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PubMed1998

Expression in Saccharomyces cerevisiae of antigenically and enzymatically active recombinant glutamic acid decarboxylase.

Glutamic acid decarboxylase (GAD) is one of the major autoantigens found in insulin-dependent (Type 1) diabetes mellitus (IDDM). A novel hybrid form of GAD was created by fusing amino acids 1-101 of the human GAD67 protein to amino acids 96-585 of the human GAD65 protein. This hybrid GAD67/65 was expressed constitutively under the control of the phosphoglycerate kinase promoter (PGK1) in the yeast Saccharomyces cerevisiae. Enzymatically active GAD was prepared from yeast lysates by a one-step purification on an affinity column using GAD-1 antibody. The purified hybrid GAD67/65 was radiolabelled with iodine-125 and tested in an immunoprecipitation assay with IDDM sera. Results obtained using the recombinant yeast hybrid GAD67/65 were very similar to those obtained using 125I-labelled porcine GAD. Recombinant yeast hybrid GAD67/65 should have utility for diagnosis and presymptomatic detection of IDDM.

Diabetes Mellitus, Type 1↗
PubMed1978

Variations among glyV-derived glycine tRNA suppressors of glutamic acid codons.

Glutamic acid codon suppressors in 18 isogenic strains of Escherichia coli have been further characterized as to map location, dominance, growth rates in various media, suppression of the GAG codon, and tRNA profiles after reversed-phase column chromatography. In general the evidence supports the conclusion that all of these suppressors are due to mutations in glyV55, the gene for a GGA/G-reading mutant form of glyV tRNA, and that they represent several different classes that may correspond to at least as many different nucleotide changes. Furthermore, 17 of the 18 suppressors can coexist in a haploid genome with a glyT suppressor that is devoid of GGA-reading ability. This result indicates the retention by those glyV suppressors of some ability to respond to GGA as well as the acquisition of the ability to read GAA, and suggests the possibility of "wobble" in the middle position of the anticodons of those tRNA's.

Chromosome Mapping↗
PubMed1994

Functional state of the beta cell affects expression of both forms of glutamic acid decarboxylase.

Glutamic acid decarboxylase (GAD) is a candidate target autoantigen involved in the pathogenesis of insulin-dependent diabetes mellitus (IDDM). The functional state of the beta cells has been suggested to play a pathogenic role in IDDM by altering beta-cell autoantigen expression. In this study, we investigated expression of GAD-65 and GAD-67 in isolated Sprague-Dawley rat islets cultured at different glucose concentrations. Using GAD isoform-specific antibodies in an immunoblot assay, we found that expression of both GAD-65 and GAD-67 in cultured islets was glucose dependent and that increased expression of both forms of GAD correlated with increased functional state of the beta cell. Our data indicate that the functional state of the beta cell influences islet cell expression of GAD. Thus, decreasing islet cell expression of GAD by suppressing beta cells activity may have a potential role in blunting the autoimmune destruction of pancreatic islet beta cells.

Animals↗
PubMed1993

Autoantibodies in IDDM primarily recognize the 65,000-M(r) rather than the 67,000-M(r) isoform of glutamic acid decarboxylase.

Glutamic acid decarboxylase autoantibodies may aid in rapid screening strategies predicting IDDM before clinical onset. Rat islets contain GAD65 and GAD67 autoantibody targets, but human islets express only GAD65, now confirmed by direct immunoprecipitation from radiolabeled rat and human islets. Because human IDDM involves beta-cell-specific autoimmunity, we tested 190 new IDDM patients and 51 healthy control subjects for antibodies to recombinant human islet GAD65, rat islet GAD67, or human insulinoma/cerebellum GAD67, each expressed separately in hamster fibroblasts. By using immunoprecipitation, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and densitometric fluorogram scanning, 132 of 190 (70%) of new IDDM patients had GAD65 autoantibodies, whereas only 17 of 190 (9%) had antibodies to rat GAD67 (P < 0.001). Of healthy control subjects, 2 of 51 (3.9%) and 1 of 51 (1.9%) had antibodies to GAD65 and GAD67, respectively. All 17 GAD67 antibody-positive patients also had GAD65 antibodies; 14 of 17 with greater GAD65 than GAD67 index. Control studies showed comparable reactivity between recombinant rat and human GAD67 and between different subcellular preparations of recombinant GAD67 of either species. In conclusion, only GAD65 is expressed in human islets, the autoantibody response is primarily to this isoform, and GAD67 antibodies add little to IDDM detection.

