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PubMed1996

Glutamic acid gamma-monohydroxamate and hydroxylamine are alternate substrates for Escherichia coli asparagine synthetase B.

Escherichia coli asparagine synthetase B (AS-B) catalyzes the synthesis of asparagine from aspartic acid and glutamine in an ATP-dependent reaction. The ability of this enzyme to employ hydroxylamine and L-glutamic acid gamma-monohydroxamate (LGH) as alternative substrates in place of ammonia and L-glutamine, respectively, has been investigated. The enzyme is able to function as an amidohydrolase, liberating hydroxylamine from LGH with high catalytic efficiency, as measured by k(cat)/K(M). In addition, the kinetic parameters determined for hydroxylamine in AS-B synthetase activity are very similar to those of ammonia. Nitrogen transfer from LGH to yield aspartic acid beta-monohydroxamate is also catalyzed by AS-B. While such an observation has been made for a few members of the trpG amidotransferase family, our results appear to be the first demonstration that nitrogen transfer can occur from glutamine analogs in a purF amidotransferase. However, k(cat)/K(M) for the ATP-dependent transfer of hydroxylamine from LGH to aspartic acid is reduced 3-fold relative to that for glutamine-dependent asparagine synthesis. Further, the AS-B mutant in which asparagine is replaced by alanine (N74A) can also use hydroxylamine as an alternate substrate to ammonia and catalyze the hydrolysis of LGH. The catalytic efficiencies (k(cat)/K(M)) of nitrogen transfer from LGH and L-glutamine to beta-aspartyl-AMP are almost identical for the N74A AS-B mutant. These observations support the proposal that Asn-74 plays a role in catalyzing glutamine-dependent nitrogen transfer. We interpret our kinetic data as further evidence against ammonia-mediated nitrogen transfer from glutamine in the purF amidotransferase AS-B. These results are consistent with two alternate chemical mechanisms that have been proposed for this reaction [Boehlein, S. K., Richards, N. G. J., Walworth, E. S., & Schuster, S. M. (1994) J. Biol. Chem. 269, 26789-26795].

Aspartate Aminotransferases↗
PubMed2003

Probing the stereochemistry of the active site of gamma-glutamyl transpeptidase using sulfur derivatives of l-glutamic acid.

Gamma-glutamyl transpeptidase (GGT) catalyses the transfer of a gamma-glutamyl moiety from a donor substrate to different acceptors, such as amino acids and water. GGT is known to display relatively low stereospecificity with respect to the alpha-stereocentre of its donor substrates. In this study we have studied its stereospecificity with respect to the stereocentre at the delta-position of different analogues of L-glutamic acid. Notably, L-methionine sulfoxide is well-recognised whereas L-methionine sulfone and L-methionine sulfoximine are not. Furthermore, when the synthetic gamma-diastereoisomers of L-methionine sulfoxide were separated and tested, it was discovered that GGT shows remarkable stereospecificity at the gamma-position, binding the S(C)S(S) diastereoisomer with a K(i) of 3.5 mM, whereas the S(C)R(S) diastereoisomer is not recognised. Finally, using a sulfoxide as a new pharmacophore for GGT, we have synthesized and tested an analogue of glutathione to obtain a very promising competitive inhibitor with a K(i) of (53 +/- 3) microM.

Animals↗
PubMed2006

Completely buried, non-ion-paired glutamic acid contributes favorably to the conformational stability of pyrrolidone carboxyl peptidases from hyperthermophiles.

