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Deletion of the glutamate carboxypeptidase II gene in mice reveals a second enzyme activity that hydrolyzes N ‐acetylaspartylglutamate
Author(s) -
Bacich Dean J.,
Ramadan Epolia,
O'Keefe Denise S.,
Bukhari Noreen,
Wegorzewska Iga,
Ojeifo Olumide,
Olszewski Rafal,
Wrenn Craige C.,
Bzdega Tomasz,
Wroblewska Barbara,
Heston Warren D. W.,
Neale Joseph H.
Publication year - 2002
Publication title -
journal of neurochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.75
H-Index - 229
eISSN - 1471-4159
pISSN - 0022-3042
DOI - 10.1046/j.1471-4159.2002.01117.x
Subject(s) - glutamate carboxypeptidase ii , biology , glutamate receptor , mutant , microbiology and biotechnology , carboxypeptidase , exon , biochemistry , enzyme , gene , receptor , genetics , prostate , cancer
Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane‐bound enzyme found on the extracellular face ofglia. The gene for this enzyme is designated FOLH1 in humans and Folh1 in mice. This enzyme has been proposed to be responsible for inactivation of the neurotransmitter N ‐acetylaspartylglutamate (NAAG) following synaptic release. Mice harboring a disruption of the gene for GCPII/ Folh1 were generated by inserting into the genome a targeting cassette in which the intron–exon boundary sequences of exons 1 and 2 were removed and stop codons were inserted in exons 1 and 2. Messenger RNA for GCPII was not detected by northern blotting or RT‐PCR analysis of RNA from the brains of –/– mutant mice nor was GCPII protein detected on western blots of this tissue. These GCPII null mutant mice developed normally to adulthood and exhibited a normal range of neurologic responses and behaviors including mating, open field activity and retention of position in rotorod tests. No significant differences were observed among responses of wild type, heterozygous mutant and homozygous mutant mice on tail flick and hot plate latency tests. Glutamate, NAAG and mRNA for metabotropic glutamate receptor type 3 levels were not significantly altered in response to the deletion of glutamate carboxypeptidase II. A novel membrane‐bound NAAG peptidase activity was discovered in brain, spinal cord and kidney of the GCPII knock out mice. The kinetic values for brain NAAG peptidase activity in the wild type and GCPII nullmutant were V max = 45 and 3 pmol/mg/min and K m = 2650 n m and 2494 n m , respectively. With the exception of magnesium and copper, this novel peptidase activity had a similar requirement for metal ions as GCPII. Two potent inhibitors of GCPII, 4,4′‐phosphinicobis‐(butane‐1,3 dicarboxilic acid) (FN6) and 2‐(phosphonomethyl)pentanedioic acid (2‐PMPA) inhibited the residual activity. The IC 50 value for 2‐PMPA was about 1 n m for wild‐type brain membrane NAAG peptidase activity consistent with its activity against cloned ratand human GCPII, and 88 n m for the activity in brain membranes of the null mutants.