Adolescent↗
PubMed2001

Glutamic acid decarboxylase and glutamate receptor changes during tolerance and dependence to benzodiazepines.

Protracted administration of diazepam elicits tolerance, whereas discontinuation of treatment results in signs of dependence. Tolerance to the anticonvulsant action of diazepam is present in an early phase (6, 24, and 36 h) but disappears in a late phase (72-96 h) of withdrawal. In contrast, signs of dependence such as decrease in open-arm entries on an elevated plus-maze and increased susceptibility to pentylenetetrazol-induced seizures were apparent 96 h (but not 12, 24, or 48 h) after diazepam withdrawal. During the first 72 h of withdrawal, tolerance is associated with changes in the expression of GABA(A) (gamma-aminobutyric acid type A) receptor subunits (decrease in gamma(2) and alpha(1); increase in alpha(5)) and with an increase of mRNA expression of the most abundant form of glutamic acid decarboxylase (GAD), GAD(67). In contrast, dl-alpha-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA) receptor GluR1 subunit mRNA and cognate protein, which are normal during the early phase of diazepam withdrawal, increase by approximately 30% in cortex and hippocampus in association with the appearance of signs of dependence 96 h after diazepam withdrawal. Immunohistochemical studies of GluR1 subunit expression with gold-immunolabeling technique reveal that the increase of GluR1 subunit protein is localized to layer V pyramidal neurons and their apical dendrites in the cortex, and to pyramidal neurons and in their dendritic fields in hippocampus. The results suggest an involvement of GABA-mediated processes in the development and maintenance of tolerance to diazepam, whereas excitatory amino acid-related processes (presumably via AMPA receptors) may be involved in the expression of signs of dependence after withdrawal.

Animals↗
PubMed1996

Influence of photoinhibition on GABA and glutamic acid levels, and on glutamate decarboxylase activity in the testis and epididymis of the golden hamster.

Gamma-aminobutyric acid (GABA) is found in the gonads and accessory reproductive organs, and a direct effect on steroidogenesis and sperm viability and motility has been described. The golden (Syrian) hamster is a seasonal breeder, and a pattern of regression-recrudescence in their reproductive organs is observed when adult animals are exposed to less than 12.5 h daylight for an extended period of time. The purpose of this study was to investigate: (1) the presence of GABA in the testis and epididymis of golden hamsters undergoing regression and spontaneous recrudescence; (2) glutamic acid levels and glutamate decarboxylase (GAD) activity in both tissues, and (3) testicular and epididymal testosterone, dihydrotestosterone and 5 alpha-androstane-3 alpha, 17 beta-diol concentrations. Adult golden hamsters were exposed to long (LP 14L:10D) or short (SP 6L:18D) photoperiods for 9, 12, 14, 16, 18 or 22 weeks. When animals were exposed to SP for 14-16 weeks, the testis and epididymis reached maximal involution. Testicular and epididymal androgen levels showed a marked decrease (p < 0.05) during the regression period, and after 18-22 weeks, values began to recover. Between 12 and 18 weeks in SP, the testicular and epididymal content of GABA and glutamic acid was reduced significantly. The concentration of GABA in both tissues showed a sharp rise (p < 0.05), while the concentration of glutamic acid diminished during the period of maximal involution (p < 0.05). In the testis, GAD activity was increased (p < 0.001) after 14 weeks in SP, with no change in the epididymis. In conclusion, glutamic acid via GAD activity could be the main source of GABA in the testis, but not in the epididymis. Furthermore, the presence of GABA in testicular cells and its subsequent photoperiodic variations might act as an important autocrine and/or paracrine modulatory signal in gonadal processes.

