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 [(3)H]2-PMPA binding assay. [(3)H]2-PMPA binding was dependent on protein concentration, saturable, and displaceable. The association (k(on)) and dissociation (k(off)) rate constants were 3x10(6) M(-1) s(-1) and 0.01 s(-1), respectively. The dissociation equilibrium constant (K(d)) determined from the ratio of the rate constants (K(d)=k(off)/k(on)) was 1 nM. Scatchard analysis revealed one binding site with K(d)=2 nM and B(max)=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 [(3)H]2-PMPA also showed tissue specificity in that tissues previously reported to be devoid of GCPII enzymatic activity were devoid of [(3)H]2-PMPA binding. [(3)H]2-PMPA binding represents an additional probe for the study of GCPII activity, and may be useful as a high throughput screening assay.
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Authors | C W Tiffany, N S Cai, C Rojas, B S Slusher |
Journal | European journal of pharmacology
(Eur J Pharmacol)
Vol. 427
Issue 2
Pg. 91-6
(Sep 14 2001)
ISSN: 0014-2999 [Print] Netherlands |
PMID | 11557259
(Publication Type: Journal Article)
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Chemical References |
- 2-(phosphonomethyl)pentanedioic acid
- Antigens, Surface
- Organophosphorus Compounds
- Tritium
- Carboxypeptidases
- FOLH1 protein, human
- Glutamate Carboxypeptidase II
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Topics |
- Animals
- Antigens, Surface
- Binding, Competitive
(drug effects)
- Brain
(metabolism)
- Carboxypeptidases
(antagonists & inhibitors, metabolism)
- Dose-Response Relationship, Drug
- Glutamate Carboxypeptidase II
- Humans
- Kidney
(metabolism)
- Liver
(metabolism)
- Male
- Membranes
(metabolism)
- Organophosphorus Compounds
(metabolism, pharmacology)
- Prostate
(metabolism)
- Rats
- Spinal Cord
(metabolism)
- Time Factors
- Tritium
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