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At least 19 recordsLinked to original sources
PubMed1978

Glutamate transport in pig heart mitochondria. Binding and structural properties of high-glutamate affinity proteolipid: reconstitution studies.

Previously, a proteolipid that can bind glutamate with high affinity has been isolated from pig heart mitochondrial membranes. A final affinity chromatography on gamma-methylglutamate-albumin coreticulated on glass fiber was necessary. This procedure includes long dialysis steps which tend to denature the high-glutamate affinity proteolipid. Here is described a new method of isolation which avoids long dialysis steps and yields greater amounts of the high-glutamate affinity proteolipid. The binding of glutamate or aspartate on high-glutamate affinity proteolipid has been studied by gel filtration, by equilibrium dialysis or by a new procedure of rapid centrifugation based on the insolubility of high-glutamate affinity proteolipid in water. The latter method permits the detection of low and high affinity sites for glutamate with a Kd 60 mM and 55 muM, respectively. Among a series of analogues, aspartate appeared to be the best competitor: Kd = 30 muM and two Ki values, 0.37 mM (at high glutamate concentration) and 3.8 muM (at low glutamate concentration). High-glutamate affinity proteolipid binds 0.4 nmol of glutamate but only 0.1 nmol of aspartate per mg protein. The sites for glutamate and aspartate appear to be different but interdependent. In the presence of high-glutamate affinity proteolipid, externally added glutamate stimulated the efflux of aspartate from preloaded liposomes. High-glutamate affinity proteolipid contains cardiolipin, phosphatidyl choline and phosphatidyl ethanolamine the distribution of which is different from that of the inner membrane. The effects of various phospholipases, trypsin, and thiol reagents were studied on the binding of glutamate. High-glutamate affinity proteolipid binds 9 nmol N-ethylmaleimide per mg protein but only 6.1 nmol in the presence of glutamate. The dissociation of high-glutamate affinity proteolipid caused by thiol reagents yielded a soluble protein fraction with higher affinity for glutamate. Electrophoresis and an immunological approach allowed the detection and titration of the glutamate dehydrogenase and aspartate aminotransferase present in high-glutamate affinity proteolipid in inhibited forms, the latter being 26-fold more concentrated than the former.

Animals↗
PubMed1978

Glutamate metabolism in relation to glutamate transport in kidney cortex mitochondria of rabbit.

1. The metabolism of glutamate was followed by measurements of phosphoenolpyruvate production, aspartate synthesis and ammonia release, whereas the transport of glutamate across the inner membrane of kidney cortex mitochondria was studied using an oxygen electrode and the swelling technique. 2. When added separately, avenaciolide and aminooxyacetate only partially inhibited both State 3 and uncoupled respiration of the mitochondria, as studied in the presence of glutamate as substrate. In contrast, the addition of both inhibitors to the reaction medium resulted in an almost complete inhibition of glutamate oxidation. 3. Swelling of kidney mitochondria in an isosmotic solution of ammonium glutamate was accelerated by uncoupler and inhibited by avenaciolide, while the swelling of mitochondria in potassium glutamate was stimulated by valinomycin and inhibited by uncoupler. 4. When glutamate was used as the sole substrate, inhibition of aspartate formation by aminooxyacetate resulted in a stimulation of both ammonia release and phosphoenolpyruvate production. In contrast, with glutamate plus malate as substrate an elevation of the rate of glutamate deamination on the addition of aminooxyacetate was accompanied by an inhibition of phosphoenolpyruvate synthesis in both State 3 and uncoupled conditions. 5. In the presence of valinomycin to induce K+-permeability a marked enhancement of glutamate deamination was accompanied by a significant inhibition of glutamate transamination. 6. Based on the presented results it was concluded that in rabbit renal mitochondria utilizing glutamate as substrate the rate of ammonia production, phosphoenolpyruvate formation and aspartate synthesis vary in response to different metabolic conditions, in which both the glutamate--H+ symport and the glutamate--aspartate exchange systems are functioning to different extents.

Aminooxyacetic Acid↗
PubMed1976

Effect of growth conditions on glutamate transport in the wild-type strain and glutamate-utilizing mutants of Escherichia coli.