Androgens↗
PubMed1978

Ranking of excitatory amino acids by the antagonists glutamic acid diethylester and D-alpha-aminoadipic acid.

A separation of the excitatory actions of the amino acids upon thalamic neurones of rats anaesthetized with urethane has been accomplished through the use of two antagonists. It has been possible to rank the excitatory compounds in their order of susceptibility to D-alpha-aminoadipic acid (DalphaAA) and L-glutamic acid diethylester (GDEE). The observation that the ranking orders of the excitants differ for these two antagonists permits an analysis of the types of receptors with which the amino acid excitants react. The results support the proposition that more than one neuronal receptor sensitive to the amino acids exists.

2-Aminoadipic Acid↗
PubMed2006

Trigeminovestibular and trigeminospinal pathways in rats: retrograde tracing compared with glutamic acid decarboxylase and glutamate immunohistochemistry.

This study identified neurons in the sensory trigeminal complex with connections to the medial (MVN), inferior (IVN), lateral (LVN), and superior (SVN) vestibular nuclei or the spinal cord. Trigeminovestibular and trigeminospinal neurons were localized by injection of retrograde tracers. Immunohistochemical processing revealed gamma-aminobutyric acid (GABA)- and glutamate-containing neurons in these two populations. Trigeminovestibular neurons projecting to the MVN and the IVN were in the caudal principal nucleus (5P), pars oralis (5o), interpolaris (5i), and caudalis (5c) and scattered throughout the rostral 5P. Projections were bilateral to the IVN, with an ipsilateral dominance to the MVN, except from the rostral 5P, which was contralateral. Neurons projecting to the LVN were numerous in the ventral caudal 5P and the 5o and less abundant in the rostral 5P, 5i, and 5c. Our results suggested that only 5P and 5o project to the dorsal LVN. Neurons projecting to the SVN were in the dorsal 5P, 5o, and 5i but not in 5c. Trigeminospinal neurons were mainly in the ventral 5o and 5i and in the lateral 5c, rarely or never in 5P. Among trigeminovestibular neurons, most of the somas were immunoreactive for glutamate, but some reacted for GABA. Among trigeminospinal neurons, the number of somas immunoreactive for each of the two amino acids was similar. Trigeminal terminals were observed in contact with vestibulospinal neurons in the IVN and LVN, giving evidence of a trigeminovestibulospinal pathway. Therefore, inhibitory and excitatory facial inputs may contribute through trigeminospinal or trigeminovestibulospinal pathways to the control of head/neck movements.

Animals↗
PubMed1994

Insulinotropic action of glutamic acid dimethyl ester.

Glutamic acid dimethyl ester (GME; 3.0-10.0 mM) enhanced insulin release evoked by 6.0-8.3 mM D-glucose, 1.0-10.0 mM L-leucine, or 5.0-10.0 mM 2-amino-bicyclo(2,2,1)heptane-2-carboxylic acid, causing a shift to the left of the sigmoidal relationship between insulin output and D-glucose concentration. In the absence of D-glucose, GME also unmasked the insulinotropic potential of glibenclamide. In islets exposed to L-leucine, the insulinotropic action of GME coincided with an early fall and later increase in 86Rb outflow and augmentation of 45Ca outflow from prelabeled islets. The measurement of O2 uptake, NH4+ output, production of 14CO2 from islets prelabeled with [U-14C]palmitate, generation of 14C-labeled amino acids and 14CO2 from the dimethyl ester of either L-[1-14C]glutamic acid or L-[U-14C]glutamic acid, and D-[2-14C]glucose as well as D-[6-14C]glucose oxidation in the presence or absence of GME indicated that the latter ester was efficiently converted to L-glutamate and its further metabolites. The overall gain in O2 uptake represented the balance between GME oxidation and its sparing action on the catabolism of endogenous fatty acids and exogenous D-glucose. It is proposed that GME might represent a new tool to bypass beta-cell defects in D-glucose transport, phosphorylation, and further metabolism and, hence, to stimulate insulin release in experiments conducted in animal models of non-insulin-dependent diabetes mellitus.

