Competitive Inhibition of N-Acetylated-α-Linked Acidic Dipeptidase Activity by N-Acetyl-L-Aspartyl-β-Linked L-Glutamate

Authors


Address correspondence and reprint requests to Dr. V. Serval, at Laboratoire de Chimie Organique Biologique, Université Pierre et Marie Curie, UA CNRS 493,4, place Jussieu, 75252 Paris cedex 05, France.

Abstract

Abstract: 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-α-linked acidic dipeptidase (NAALADase). Therefore, some properties of NAA-LADase 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 μM; 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 μM), and competitively by N-acetyl-L-aspartyl-β-linked L-glutamate (β-NAAG; Ki= 0.70 ± 0.05 μM). To determine whether glutamate-containing dipeptides, such as NAAG, β-NAAG, N-acetyl-L-aspartyl-D-glutamate, L-aspartyl-L-glutamate, L-alanyl-L-glutamate, L-glutamyl-L-glutamate, and L-glutamyl-γ-linked L-glutamate, were substrates of NAA-LADase, 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. β-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 β-NAAG), which, in the case of L-alanyl-L-glutamate, for instance, accounted for all observed hydrolysis.

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