Distribution of alpha 1 and alpha 2 GABAA receptor subunits in developing chick optic tectum (original) (raw)
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Poster sessions AP11: Neurotransmitters, Transporter and Enzymes
Journal of Neurochemistry, 2008
Glutamate carboxypeptidase II (GCPII, EC 3.4.17.21) is a membrane peptidase expressed in a number of tissues such as kidney, prostate and brain. The brain form of GCPII (also known as N-acetylated-a-linked-acidic dipeptidase, NAALADase) cleaves N-acetyl-aspartyl glutamate to yield free glutamate. Animal model experiments show that inhibition of GCPII prevents neuronal cell death during experimental ischaemia. GCPII thus represents an important target for the treatment of neuronal damage caused by excess glutamate. We report the mapping of the entire coding region of GCPII and identification of the region sufficient and necessary for the production of active recombinant protein. Extracellular portion of human glutamate carboxypeptidase II (amino acids 44-750) was expressed in Drosophila Schneider's cells and purified to homogeneity. A novel assay for hydrolytic activity of GCPII, based on fluorimetric detection of released alpha-amino groups was established, and used for enzymological characterization of GCPII. The potential of this assay for high-throughput inhibitor testing was evaluated and pH dependence for the enzymatic activity have been analysed. Using a complete set of protected dipeptides, substrate specificity of recombinant GCPII was elucidated. Ac-Glu-Met, Ac-Asp-Met and surprisingly Ac-Ala-Met were identified as novel substrates for GCPII. The glycosylation has been found indispensable for the activity of the enzyme. A series of point mutants of the enzyme has been expressed and purified and the glycosylation sites critical for the proteolytic activity have been identified.
Journal of Neurochemistry, 1965
THE activities of a great many enzymes increase rapidly in the period in which the brain is growing fast and the electrical activity is attaining a mature pattern (HIMWICH, 1962; FLEXNER, 1955). Very few studies deal with the behaviour of closely related enzymes in this period of brain development. In view of the current opinion on the important function of y-aminobutyric acid (GABA) in nervous activity, we studied the levels of glutamate decarboxylase (GAD) and y-aminobutyric transaminase (GABAT) during the development of the brain of rats, rabbits and chickens. SISKEN, SANO and ROBERTS (1961) determined both enzymes in the optic lobe of the chicken from 10 days after incubation onwards. The activities of both enzymes seem to rise in a similar manner. In an earlier publication (VAN KEMPEN, VAN DEN BERG, VAN DER HELM and VELDSTRA, 1965) we reported some studies on the intracellular localization of GAD, GABAT and other enzymes in brain from adult rat, studied by centrifugation of mitochondria1 preparations on continuous sucrose gradients. This report describes the results of similar experiments with brains from immature rats. It was suspected that there might be changes in the intracellular localization of enzymes at the time when maximal changes in morphological, physiological and biochemical parameters occur. METHODS The animals were obtained from Centraal Proefdieren Bedrijf T.N.O., Bilthoven. Centrifugation methods and enzyme assays are as described in a previous publication (VAN KEMPEN et al., 1965). RESULTS Levels of GAD and GABAT The levels of GAD, GABAT and the GABATIGAD ratio in all the experiments are presented in Tables 1 , 2 and 3. In all instances, except for spinal cord from the rabbit, there is a sharp increase of both enzyme activities, in the rat after the 5th day, in the rabbit between 0-5th day and in the chicken after the 14th day, following incubation. The GABAT/GAD ratio is fairly constant during the whole period studied in the
Gamma-aminobutyric acid (GABA) metabolism in mammalian neural and nonneural tissues
Comparative Biochemistry and Physiology Part A: Physiology, 1995