Animals↗
PubMed1978

Action of glutamic acid and of some glutamate analogues on the molluscan central neurones.

The effects of L-glutamic acid and of some glutamate analogues have been studied on the central nervous system of the snail Heobania vermiculata, using conventional electrophysiological techniques. The glutamate H-response had the mean equilibrium value of -(57 +/- 4) mV and was associated with a Cl- conductance change. The D-response to glutamate application showed an involvement of sodium ions. Aspartate was agonist of glutamate action and displayed similar equilibrium value of the H-response, whereas quisqualate H-response was 'non-invertible'.

Animals↗
PubMed1985

Sequencing of an active-site peptide of angiotensin I-converting enzyme containing an essential glutamic acid residue.

A glutamic acid residue at the active site of bovine lung angiotensin I-converting enzyme, a zinc-metallo peptidyl dipeptidase, was esterified with p-[N,N-bis(chloroethyl)amino]phenylbutyryl-L-[U-14C]proline (chlorambucyl-L-[U-14C]-L-proline), an affinity label for this enzyme (Harris, R.B., and Wilson, I.B. (1983) J. Biol. Chem. 258, 1357-1362). The radiolabeled enzyme was digested with BrCN and only 1 of the 30 cleavage peptides resolved by reverse-phase high performance liquid chromatography (HPLC) contained the bound radiolabel. This active-site peptide (Mr = 16,000) was digested with trypsin and the labeled peptide formed (T-2) was further degraded with thermolysin. The thermolytic peptides were resolved by reverse-phase HPLC. Only 1 of the 5 peptides obtained (Th-1, Mr = 1290) contained the bound radiolabel. Th-1 (12 residues) was subjected to manual Edman degradation and the following partial sequence was determined: H2N-Phe-Thr-Glu-Leu-Ala-Asp-Ser-Glu... The radiolabel was released at cycle 3 and the amount recovered was equivalent to the amount of phenylthiohydantoin-Glu detected on HPLC. Thus, glutamic acid is esterified with chlorambucyl-L-[U-14C]proline in confirmation of our earlier findings. The sequence determined is homologous in 5 residues with the corresponding sequences of bovine carboxypeptidase A and B, two other mammalian zinc proteases. There is little sequence homology with thermolysin, a bacterial zinc protease that also contains an essential active-site glutamic acid residue.

Amino Acid Sequence↗
PubMedDate not supplied

On the mechanism of enhanced ATP formation in hypoxic myocardium caused by glutamic acid.

The effect of glutamic acid on the cardiac contractile function and sources of anaerobic ATP formation in hypoxic myocardium was studied in isovolumic rat hearts. The presence of glutamic acid (5 mM) in the perfusate significantly diminished an increment in diastolic pressure caused by 60 min hypoxia, and facilitated its complete recovery during 30 min reoxygenation. This effect was combined with the maintenance of a higher ATP level during hypoxia and reoxygenation. The total content of lactate in the heart-perfusate system rose exactly as during hypoxia without glutamic acid, while pyruvate content decreased due to increased alanine formation. Restoration of tissue content of glutamate and aspartate in the presence of exogenous glutamic acid was accompanied by a more than 2-fold increase in succinate formation, the end-product of the Krebs' cycle under anaerobic conditions. The products of glutamic acid transamination with oxaloacetic acid, aspartic and alpha-ketoglutaric acids (5mM each), induced the same functional and metabolic alterations as glutamic acid. Amino-oxyacetic acid, a tramsaminase inhibitor, eliminated the effects caused by glutamic acid. Moreover, the inhibition of transamination was accompanied by a decreased succinate and alanine synthesis as well as insignificantly increased lactate formation compared to hypoxia without additives. The results suggest that the beneficial effect of glutamic acid is due to the activation of anaerobic ATP formation in the mitochondria rather than stimulation of glycolysis.