The effects of growth conditions on the glutamate transport activity of intact cells and membrane vesicles and on the levels of glutamate-binding protein in wild-type Escherichia coli K-12 CS101 and in two glutamate-utilizing mutants, CS7 and CS2TC, were studied. Growth of CS101 on aspartate as the sole source of carbon or nitrogen resulted in a severalfold increase in glutamate transport activity of intact cells and membrane preparations to levels characteristic of the operator-constitutive mutant CS7. The high glutamate transport activity of mutant CS7 was not depressed further by growth on aspartate. Synthesis of glutamate-binding protein was not enhanced by aspartate in either strain. Mutant CS2TC produces a heat-labile repressor of glutamate permease synthesis and is therefore able to grow on glutamate at 42 C but not at 30 C. CS2TC cells grown in a glycerol-minimal medium at the restrictive temperature (30 C) exhibit low glutamate transport activity. Growth on aspartate at 30 C results in derepressed synthesis of glutamate permease. Cells grown on glycerol at 42 C have high glutamate transport activity. No further derepression is obtained upon growth on aspartate. Growth of CS101 and CS7 in "rich broth" greatly reduces the levels of glutamate-binding protein but does not appreciably affect glutamate transport by whole cells or membrane preparations. The identity of the carrier and the role of the binding protein in glutamate transport are discussed in the light of these findings.

Aspartic Acid↗
PubMed1978

Purification and properties of glutamate synthase and glutamate dehydrogenase from Bacillus megaterium.

Bacillus megaterium N.C.T.C. no. 10342 exhibits glutamate synthetase (EC 2.6.1.53) and glutamate dehydrogenase (EC 1.4.1.4) activities. Concentrations of glutamate synthase were high when the bacteria were grown on 3mM-NH4Cl and low when they were grown on 100mM-NH4Cl, whereas glutamate dehydrogenase concentrations were higher when the bacteria were grown on 100mM-NH4Cl than on 3mM-NH4Cl. Glutamate synthase and glutamate dehydrogenase were purified to homogeneity from B. megaterium grown in 10mM-glucose/10mM-NH4Cl. The purified enzymes had mol.wts. 840000 and 270000 for glutamate synthase and glutamate dehydrogenase respectively. The Km values for substrates with NADPH and coenzyme were (glutamate synthase activity shown first) 9 micron and 360 micron for 2-oxoglutarate, 7.1 micron and 8.7 micron for NADPH, and 0.2 mM for glutamine and 22 mM for NH4Cl, similar values to those of enzymes from Escherichia coli. Glutamate synthase contained NH3-dependent activity (different from authentic glutamate dehydrogenase), which was enhanced 4-fold during treatment at pH 4.6 NH3-dependent activity was generally about 2% of the glutamine-dependent activity. Amidination of glutamate synthase by the bi-functional cross-linking reagent dimethyl suberimidate inactivated glutamine-dependent glutamate synthase activity, but increased NH3-dependent activity. A cross-linked structure of mol.wt. approx 200000 was the main product formed.

Amino Acids↗
PubMed1978

The absorption by human volunteers of glutamic acid from monosodium glutamate and from a partial enzymic hydrolysate of casein.

Peripheral plasma concentrations of glutamic and aspartic acids and alanine were measured after ingestion of monosodium glutamate or a pancreatic hydrolysate of casein by human volunteers. The doses of each material were such that they contained similar amounts of glutamic acid. Plasma glutamic acid concentrations rose promptly after the monosodium glutamate but mean peak concentrations were well below those likely to cause neurological damage. Plasma aspartic acid concentrations also rose after the monosodium glutamate but the behaviour of plasma alanine concentrations suggested that intestinal transamination of glutamic acid was insufficient to cause an appreciable rise in alanine concentration in the peripheral plasma. Significant increments in plasma glutamic acid concentrations did not occur after the pancreatic hydrolysate of casein and it is probable that competition for absorptive mechanisms by other amino acids, both free and peptide-bound, causes absorption of glutamic acid to be slower from mixtures of peptides and amino acids than from monosodium glutamate itself.

Absorption↗
PubMed1978

Enzymes of the glutamate and aspartate synthetic pathways in a glutamate-producing bacterium, Brevibacterium flavum.

