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

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

Research output: Contribution to journalArticle

Abstract

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
Volume427
Issue number2
DOIs
StatePublished - Sep 14 2001
Externally publishedYes

Fingerprint

Glutamate Carboxypeptidase II
Membranes
Brain
High-Throughput Screening Assays
Quisqualic Acid
Organ Specificity
Neuropeptides
2-(phosphonomethyl)pentanedioic acid
Glutamic Acid
Ischemia
Phosphates
Binding Sites
Pharmacology
Enzymes

Keywords

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

ASJC Scopus subject areas

  • Cellular and Molecular Neuroscience
  • Pharmacology

Cite this

Binding of the glutamate carboxypeptidase II (NAALADase) inhibitor 2-PMPA to rat brain membranes. / Tiffany, Carol W; Cai, Ning S.; Rojas, Camilo; Slusher, Barbara.

In: European Journal of Pharmacology, Vol. 427, No. 2, 14.09.2001, p. 91-96.

Research output: Contribution to journalArticle

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