Adenosine Triphosphate↗
PubMed2005

Synthesis of N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid as dual inhibitors of dihydrofolate reductase and thymidylate synthase and as potential antitumor agents.

Two novel classical antifolates N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 3 and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic acid 4 were designed, synthesized, and evaluated as antitumor agents. Compounds 3 and 4 were obtained from 2,4-diamino-5-methylpyrrolo[2,3-d]pyrimidine 7 and 2-amino-4-oxo-5-methylpyrrolo[2,3-d]pyrimidine 12, respectively, in a concise three-step sequence. Compound 3 is the first example, to our knowledge, of a 2,4-diamino classical antifolate that has potent inhibitory activity against both human dihydrofolate reductase (DHFR) and human thymidylate synthase (TS). Compound 4 was a dual DHFR-TS inhibitor against the bifunctional enzyme derived from Toxoplasma gondii (tg). Further evaluation of the mechanism of action of 3 implicated DHFR as its primary intracellular target. Both 3 and 4 were folylpolyglutamate synthetase (FPGS) substrates. Compound 3 also inhibited the growth of several human tumor cell lines in culture with GI50 < 10(-8) M. This study shows that the pyrrolo[2,3-d]pyrimidine scaffold is conducive to dual DHFR-TS and tumor inhibitory activity, and the potency is determined by the 4-position substituent.

Animals↗
PubMed1981

Ibotenic acid analogues as inhibitors of [3H]glutamic acid binding to cerebellar membranes.

The L-[3H]glutamic acid binding capability of rat cerebellar membranes prepared with or without preincubation at 37 degrees C followed by washing was investigated. The two preparations (KD = 820 nM, Bmax = 54.5 pmol/mg protein; KD = 509 nM, Bmax = 13.0 pmol/mg protein) showed no difference in specificity of the binding of the ibotenic acid analogues, consistent with the removal of an endogenous inhibitor by the preincubation at 37 degrees C followed by washing. The order of potency of the ibotenic acid analogues as inhibitors of L-[3H]glutamic acid binding is different from the order of potency in vivo, suggesting that the binding sites found are different from the physiological glutamic acid receptor.

Animals↗
PubMed1995

Targeting of the 67-kDa isoform of glutamic acid decarboxylase to intracellular organelles is mediated by its interaction with the NH2-terminal region of the 65-kDa isoform of glutamic acid decarboxylase.

The two isoforms of glutamic acid decarboxylase (GAD), GAD67 and GAD65, synthesize the neurotransmitter gamma-aminobutyric acid in neurons and pancreatic beta-cells. Previous studies suggest that GAD67 is a soluble cytosolic protein, whereas GAD65 is membrane-associated. Here, we study the intracellular distribution of GAD67 in neurons, pancreatic beta-cells, and fibroblasts transfected either with GAD65 and GAD67 together or with GAD67 alone. Neuronal GAD67 is partially recovered with GAD65 in membrane-containing pellet fractions and Triton X-114 detergent phases. The two proteins co-immunoprecipitate from extracts of brain and GAD65-GAD67 co-transfected fibroblasts, but not when extracts of GAD65 and GAD67 transfected fibroblasts were mixed and used as a starting material for immunoprecipitation. GAD67 is concentrated in the Golgi complex region in GAD65-GAD67 co-transfected fibroblasts, but not in fibroblasts transfected with GAD67 alone. A pool of neuronal GAD67 co-localizes with GAD65 in the Golgi complex region and in many synapses. The two proteins also co-localize in the perinuclear region of some pancreatic beta-cells. GAD67 interacts with the NH2-terminal region of GAD65, even in the absence of palmitoylation of this region of GAD65. Taken together, our results indicate that GAD65-GAD67 association occurs in vivo and is required for the targeting of GAD67 to membranes.

Animals↗