4-Aminobutyric acid (GABA), a major inhibitory neurotransmitter of mammalian central nervous system, is found in a wide range of organisms, from prokaryotes to vertebrates. GABA is widely distributed in nonneural tissue including peripheral nervous and endocrine systems. GABA acts on GABAA and GABA, receptors. GABA, receptors are ligand-gated chloride channels modulated by a variety of drugs. GABA, receptors are essentially presynaptic, usually coupled to potassium or calcium channels, and they function via a GTP binding protein. In neural and nonneural tissues, GABA is metabolized by three enzymes-glutamic acid decarboxylase (GAD), which produces GABA from glutamic acid, and the catabolic enzymes GABA-transaminase (GABA-T) and succinic semialdehyde dehydrogenase (SSADH). Production of succinic acid by SSADH allows entry of the GABA carbon skeleton into the tricarboxylic acid cycle. Alternate sources of GABA include putrescine, spermine, spermidine and ornithine, which produce GABA via deamination and decarboxylation reactions, while L-glutamine is an additional source of glutamic acid via deamination. GAD from mammalian brain occurs in two molecular forms, GAD,, and GAD, (referring to subunit relative molecular weight (M,) in kilodaltons). These different forms of GAD are the product of different genes, differing in nucleotide sequence, immunoreactivity and subcellular localization. The presence and characteristics of GAD have been investigated in a wide variety of nonneural tissues including liver, kidney, pancreas, testis, ova, oviduct, adrenal, sympathetic ganglia, gastrointestinal tract and circulating erythrocytes. In some tissues, one form (GAD, or GAD,,) predominates. GABA-T has been located in most of the same tissues, primarily through histochemical and/or immunochemical methods; GABA-T is also present in a variety of circulating cells, including platelets and lymphocytes. SSADH, the final enzyme GABA catabolism, has been detected in some of the tissues in which GAD and GABA-T have been identified, although the presence of this enzyme has not been demonstrated in mammalian pancreas, ova, oviduct, testis or sympathetic ganglia.
Journal of Neurochemistry, 2002
Glutamate carboxypeptidase II (GCPII, EC 3.14.17.21) is a membrane-bound enzyme found on the extracellular face of glia. 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 null mutant were V max ¼ 45 and 3 pmol/mg/min and K m ¼ 2650 nM and 2494 nM, 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) 2 inhibited the residual activity. The IC 50 value for 2-PMPA was about 1 nM for wild-type brain membrane NAAG peptidase activity consistent with its activity against cloned rat and human GCPII, and 88 nM for the activity in brain membranes of the null mutants. Keywords: N-acetylaspartylglutamate (NAAG), N-acetylaspartylglutamate-peptidase (NAAG-peptidase), glutamate carboxypeptidase II metabotropic glutamate receptor. 3
Biochemical correlates of GABA function in rat cortical neurons in culture
Brain Research, 1980
Key words: GABA --cortical neurons --neuron cell culture --[aH]muscimol binding SUMMARY Serial biochemical studies of a rat cortical tissue culture system in which synapses regularly form showed that ),-aminobutyric acid (GABA) is present in the cultures and increases with their maturation. The tissue GABA concentration in mature cultures is similar to that of adult rat cortex in vivo. The synthetic enzyme, glutamate decarboxylase, also increases with age as does high affinity GABA uptake. GABA uptake was blocked by L-2,4-diaminobutyrate (DABA) and had the properties of neuronal GABA uptake. Specific release by depolarizing media of both exogenous [aH]GABA and GABA synthesized from D-[U-14C]glucose was demonstrated. The GABA released by high potassium media had higher specific activity and a greater contribution from glucose (as compared to acetate) than GABA found in the medium in the absence of depolarization. Calcium dependency of evoked GABA release could be shown only after pretreatment of cultures with ethyleneglycol-bis-(fl-aminoethyl ether)-N,N'-tetraacetic acid or EGTA. Synaptosomes may exhibit greater calcium dependence of evoked transmitter release than intact cells in culture because their intracellular calcium stores are depleted during preparation. Glycine uptake by the cultures was much less in amount than was GABA uptake, and specific release of glycine could not be demonstrated.