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J. Biol. Chem., Vol. 262, Issue 30, 14498-14506, Oct, 1987
MB Robinson, RD Blakely, R Couto and JT Coyle
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.
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
Department of Neuroscience, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205.
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