Binding of the glutamate carboxypeptidase II (NAALADase) inhibitor 2-PMPA to rat brain membranes

2-Phosphonomethyl pentanedioic acid (2-PMPA) is a potent and selective inhi bitor of glutamate carboxypeptidase II (NAALADase), and has shown robust ne uroprotective activity in both in vitro and in vivo models of ischemia. In the brain, glutamate carboxypeptidase II (GCPII) (EC3.4.17.21) hydrolyzes t he neuropeptide N-acetylaspartylglutamate (NAAG) to glutamate and N-acetyla spartate. We report the development and characterization of a [H-3]2-PMPA b inding assay. [H-3]2-PMPA binding was dependent on protein concentration, s aturable, and displaceable. The association (k(on)) and dissociation (k(off )) rate constants were 3 X 10(6) M-1 s(-1) and 0.01 s(-1), respectively. Th e dissociation equilibrium constant (Kd) determined from the ratio of the r ate constants (K-d = k(off)/k(on)) was 1 nM. Scatchard analysis revealed on e binding site with K-d = 2 nM and B-max = 0.7 pmol/mg. Binding exhibited s imilar pharmacological properties to GCPII enzyme activity, including chlor ide dependency, cobalt stimulation and inhibition by phosphate and quisqual ate. The binding of [H-3]2-PMPA also showed tissue specificity in that tiss ues previously reported to be devoid of GCPII enzymatic activity were devoi d of [H-3]2-PMPA binding. [H-3]2-PMPA binding represents an additional prob e for the study of GCPII activity, and may be useful as a high throughput s creening assay. (C) 2001 Elsevier Science B.V. All rights reserved.