Asensio Aaron C, Rodríguez-Ferrer Carmen R, Castañeyra-Perdomo Agustín, Oaknin Sol, Rotllán Pedro
Departamentos de Bioquímica y Biol. Molecular, Universidad de La Laguna, 38206 La Laguna, Tenerife, Spain.
Neurochem Int. 2007 Mar;50(4):581-90. doi: 10.1016/j.neuint.2006.11.006. Epub 2006 Dec 21.
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 microM range. Diadenosine polyphosphates and heparin behaved as competitive inhibitors in the enzymatic hydrolysis of diethenoadenosine polyphosphates and AMP, ATP, alpha,beta-methyleneADP, ADPbetaS ATPgammaS, beta,gamma-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.
从大鼠脑中获得的突触体和质膜表现出胞外酶水解活性，负责神经递质/神经调节核苷酸二腺苷多磷酸的水解。通过蔗糖梯度超速离心从几个脑区（下丘脑、海马体、颞叶皮质、额叶皮质纹状体和小脑）分离得到的完整突触体以及质膜和突触膜，将荧光底物二乙烯腺苷多磷酸降解为副产物乙烯腺苷。纯化的胞外酶裂解底物时总是释放出单核苷酸部分5'-磷酸乙烯腺苷和相应的(n-1)5'-磷酸乙烯腺苷。胞外酶水解在pH 9.0（pH范围6.5 - 9.0）时达到最大活性，并被Ca(2+)和Mg(2+)离子激活，在阳离子浓度约为2.0 mM时效果最佳。EDTA显著降低活性，酶重新激活需要Zn(2+)。底物水解遵循双曲线动力学，K(m)值在3 - 10 microM范围内。二腺苷多磷酸和肝素在二乙烯腺苷多磷酸的酶促水解中表现为竞争性抑制剂，AMP、ATP、α,β-亚甲基ADP、ADPβS、ATPγS、β,γ-亚甲基ATP、苏拉明和焦碳酸二乙酯也是抑制剂。胞外酶活性具有胞外核苷酸焦磷酸酶/磷酸二酯酶（E-NPP）家族成员的典型特征，抑制数据表明NPP1胞外酶参与脑内细胞外二腺苷多磷酸的裂解。小脑、下丘脑和海马体的突触膜活性最高，年轻和老年动物之间未观察到活性差异。然而，质膜显示出胞外酶活性分布更均匀，但在老年动物中检测到普遍增加。老年动物质膜中发现的胞外酶二腺苷多磷酸裂解活性增强可能通过限制细胞外四磷酸二腺苷的神经保护作用而在老年脑中发挥有害作用。