Pyrrolidone carboxyl peptidases (PCPs) from hyperthermophiles have a structurally conserved and completely buried Glu192 in the hydrophobic core; in contrast, the corresponding residue in the mesophile protein is a hydrophobic residue, Ile. Does the buried ionizable residue contribute to stabilization or destabilization of hyperthermophile PCPs? To elucidate the role of the buried glutamic acid in stabilizing PCP from hyperthermophiles, we constructed five Glu192 mutants of PCP-0SH (C142S/C188S, Cys-free double mutant of PCP) from Pyrococcus furiosus and examined their thermal and pH-induced unfolding and crystal structures and compared them with those of PCP-0SH. The stabilities of apolar (E192A/I/V) and polar (E192D/Q) mutants were less than PCP-0SH at acidic pH values. In the alkaline region, the mutant proteins, except for E192D, were more stable than PCP-0SH. The thermal stability data and theoretical calculations indicated an apparent pKa value > or = 7.3 for Glu192. Present results confirmed that the protonated Glu192 in PCP-0SH forms strong hydrogen bonds with the carbonyl oxygen and peptide nitrogen of Pro168. New intermolecular hydrogen bonds in the E --> A/D mutants were formed by a water molecule introduced into the cavity created around position 192, whereas the hydrogen bonds disappeared in the E --> I/V mutants. Structure-based empirical stability of mutant proteins was in good agreement with the experimental results. The results indicated that (1) completely buried Glu192 contributes to the stabilization of PCP-0SH because of the formation of strong intramolecular hydrogen bonds and (2) the hydrogen bonds by the nonionized and buried Glu can contribute more than the burial of hydrophobic groups to the conformational stability of proteins.

Calorimetry, Differential Scanning↗
PubMed1998

Effects of glucose and glycerol on gamma-poly(glutamic acid) formation by Bacillus licheniformis ATCC 9945a.

Bacillus licheniformis ATCC 9945a is one of the bacterial strains that produce gamma-poly(glutamic acid) (gamma-PGA). The use of carbohydrate medium components for gamma-PGA production was explored. Cells were grown in shake flasks or in controlled pH fermentors using medium formulations that contain different carbon sources. During the cultivations, aliquots were removed to monitor cell growth, carbon utilization, polymer production, and polymer molecular weight. Glucose was a better carbon source than glycerol for cell growth. Furthermore, glucose was utilized at a faster rate than glycerol, citrate, or glutamate. However, by using mixtures of glucose and glycerol in medium formulations, the efficiency of gamma-PGA production increased. For example, by increasing the glycerol in medium formulations from 0 to 40 g/L, the gamma-PGA broth concentration after 96 h increased from 5.7 to 20.5 g/L. Considering that glycerol utilization was low for the glucose/glycerol mixtures studied, it was unclear as to the mechanism by which glycerol leads to enhanced product formation. Cell growth and concomitant gamma-PGA production (12 g/L) at pH 6.5 was possible using glucose as a carbon source if trace amounts (0.5 g/L each) of citrate and glutamate were present in the medium. We suggested that citrate and glutamate were useful in preventing salt precipitation from the medium. In addition, glutamate may be preferred relative to ammonium chloride as a nitrogen source. The conversion of glucose to gamma-PGA by the strain ATCC 9945a was believed to occur by glycolysis of glucose to acetyl-CoA and tricarboxylic acid (TCA) cycle intermediates that were then metabolized via the TCA cycle to form alpha-ketoglutarate, which is a direct glutamate precursor.

Bacillus↗
PubMed2005

Preparation and reactivity of aminoacyl pyroglutamates. Facile synthesis of 10-membered-ring cyclic dipeptides derived from 1,4-diaminobutyric and glutamic acids.

A number of protected proline-containing dipeptides Boc-Xaa-Pro-OBu(t) were converted via epimerization-free oxidation with RuO4 to dipeptides with an internal pyroglutamic acid residue, Boc-Xaa-Glp-OBu(t). The latter were subjected to oxidative Hoffman-type rearrangement induced by PhI[OC(O)CF3]2 to give N-(aminoacyl)-pyroglutamates. The behavior of these derivatives under basic conditions was studied, and for two such a derivatives an aminoacyl incorporation reaction was observed, producing otherwise poorly accessible 10-membered-ring dilactams derived from 1,4-diaminobutyric and glutamic acids in practicable yields.

Aminoacylation↗
PubMed2004

The last C-terminal residue of VP3, glutamic acid 257, controls capsid assembly of infectious bursal disease virus.

