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

Carol W. Tiffany, Ning S. Cai, Camilo Rojas, Barbara S. Slusher

Research output: Contribution to journalArticlepeer-review


2-Phosphonomethyl pentanedioic acid (2-PMPA) is a potent and selective inhibitor of glutamate carboxypeptidase II (NAALADase), and has shown robust neuroprotective activity in both in vitro and in vivo models of ischemia. In the brain, glutamate carboxypeptidase II (GCPII) (EC3.4.17.21) hydrolyzes the neuropeptide N-acetylaspartylglutamate (NAAG) to glutamate and N-acetylaspartate. We report the development and characterization of a [3H]2-PMPA binding assay. [3H]2-PMPA binding was dependent on protein concentration, saturable, and displaceable. The association (kon) and dissociation (koff) rate constants were 3 × 106 M-1 s-1 and 0.01 s-1, respectively. The dissociation equilibrium constant (Kd) determined from the ratio of the rate constants (Kd = koff/kon) was 1 nM. Scatchard analysis revealed one binding site with Kd = 2 nM and Bmax = 0.7 pmol/mg. Binding exhibited similar pharmacological properties to GCPII enzyme activity, including chloride dependency, cobalt stimulation and inhibition by phosphate and quisqualate. The binding of [3H]2-PMPA also showed tissue specificity in that tissues previously reported to be devoid of GCPII enzymatic activity were devoid of [3H]2-PMPA binding. [3H]2-PMPA binding represents an additional probe for the study of GCPII activity, and may be useful as a high throughput screening assay.

Original languageEnglish (US)
Pages (from-to)91-96
Number of pages6
JournalEuropean Journal of Pharmacology
Issue number2
StatePublished - Sep 14 2001


  • Glutamate carboxypeptidase
  • Glutamate carboxypeptidase II (NAALADase)
  • N-acetylaspartylglutamate (NAAG)
  • Prostate specific membrane antigen
  • [H]2-(phosphonomethyl) pentanedioic acid ([H]2-PMPA)

ASJC Scopus subject areas

  • Pharmacology


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