Ionotropic glutamate receptors.

The glutamate-binding sites of ionotropic glutamate receptors are formed from two extracellular domains of a single subunit. Conformational changes induced by agonist binding produce mechanical processes that are translated into ion gating and receptor desensitization. The interactions between macromolecular assemblies of synaptic proteins and ionotropic glutamate receptors, and their subsequent roles in receptor clustering and specificity are being elucidated. Kainate receptor pharmacology is finally revealing its secrets as a result of the availability of selective pharmacological agents.     

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.     

Spectra of glutamate dehydrogenase with diethylstilbestrol.

Glutamate dehydrogenase displays hyperchromicity at 256 nm and at 276 nm upon binding of diethylstilbestrol. Increase in absorbancy is linear at both regions up to 250 micrometer DES, and becomes parabolic at higher concentration of DES. ADP in the presence of DES causes decrease in absorbancy at 256 nm; absorbancy at 276 nm increased by DES is not affected by ADP. DES prevents spectral effects produced by GTP (decrease in absorbancy at 254 nm and at 276 nm). ADP still decreases absorbancy at 254 nm, leaving the 276 nm region unchanged. ADP enhances spectral effects produced by GTP. GTP, however, prevents changes produced by ADP.     

Spider toxin and the glutamate receptors.

A neurotoxin (JSTX) was isolated from the venom of spider (Nephila clavata). JSTX blocked both the excitatory postsynaptic (EPSPs) and glutamate-induced potentials in lobster neuromuscular synapse and squid giant synapse. In mammalian central nervous system, JSTX blocked the EPSPs in CA1 pyramidal neurons resulting from stimulation of Schaffer collateral/commissure input. Pharmacological investigation showed that JSTX preferentially suppressed quisqualate/kainate receptor subtypes but was much less effective on NMDA receptor. Using synthesized spider toxins we studied the structure-activity relationship and found that the 2,4 dihydroxyphenylacetyl asparagine in the toxin structure was responsible for suppressive action, while the remaining part containing a polyamine was related to the agonist binding site with the polycationic part enhancing the toxic activity. Labeling of synthesized JSTX was used for histochemical as well as biochemical studies. Using autoradiography, 125I-JSTX-3 was found to bind at the lobster neuromuscular synapse. Histochemical study utilizing the interaction of biotinylated JSTX-3 with avidin showed specific binding of the toxin in rat cerebellum and hippocampus. JSTX-3-binding protein was purified from rat brain by affinity chromatography. SDS-PAGE of the affinity purified protein showed at least 4 bands ranging from 40 to 70 kDa.     

Spin-labeling studies of beef-liver glutamate dehydrogenase.

Bovine liver glutamate dehydrogenase was spin labeled with a nitroxide derivative of parachloromercuribenzoate. The ESR spectrum was of the immobilized type and the labeling yield 0.6 mole of spin label bound per mole of protomer under standard conditions. The specific activity of the labeled enzyme was not modified but the activation by ADP abolished. Inhibition by GTP was not altered but the ESR spectrum showed that the bound spin label was further immobilized in the presence of GTP and NADPH. In the presence of the coenzyme NADPH, the labeling yield decreased to half its initial value. Such a protection effect was observed neither with NADH nor with ADP.     

Three-dimensional models of non-NMDA glutamate receptors.

Structural models have been produced for three types of non-NMDA inotropic glutamate receptors: an AMPA receptor, GluR1, a kainate receptor, GluR6; and a low-molecular-weight kainate receptor from goldfish, GFKAR alpha. Modeling was restricted to the domains of the proteins that bind the neurotransmitter glutamate and that form the ion channel. Model building combined homology modeling, distance geometry, molecular mechanics, interactive modeling, and known constraints. The models indicate new potential interactions in the extracellular domain between protein and agonists, and suggest that the transition from the "closed" to the "open" state involves the movement of a conserved positive residue away from, and two conserved negative residues into, the extracellular entrance to the pore upon binding. As a first approximation, the ion channel domain was modeled with a structure comprising a central antiparallel beta-barrel that partially crosses the membrane, and against which alpha-helices from each subunit are packed; a third alpha-helix packs against these two helices in each subunit. Much, but not all, of the available data were consistent with this structure. Modifying the beta-barrel to a loop-like topology produced a model consistent with available data.     

Folates as inhibitors of glutamate dehydrogenase.

Folates and tetrahydrofolates inhibit beef liver glutamate dehydrogenase (EC 1.4.1.2). Double reciprocal plats indicate a competitive inhibition for alpha-ketoglutarate-glutamate by folic acid and methotrexate and a complex or mixed type for NAD-NADH site. Pteroic acid is not inhibitory at the concentrations studied. The addition of up to four gamma-linked glutamyl residues to folic and tetrahydrofolic acids increases the inhibition. Further chain elongation of the gamma-peptide had no effect on the inhibitory activity. The p-aminobenzoate poly-gamma-glutamates were less inhibitory than the corresponding folyl polyglutamates.     

NAD-linked glutamate dehydrogenase in Trypanosoma cruzi.

1. Epimastigotes of Trypanosoma cruzi, Tulahuén strain, contained a NAD-linked glutamate dehydrogenase (EC 1.4.1.3), in addition to the already known NADP-linked enzyme enzyme (EC 1.4.1.4). 2. The partially purified NAD-linked enzyme had a higher molecular weight and was much more labile than the NADP-linked enzyme, and was inhibited by purine nucleotides. 3. These results further emphasize the difference in glutamate metabolism between the parasite and its mammalian host.     