Infectious bursal disease virus (IBDV) is a nonenveloped virus with an icosahedral capsid composed of two proteins, VP2 and VP3, that derive from the processing of the polyprotein NH(2)-pVP2-VP4-VP3-COOH. The virion contains VP1, the viral polymerase, which is both free and covalently linked to the two double-stranded RNA (dsRNA) genomic segments. In this study, the virus assembly process was studied further with the baculovirus expression system. While expression of the wild-type polyprotein was not found to be self-sufficient to give rise to virus-like particles (VLPs), deletion or replacement of the five C-terminal residues of VP3 was observed to promote capsid assembly. Indeed, the single deletion of the C-terminal glutamic acid was sufficient to induce VLP formation. Moreover, fusion of various peptides or small proteins (a green fluorescent protein or a truncated form of ovalbumin) at the C terminus of VP3 also promoted capsid assembly, suggesting that assembly required screening of the negative charges at the C terminus of VP3. The fused polypeptides mimicked the effect of VP1, which interacts with VP3 to promote VLP assembly. The C-terminal segment of VP3 was found to contain two functional domains. While the very last five residues of VP3 mainly controlled both assembly and capsid architecture, the five preceding residues constituted the VP1 (and possibly the pVP2/VP2) binding domain. Finally, we showed that capsid formation is associated with VP2 maturation, demonstrating that the protease VP4 is involved in the virus assembly process.

Amino Acid Sequence↗
PubMed1997

Distribution and projection of the medullary cardiovascular control neurons containing glutamate, glutamic acid decarboxylase, tyrosine hydroxylase and phenylethanolamine N-methyltransferase in rats.

This study was aimed at showing the distribution and projection of the medullary cardiovascular control neurons that contain a standard neurotransmitter or a related enzyme in the rat. A small amount of HRP was injected into either the depressor area of the caudal ventrolateral medulla (D-CVLM) or the pressor area of the rostral ventrolateral medulla (P-RVLM). Using an immunohistochemical method, we identified HRP-labelled neurons which were stained with antiserum to glutamate (Glu), glutamic acid decarboxylase (GAD), tyrosine hydroxylase (TH) or phenylethanolamine N-methyltransferase (PNMT). Our findings are summarized as follows. (1) The Glu-containing neurons in the nucleus tractus solitararii (NTS) project to the D-CVLM (n = 279, 100% assumed as a standard value) and P-RVLM (n = 225, 81% against the standard), indicating divergent excitatory projection. (2) The GAD-containing neurons in the NTS (n = 74, 27% against the standard) project to the P-RVLM, indicating the convergent inhibitory projection. (3) The projections of the TH-containing neurons from the NTS (n = 19, 7% against the standard) and CVLM (n = 4, 1% against the standard) to the P-RVLM are weaker than those of the GAD-containing neurons, suggesting that the catecholaminergic neurons play a minor role in inhibition of the sympathetic activity of the P-RVLM neurons. These results suggest that the glutamatergic NTS neurons excite both the P-RVLM and D-CVLM neurons, and the gamma-aminobutyric acid (GABA)ergic NTS and CVLM neurons inhibit the sympathetic activity of the P-RVLM neurons.

Animals↗
PubMed2001

Use of L-[(15)N] glutamic acid and homoglutathione to determine both glutathione synthesis and concentration by gas chromatography-mass spectrometry (GCMS).

A method for simultaneous measurement of both glutathione enrichment and concentration in a biological sample using gas chromatography mass spectrometry is described. The method is based on the preparation of N,S-ethoxycarbonylmethyl ester derivatives of glutathione, and the use of homoglutathione (glutamyl-cysteinyl--alanine) as an internal standard. A procedure for determination of glutamate concentration and enrichment is also reported. Both methods have within-day and day-to-day inter-assay coefficients of variation less than 5%, and recoveries of known added amounts of glutathione and glutamate are close to 100%. Taken together, these methods allowed determination of glutathione concentration and fractional synthesis rate in red blood cells using L-[(15)N] glutamic acid infusion. This approach was applied in vivo to investigate the effects of a 72 h fast, compared with a control overnight fast, on erythrocyte glutathione in a single dog. The 72 h fast was associated with a 39% decline in erythrocyte glutathione level, (2.9 +/- 0.4 versus 4.7 +/- 0.5 mmol l(-1), fasting versus control) with no change in glutathione fractional synthesis (67.4 versus 71.3% d(-1), fasting versus control).

