Serval V, Barbeito L, Pittaluga A, Cheramy A, Lavielle S, Glowinski J
Laboratoire de Chimie Organique Biologique, Université Pierre et Marie Curie, UA CNRS 493, Paris, France.
J Neurochem. 1990 Jul;55(1):39-46. doi: 10.1111/j.1471-4159.1990.tb08818.x.
The endogenous neuropeptide N-acetyl-L-aspartyl-L-glutamate (NAAG) fulfills several criteria required to be accepted as a neurotransmitter. NAAG inactivation may proceed through enzymatic hydrolysis into N-acetyl-L-aspartate and glutamate by an N-acetylated-alpha-linked acidic dipeptidase (NAALADase). Therefore, some properties of NAALADase activity were investigated using crude membranes from the rat forebrain. Kinetic parameters of the hydrolysis of [Glu-3H]NAAG were determined first (Km = 0.40 +/- 0.05 microM; Vmax = 155 +/- 20 pmol/min/mg of protein). The enzymatic activity, i.e., NAALADase, was inhibited noncompetitively by the glutamatergic agonist quisqualate (Ki = 1.9 +/- 0.3 microM), and competitively by N-acetyl-L-aspartyl-beta-linked L-glutamate (beta-NAAG; Ki = 0.70 +/- 0.05 microM). To determine whether glutamate-containing dipeptides, such as NAAG, beta-NAAG, N-acetyl-L-aspartyl-D-glutamate, L-aspartyl-L-glutamate, L-alanyl-L-glutamate, L-glutamyl-L-glutamate, and L-glutamyl-gamma-linked L-glutamate, were substrates of NAALADase, rat brain membranes were immobilized on a C-8 column. Thus, endogenous trapped glutamate was washed away and formation of unlabelled glutamate could be estimated using an o-phthaldialdehyde/reverse-phase HPLC detection procedure. beta-NAAG was shown to be a nonhydrolyzable competitive inhibitor of NAALADase. L-Aspartyl-L-glutamate was hydrolyzed faster than NAAG, suggesting that the acetylated moiety is not essential for NAALADase specificity. Rat brain membranes also contained nonspecific peptidase activities (insensitive to both quisqualate and beta-NAAG), which, in the case of L-alanyl-L-glutamate, for instance, accounted for all observed hydrolysis.
内源性神经肽N-乙酰-L-天冬氨酰-L-谷氨酸（NAAG）符合被认定为神经递质所需的多项标准。NAAG的失活可能通过N-乙酰化-α-连接酸性二肽酶（NAALADase）将其酶解为N-乙酰-L-天冬氨酸和谷氨酸来进行。因此，利用大鼠前脑的粗制膜片对内源性N-乙酰-L-天冬氨酰-L-谷氨酸酰胺酶（NAALADase）活性的一些特性进行了研究。首先测定了[Glu-3H]NAAG水解的动力学参数（Km = 0.40±0.05微摩尔；Vmax = 155±20皮摩尔/分钟/毫克蛋白质）。谷氨酸能激动剂quisqualate对该酶活性即NAALADase有非竞争性抑制作用（Ki = 1.9±0.3微摩尔），而N-乙酰-L-天冬氨酰-β-连接L-谷氨酸（β-NAAG；Ki = 0.70±0.05微摩尔）有竞争性抑制作用。为了确定含谷氨酸的二肽，如NAAG、β-NAAG、N-乙酰-L-天冬氨酰-D-谷氨酸、L-天冬氨酰-L-谷氨酸、L-丙氨酰-L-谷氨酸、L-谷氨酰-L-谷氨酸和L-谷氨酰-γ-连接L-谷氨酸是否为NAALADase的底物，将大鼠脑膜固定在C-8柱上。这样，内源性截留的谷氨酸就被洗脱，未标记谷氨酸的生成可采用邻苯二甲醛/反相高效液相色谱检测方法进行估算。结果表明，β-NAAG是NAALADase的一种不可水解的竞争性抑制剂。L-天冬氨酰-L-谷氨酸比NAAG水解得更快，这表明乙酰化部分对NAALADase的特异性并非必不可少。大鼠脑膜还含有非特异性肽酶活性（对quisqualate和β-NAAG均不敏感），例如对于L-丙氨酰-L-谷氨酸，这种非特异性肽酶活性可解释所有观察到的水解现象。