Glutamate-auxotrophic mutants lacking phosphoenolpyruvate carboxylase(PC), citrate synthase (CS) or glutamate dehydrogenase (GD), an aspartate auxotroph lacking aspartate aminotransferase (TA), and a glutamate-aspartate double auxotroph lacking both aconitase (AH) and TA were obtained from Brevibacterium flavum No. 2247, a glutamate-producing bacterium. Prototrophic revertants further derived from the CS- and GD-lacking auxotrophs concomitantly recovered the enzyme activities that their parents had lost. These results indicate involvement of the tricarboxylic acid (TCA) cycle and GD in glutamate biosynthesis, that of PC in the biosynthesis of the TCA cycle intermediates and that of TA in aspartate biosynthesis. The CS-deficient mutants accumulated large amounts of acetate and small amounts of pyruvate, aspartate and alanine, while the GD-deficient strains accumulated large amounts of 2-oxo-glutarate and small amounts of citrate. Synthesis of PC was repressed by either glutamate or aspartate and those of CS and GD were repressed by glutamate, whereas those of pyruvate dehydrogenase (PD), AH, and isocitrate dehydrogenase were not affected significantly by glutamate; that of TA was also not affected by aspartate or by glutamate. The specific activities of PD and AH gave peaks during the cellular cultivation, related to the temporary accumulation of their substrates, pyruvate and citrate, respectively. These and previous results on the regulation of the enzymatic activities provide a definite regulatory mechanism for glutamate and aspartate syntheses.

Aconitate Hydratase↗
PubMed1979

Glutamate as a CNS transmitter. I. Evaluation of glucose and glutamine as precursors for the synthesis of preferentially released glutamate.

Slices of the molecular layer of the dentate gyrus of the hippocampal formation were incubated with either [14C]glucose, [14C]pyruvate or 14C glutamine and the efflux of endogenous and radioactive glutamate was monitored under various conditions. After prelabeling with either [14C]glutamine or [14C]glucose elevation of K+ concentration to 56 mM (Ca2+ free) increased efflux of endogenous and [14C]glutamate. Introduction of Ca2+ into the elevated K+ medium further increased the efflux of endogenous glutamate and radioactive glutamate derived from any of the precursors tested. In glutamine containing media, the increase in glutamate efflux as well as basal efflux was considerably higher than in the absence of glutamine and the specific activity of glutamate release was higher than that in tissue. Thus glutamine was superior to glucose or pyruvate as precursor and most specifically labeled the putative transmitter pool of glutamate. Similar experiments were carried out 4 and 14 days after a unilateral lesion in the entorhinal cortex which provides about 60% of the total synaptic input to the dentate granule cells. The Ca2+ dependent release of glutamate derived from either glucose or glutamine was markedly reduced on the operated side. This result suggests that the transmitter pool of glutamate is in perforant path terminals and can be synthesized from glucose or glutamine.

Animals↗
PubMed1979

Serum glutamic oxalacetic transaminase, glutamic pyruvic transaminase, gamma-glutamyl transpeptidase and glutamic dehydrogenase levels in favism.

Serum GOT, GPT, gamma-GT and GLDH were determined in 15 G-6-PD-deficient subjects during a hemolytic crisis and hemoglobinuria due to ingestion of fresh fava beans. The same G-6-PD-deficient subjects were studied again 2 months after the crisis, when they were asymptomatic. 15 normal healthy children served as controls. A statistically significant increase, above normal, in serum GOT, GLDH and gamma-GT was observed in the favic subjects during the crisis. All the values reverted to normal in the asymptomatic period.

Alanine Transaminase↗
PubMed1976

Glutamate transport in membrane vesicles of the wild-type strain and glutamate-utilizing mutants of Escherichia coli.

A highly specific energy-dependent glutamate transport system was demonstrated in membrane vesicles of glutamate-utilizing Escherichia coli K-12 mutants. The glutamate transport activity of membranes from the parent strain, unable to grow on glutamate, was very low. With ascorbate-phenazine methosulfate as the electron donor, mutant preparations displayed 17 to 20 times higher activity than did the wild type. However, the affinity of the mutant carrier for L-glutamate remained the same as in the parent strain. Comparative inhibition analysis of glutamate transport in whole cells and membrane vesicles and of in vitro binding of glutamate to a specific periplasmic-binding protein suggests that under certain conditions the latter may be a component of the E. coli K-12 glutamate transport system.

Amino Acids↗
PubMed1976

The interaction of four putative glutamate antagonists with glutamate and their effects on the toad spinal cord.

