed between young aged animals. However, showed a more homogeneous distribution but general increase detected Enhancement polyphosphate cleaving found old animals could play deleterious role limiting neuroprotective reported tetraphosphate.