Animals↗
PubMed1998

Involvement of glutamic acid 278 in the redox reaction of the cytochrome c oxidase from Paracoccus denitrificans investigated by FTIR spectroscopy.

The molecular processes concomitant with the redox reactions of wild-type and mutant cytochrome c oxidase from Paracoccus denitrificans were analyzed by a combination of protein electrochemistry and Fourier transform infrared (FTIR) difference spectroscopy. Oxidized-minus-reduced FTIR difference spectra in the mid-infrared (4000-1000 cm-1) reflecting full or stepwise oxidation and reduction of the respective cofactor(s) were obtained. In the 1800-1000 cm-1 range, these FTIR difference spectra reflect changes of the polypeptide backbone geometry in the amide I (ca. 1620-1680 cm-1) and amide II (ca. 1560-1540 cm-1) region in response to the redox transition of the cofactor(s). In addition, several modes in the 1600-1200 cm-1 range can be tentatively attributed to heme modes. A peak at 1746 cm-1 associated with the oxidized form and a peak at 1734 cm-1 associated with the reduced form were previously discussed by us as proton transfer between Asp or Glu side chain modes in the course of the redox reaction of the enzyme [Hellwig, P., Rost, B., Kaiser, U., Ostermeier, C., Michel, H., and Mäntele, W. (1996) FEBS Lett. 385, 53-57]. These signals were resolved into several components associated with the oxidation of different cofactors. For a stepwise potential titration from the fully reduced state (-0.5 V) to the fully oxidized state (+0.5 V), a small component at 1738 cm-1 develops in the potential range of approximately +0.15 V and disappears at more positive potentials while the main component at 1746 cm-1 appears in the range of approximately +0.20 V (all potentials quoted vs Ag/AgCl/3 M KCl). This observation clearly indicates two different ionizable residues involved in redox-induced proton transfer. The major component at 1746 cm-1 is completely lost in the FTIR difference spectra of the Glu 278 Gln mutant enzyme. In the spectrum of the subunit I Glu 278 Asp mutant enzyme, the major component of the discussed difference band is lost. In contrast, the complete difference signal of the wild-type enzyme is preserved in the Asp 124 Asn, Asp 124 Ser, and Asp 399 Asn variants, which are critical residues in the discussed proton pump channel as suggested from structure and mutagenesis experiments. On the basis of these difference spectra of mutants, we present further evidence that glutamic acid 278 in subunit I is a crucial residue for the redox reaction. Potential titrations performed simultaneously for the IR and for the UV/VIS indicate that the signal related to Glu 278 is coupled to the electron transfer to/from heme a; however, additional involvement of CuB electron transfer cannot be excluded.

Asparagine↗
PubMedDate not supplied

[Accumulation of glutamic acid and glycine in human cerebral tumors in vitro].

Preliminary results are given on transport of glycine and L-glutamate into human cerebral tissue, normal and tumoral. In comparison with normal tissue, glycine transport is diminished in meningioma and oligodendroblastoma, unaffected in neurinoma, sharply increased in medulloblastoma. Glutamic acid transport is lowered in neurinoma and oligodendroblastoma; increased in medulloblastoma. Such preliminary observations are briefly discussed.

Brain↗
PubMed2000

Cholinergic endings on various neurones containing calcium binding proteins and glutamic acid decarboxylase in the hippocampus of the rat.

Immunohistochemical study of the cholinergic innervation of the hippocampal cells containing glutamic acid decarboxylase (GAD) and calcium binding proteins: parvalbumin (PV), calbindin D28k (CB) and calretinin (CR) was conducted on 5 adult rat brains. Analysis of sections with double immunostaining for vesicular acetylcholine transporter (VAChT; the marker of cholinergic cells, fibres and terminals) and respectively either GAD or PV, CB, CR, using confocal laser-scanning microscope shows that the intensive cholinergic innervations receive GAD, PV and CB-positive hippocampal cells. Cholinergic afferentiations of the CR-positive neurones are considerably fewer.

Acetylcholine↗
PubMed1985

Estrogen effects on nigral glutamic acid decarboxylase activity: a possible role for catecholestrogen.