1. Four putative glutamate antagonists (L-glutamate diethyl ester, L-glutamate dimethyl ester, L-proline and 1-hydroxy-3-amino-pyrrolidone-2) were tested on the isolated hemisected toad spinal cord. 2. 1-hydroxy-3-amino-pyrrolidone-2 (10(-3)-10(-2) M) selectively antagonized the depolarizations evoked in both dorsal and ventral roots by applications of L-glutamate (5 X 10(-4) M). 3 1-hydroxy-3-amino-pyrrolidone-2 also antagonized the depolarizations evoked in both dorsal and ventral roots by stimulation of the adjacent dorsal root. 4. The dimethyl and diethyl esters of L-glutamate and L-proline had their own depolarizing actions on the dorsal and ventral roots, and neither potentiated nor antagonized the effects of L-glutamate. 5. The results with 1-hydroxy-3-amino-pyrrolidone-2 offer further evidence for the involvement of L-glutamate and L-aspartate in synaptic transmission in the amphibian spinal cord.

Animals↗
PubMed1977

Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. I. Subcellular localization, biosynthesis, and intracellular translocation of glutamate dehydrogenase.

1. The presence of glutamate dehydrogenase in the microsomal fraction of rat liver was confirmed. The identities of mitochondrial and microsomal glutamate dehydrogenases were proved by immunochemical methods and by SDS polyacrylamide gel electrophoresis of purified enzymes. 2. Synthesis of glutamate dehydrogenase by the membrane-bound ribosomes of rough endoplasmic reticulum was determined. Newly synthesized enzyme molecules were discharged on the cytoplasmic surface of endoplasmic reticulum membranes. 3. A precursor-product relationship was found between microsomal and mitochondrial glutamate dehydrogenases. About six hours were needed for the transport of glutamate dehydrogenase from the site of synthesis to mitochondria. 4. The half-life of glutamate dehydrogenase was about 5.5 days, which was somewhat longer than that of mitochondrial total protein determined in the same experiment. 5. Mitochondrial-type malate dehydrogenase was also present in the microsomal fraction. Subfractionation of smooth microsomes revealed the existence of particular light microsomal vesicles in which both glutamate dehydrogenase and malate dehydrogenase were concentrated. These vesicles may participate in intracellular transport of matrix enzymes from microsomes to mitochondria.

Animals↗
PubMed1977

Biogenesis of the mitochondrial matrix enzyme, glutamate dehydrogenase, in rat liver cells. II. Significance of binding of glutamate dehydrogenase to microsomal membrane.

1. Glutamate dehydrogenase and malate dehydrogenase solubilized from liver microsomes were able to rebind to microsomal vesicles while the corresponding dehydrogenases extracted from mitochondria showed no affinity for microsomes. 2. Competition was noticed between microsomal glutamate dehydrogenase and microsomal malate dehydrogenase in the binding to microsomal membranes. Mitochondrial malate dehydrogenase or bovine serum albumin did not inhibit the binding of microsomal glutamate dehydrogenase to microsomes. 3. Binding of microsomal glutamate dehydrogenase to microsomal membranes decreased when microsomes was preincubated with trypsin. 4. Rough microsomal glutamate dehydrogenase was more efficiently bound to rough microsomes than smooth microsomes. Conversely, smooth microsomal glutamate dehydrogenase had higher affinity for smooth microsomes than for rough microsomes. 5. A difference was noticed among the glutamate dehydrogenase isolated from rough and smooth microsomes, and from mitochondria, which suggested the possibility of minor post-translational modification of enzyme molecules in the transport from the site of synthesis to mitochondria.

Animals↗
PubMed1978

Inactivation of glutamate dehydrogenase and glutamate synthase from Bacillus megaterium by phenylglyoxal, butane-2,3-dione and pyridoxal 5'-phosphate.

Reaction of phenylglyoxal with glutamate dehydrogenase (EC 1.4.1.4), but not with glutamate synthase (EC 2.6.1.53), from Bacillus megaterium resulted in complete loss of enzyme activity. NADPH alone or together with 2-oxoglutarate provided substantial protection from inactivation by phenylglyoxal. Some 2mol of [14C]Phenylglyoxal was incorporated/mol of subunit of glutamate dehydrogenase. Addition of 1mM-NADPH decreased incorporation by 0.7mol. The Ki for phenylglyoxal was 6.7mM and Ks for competition with NADPH was 0.5mM. Complete inactivation of glutamate dehydrogenase by butane-2,3-dione was estimated by extrapolation to result from the loss of 3 of the 19 arginine residues/subunit. NADPH, but not NADH, provided almost complete protection against inactivation. Butane-2,3-dione had only a slight inactivating effect on glutamate synthase. The data suggest that an essential arginine residue may be involved in the binding of NADPH to glutamate dehydrogenase. The enzymes were inactivated by pyridoxal 5'-phosphate and this inactivation increased 3--4-fold in the borate buffer. NADPH completely prevented inactivation by pyridoxal 5'-phosphate.

