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

Abstract

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