Repeated but not single injections of estradiol benzoate significantly reduced nigral glutamic acid decarboxylase (GAD, EC 4.1.1.15). A single injection of the catecholestrogen 2-hydroxyestradiol produced similar results. Tolerance developed to the latter effect, as reflected by the lack of nigral GAD activity changes in rats repeatedly injected with 2-hydroxyestradiol. Repeated injection of the antiestrogen tamoxifen not only failed to antagonize the action of estradiol benzoate but itself reduced nigral GAD activity. Hypophysectomy, which itself decreased nigral GAD activity prevented the lowering effects of either repeated estradiol benzoate administration or single 2-hydroxyestradiol injection on the enzymatic activity.

Animals↗
PubMed1993

Hypothalamic angiotensin release in response to AII or glutamic acid stimulation of the SFO in rats.

Recent evidence from our laboratory suggests that angiotensin II (AII) is synthesized, stored within cells in the paraventricular nucleus (PVN) of the hypothalamus, and upon appropriate stimulation, released and rapidly converted to angiotensin III (AIII). The present investigation extends these observations by first employing a retrograde tracer to confirm a direct connection from the subfornical organ (SFO) to the PVN, and then showing that microinfusion of AII or glutamic acid into the SFO provokes release of endogenous angiotensin within the PVN. Potentially it is this release that contributes to the elevations in blood pressure and drinking that have been reported to occur with electrical and chemical stimulation of the SFO. These results represent the first evidence of releasable angiotensin provoked by the chemical activation of a neural pathway that has been histochemically demonstrated to link the SFO with the PVN and brain stem structures concerned with cardiovascular functioning.

Angiotensin II↗
PubMed1981

Effects of variations in glutamic acid decarboxylase activity on acute oxygen poisoning.

A study was made to test the influence of rapid variations in glutamic acid decarboxylase (GAD) activity on the susceptibility of rats to hyperbaric oxygen (HBO). GAD was inhibited by the convulsant drug unsymmetrical dimethylhydrazine (UDMH) and reactivated by pyridoxine (PYR) after onset of convulsive activity. There was a relatively long induction period after UDMH injection until the onset of convulsions and the predictable interictal periods between successive periodic convulsions made it possible to study the impact of variations in GAD activity on survival rates, suspectibility to HBO and brain glycogen levels in a time sequence after UDMH administration. The experiments showed that UDMH interferes with aerobic metabolism in brain in such a way that profound alterations in resistance to acute oxygen poisoning resulted. An accumulation of substrate proximal to the enzyme block is assumed to develop during UDMH poisoning. The protective effect against HBO toxicity that was achieved after reactivation of GAD by PYR injection, as well as the rapid re-establishment of glycogen levels, is believed to speak in favour of this hypothesis.

Animals↗
PubMed1996

Antibody to the M(r) 65,000 isoform of glutamic acid decarboxylase are detected in non-insulin-dependent diabetes in Japanese.

It was recently reported that antibodies against glutamic acid decarboxylase (GADAb) have a high positive predictive value for insulin-dependency in non-insulin-dependent diabetic (NIDDM) patients. We studied 289 patients classified at onset as having NIDDM. Patients positive for GAD65Ab had a disease onset at a younger age, lower body mass index (BMI) and lower serum C-peptide concentration, and were more often treated with insulin. Among 73 insulin-treated patients, patients with lower C-peptide level (n = 30, C-peptide < or = 0.9 ng/ml) showed a higher frequency of GAD65Ab (46.7%) than patients with normal C-peptide (n = 53, C-peptide > 0.9; 7.5%, P < 0.0001). The 206 remaining non-insulin-treated patients were divided into 48 short-duration (less than 5 years from diagnosis) and 158 long-duration patients. Frequency of GAD65Ab in short-duration patients (10.4%) was significantly higher than that in long-duration patients (3.2%, P < 0.05). Among short-duration patients, there was no significant difference in C-peptide levels between GAD65-positive and negative patients (2.175 and 2.226 ng/ml). In conclusion, GAD65Ab, a marker of insulin deficiency, may predict the development of insulin dependency in non-insulin-dependent Japanese diabetic patients before serum C-peptide concentration decreases.

Adolescent↗