title: 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.
abstract: 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.
authors: Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
authors: M B
authors: MB
authors: Robinson
authors: R D
authors: RD
authors: Blakely
authors: R
authors: R
authors: Couto
authors: J T
authors: JT
authors: Coyle
mesh_terms: D000818:Animals; D001921:Brain / Q000201:enzymology*; D002712:Chlorides / Q000494:pharmacology; D004150:Dipeptidases / Q000037:antagonists & inhibitors / Q000302:isolation & purification*; D004151:Dipeptides / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D006868:Hydrolysis; D007700:Kinetics; D008670:Metals / Q000494:pharmacology; D051381:Rats; D011919:Rats, Inbred Strains; D013329:Structure-Activity Relationship
chemical_list: D002712:Chlorides; D004151:Dipeptides; D008670:Metals; C027172:isospaglumic acid; D004150:Dipeptidases; C038937:dipeptidase