Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain.
Hydrolysis of the brain dipeptide N-acetyl-L-aspartyl-L-glutamate. Identification and characterization of a novel N-acetylated alpha-linked acidic dipeptidase activity from rat brain.
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DOI：
10.1016/s0021-9258(18)47823-4
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发表时间：
1987-10
期刊：
影响因子：
--
通讯作者：
M. Robinson;R. Blakely;R. Couto;J. Coyle
中科院分区：
文献类型：
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作者：
M. Robinson;R. Blakely;R. Couto;J. Coyle
高效液相色谱研究记录了大鼠脑细胞膜中存在一种酶活性，N-乙酰化α-连接酸性二肽酶（NAALA二肽酶），可将内源性脑二肽N-乙酰基-L-乙酰基-L-谷氨酸裂解为N-乙酰基-天冬氨酸和谷氨酸。用离子交换色谱法定量分离[3，4 - 3 H]谷氨酸和N-乙酰-L-乙酰-L-[3，4 - 3 H]谷氨酸，我们发现NAALA二肽酶活性基本上局限于神经组织和肾脏。我们的特点是NAALA二肽酶活性裂解突触体膜从大鼠前脑。膜结合NAALA二肽酶活性在37 ℃时在pH 6.0和7.4之间是最佳的。Eadie-Hofstee动力学数据分析显示，N-乙酰基-L-乙酰基-L-谷氨酸具有相当高的表观亲和力，Km = 540 nM，Vmax = 180 nM/mg蛋白质/min。而NAALA二肽酶显示需要一价阴离子，如Cl-，多价阴离子磷酸盐和硫酸盐分别在100 μ M和1 mM时抑制酶活性50%。二价金属离子螯合剂EGTA，EDTA和邻菲咯啉完全废除活动，这是部分恢复锰。用1 mM二硫苏糖醇处理膜可消除NAALA二肽酶活性。NAALA二肽酶活性也敏感的氨肽酶抑制剂bestatin和嘌呤霉素，虽然不是选择性氨肽酶A抑制剂amastatin。从抑制剂研究推断的结构-活性关系表明，这种酶对N-乙酰化α-连接的酸性二肽具有特异性。NAALA二肽酶也被兴奋性氨基酸激动剂L-使君子酸有效抑制。NAALA二肽酶的性质与先前表征的酶的性质的比较表明，这是一种新的肽酶，其可能参与N-乙酰基-L-乙酰基-L-谷氨酸的突触降解。
High performance liquid chromatography studies documented the presence of an enzyme activity, N-acetylated alpha-linked acidic dipeptidase (NAALA dipeptidase), in rat brain membranes that cleaves the endogenous brain dipeptide, N-acetyl-L-aspartyl-L-glutamate to N-acetyl-aspartate and glutamate. With ion exchange chromatography, which quantitatively separated [3,4-3H]glutamate from N-acetyl-L-aspartyl-L-[3,4-3H]glutamate, we found that NAALA dipeptidase activity was essentially restricted to nervous tissue and kidney. We characterized NAALA dipeptidase activity in lysed synaptosomal membranes obtained from rat forebrain. Membrane-bound NAALA dipeptidase activity was optimal between pH 6.0 and 7.4 at 37 degrees C. Eadie-Hofstee analysis of kinetic data revealed a rather high apparent affinity for N-acetyl-L-aspartyl-L-glutamate with a Km = 540 nM and a Vmax = 180 nM/mg of protein/min. While NAALA dipeptidase showed a requirement for monovalent anions such as Cl-, the polyvalent anions phosphate and sulfate inhibited enzyme activity 50% at 100 microM and 1 mM, respectively. The divalent metal ion chelators EGTA, EDTA, and o-phenanthroline completely abolished activity, which was partially restored by manganese. Treatment of membranes with 1 mM dithiothreitol abolished NAALA dipeptidase activity. NAALA dipeptidase activity was also sensitive to the aminopeptidase inhibitors bestatin and puromycin, although not to the selective aminopeptidase A inhibitor amastatin. Structure-activity relationships inferred from inhibitor studies suggest that this enzyme shows specificity for N-acetylated alpha-linked acidic dipeptides. NAALA dipeptidase was also potently inhibited by the excitatory amino acid agonist L-quisqualate. Comparison of the properties of NAALA dipeptidase to those of previously characterized enzymes suggests that this is a novel peptidase which may be involved in the synaptic degradation of N-acetyl-L-aspartyl-L-glutamate.