Aldehydes↗
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↗
PubMed1979

Effect of ammonia on the glutamate dehydrogenase catalyzed oxidative deamination of L-glutamate. The steady state.

Ammonia is known to inhibit the steady-state rate of oxidation of L-glutamate catalyzed by glutamate dehydrogenase. We reported previously [Brown, A., Colen, A. H., & Fisher, H. F. (1978) Biochemistry 17, 2031] kinetic evidence supporting the formation in the initial rapid phase of a complex which is composed of enzyme, reduced coenzyme, alpha-ketoglutarate, and ammonia. We show here that the effects of ammonia on the steady-state reaction can be correlated with transient-state kinetic effects related to the concentration of that ammonia-containing complex. These results indicate the existence of alternate reaction pathways which become important at high ammonia concentrations. These new pathways provide an additional route for the release of NADPH from the enzyme surface. The expanded mechanism shows that the noncompetitive product inhibition by ammonia can occur without the simultaneous presence of ammonia and L-glutamate on the enzyme. This mechanism also accommodates the observed substrate inhibition by L-glutamate.

Ammonia↗
PubMed1977

Identification of glutamate chain lengths of endogenous folylpoly-gamma-glutamates in rat tissues.

A simplified procedure for the determination of the glutamate chain lengths of endogenous tissue folate is described. Natural pteroylpoly-gamma-glutamates in tissue extracts, irrespective of their one-carbon moiety, were converted by a reductive cleavage procedure to a homologous series of p-aminobenzoylpoly-gamma-glutamates, differing only in glutamate chain length. Desalting and concentration of the extracts were achieved by absorbing the derivatives on active charcoal followed by their elution with ethanol:ammonia. Aminobenzoylpolyglutamates were separated by DEAD-cellulose chromatography and quantitated by a colorimetric procedure for primary aromatic amines. The major endogenous folates in rat liver and kidney were pteroylpentaglutamate derivatives with smaller amounts of pteroyltetra- and hexaglutamate.

Animals↗
PubMedDate not supplied

Helix-coil stability constants for the naturally occurring amino acids in water. IX. Glutamic acid parameters from random poly(hydroxybutylglutamine-co-L-glutamic acid).

The synthesis and characterization of water-soluble random copolymers containing L-glutamic acid with N5-(4-hydroxybutyl)-L-glutamine and the thermally induced helix-coil transitions of these copolymers in water and in 0.1 N KCl are described. The incorporation of L-glutamic acid was found to increase the helix content of the polymer at low pH and to decrease it at high pH even though the presence of 0.1 N KCl effectively eliminated the difference between the electrostatic free energies of the helix and the coil. The Zimm-Bragg parameters sigma and s for the helix-coil transition in poly(L-glutamic acid) in water and in 0.1 N KCl were deduced from an analysis of the melting curves of the copolymers in the manner described in earlier papers. The synthesis of N-acetyl-N'-methylglutamic acid amide and its titration, as well as that of the copolymers and poly(L-glutamic acid), in 0.1 N KCl are described.

Butanols↗
PubMedDate not supplied

[Content of N-acetyl-L-asparate, N-acetyl-L-glutamate and N-acetyl-L-aspartyl-L-glutamate in the brain of mammals at increased oxygen tension].

The contents of N-acetyl-l-asparate and N-acetyl-l-glutamate, N-acetyl-l-aspartyl-l-glutamate were studied in the brain of rats of six age groups: newborns and on the 1st, 7th, 14th, and 30th day after birth. The amount of N-acetyl-l-asparate, N-acetyl-l-glutamate and peptide in the rat brain for 30 days of the postanal life is 8, 3.5 and 14.3 times as high, respectively. Under hyperoxic the amount of peptide, N-acetyl-l-asparate and N-acetyl-l-glutamate in the brain of rats of all the examined groups decreases especially in 14-, 21- and 30-day animals.

Animals↗