Partial purification of Drosophila glutamate decarboxylase.

Abstract— A 600-fold purification of l -glutamatc decarboxylasc from Drosophila melanogaster Oregon R Wild Type has been achieved. The purification procedures involve the initial homogenization of whole flies in dilute potassium phosphate buffer containing dithiothreitol and phenylmethyl sulfonyl fluoride as protectors, followed by a series of column chromatography with hydroxylapatite, Sephadex G-150 and DEAE-Sephadex. The purified enzyme has an apparent K_m of 11 mm for l -glutamate and requires a 40mm -K⁺ for maximum activity. The purified enzyme shows only 1 pH optimum around pH 7.5, while crude preparations of the inset display 2 pH optima, pH 4.8–5.2. and 7.5. The significance and possible application of this study are also discussed.     

Brain glutamate decarboxylase and pyrroloquinoline quinone.

Porcine brain glutamate decarboxylase was examined for the presence of covalently bound pyrroloquinoline quinone (PQQ). HPLC analysis of pure glutamate decarboxylase subjected to the hexanol extraction procedure gave negative results when monitored at 320 nm, the maximum of absorbance of 4-hydroxy-5-hexoxy-PQQ. Resolved glutamate decarboxylase exhibits a structureless absorption band at wavelengths longer than 300 nm which cannot be attributed to PQQ. The holoenzyme is not a pyridoxal-quinoprotein; its catalytic mechanism involves the participation of only one cofactor, i.e. pyridoxal-5-P. Free PQQ is a strong inhibitor of the decarboxylase (Ki = 13 microM) and the reaction with the protein results in spectral changes resembling those of polylysine treated with PQQ. If the concentration of free PQQ in some regions of the brain reaches the micromolar level, then PQQ might play a role in the regulation of glutamate decarboxylase activity.     

A family of metabotropic glutamate receptors.

Three cDNA clones, mGluR2, mGluR3, and mGluR4, were isolated from a rat brain cDNA library by cross-hybridization with the cDNA for a metabotropic glutamate receptor (mGluR1). The cloned receptors show considerable sequence similarity with mGluR1 and possess a large extracellular domain preceding the seven putative membrane-spanning segments. mGluR2 is expressed in some particular neuronal cells different from those expressing mGluR1 and mediates an efficient inhibition of forskolin-stimulated cAMP formation in cDNA-transfected cells. The mGluRs thus form a novel family of G protein-coupled receptors that differ in their signal transduction and expression patterns.     

Transaminations catalysed by brain glutamate decarboxylase.

In addition to normal decarboxylation of glutamate to 4-aminobutyrate, glutamate decarboxylase from pig brain was shown to catalyse decarboxylation-dependent transamination of L-glutamate and direct transamination of 4-aminobutyrate with pyridoxal 5'-phosphate to yield succinic semialdehyde and pyridoxamine 5'-phosphate in a 1:1 stoichiometric ratio. Both reactions result in conversion of holoenzyme into apoenzyme. With glutamate as substrate the rates of transamination differed markedly among the three forms of the enzyme (0.008, 0.012 and 0.029% of the rate of 4-aminobutyrate production by the alpha-, beta- and gamma-forms at pH 7.2) and accounted for the differences among the forms in rates of inactivation by glutamate and 4-aminobutyrate. Rates of transamination were maximal at about pH 8 and varied in parallel with the rate constants for inactivation from pH 6.5 to 8.0. Rates of transamination of glutamate and 4-aminobutyrate were similar, suggesting that the decarboxylation step is not entirely rate-limiting in the normal mechanism. The transamination was reversible, and apoenzyme could be reconstituted to holoenzyme by reverse transamination with succinic semialdehyde and pyridoxamine 5'-phosphate. As a major route of apoenzyme formation, the transamination reaction appears to be physiologically significant and could account for the high proportion of apoenzyme in brain.     

Role of glutamate 144 and glutamate 164 in the catalytic mechanism of enoyl-CoA hydratase.

The role of two glutamate residues (E164 and E144) in the active site of enoyl-CoA hydratase has been probed by site-directed mutagenesis. The catalytic activity of the E164Q and E144Q mutants has been determined using 3'-dephosphocrotonyl-CoA. Removal of the 3'-phosphate group reduces the affinity of the substrate for the enzyme, thereby facilitating the determination of K(m) and simplifying the analysis of the enzymes' pH dependence. k(cat) for the hydration of 3'-dephosphocrotonyl-CoA is reduced 7700-fold for the E144Q mutant and 630000-fold for the E164Q mutant, while K(m) is unaffected. These results indicate that both glutamate residues play crucial roles in the hydration chemistry catalyzed by the enzyme. Previously, we reported that, in contrast to the wild-type enzyme, the E164Q mutant was unable to exchange the alpha-proton of butyryl-CoA with D(2)O [D'Ordine, R. L., Bahnson, B. J., Tonge, P. J. , and Anderson, V. E. (1994) Biochemistry 33, 14733-14742]. Here we demonstrate that E144Q is also unable to catalyze alpha-proton exchange even though E164, the glutamate that is positioned to abstract the alpha-proton, is intact in the active site. The catalytic function of each residue has been further investigated by exploring the ability of the wild-type and mutant enzymes to eliminate 2-mercaptobenzothiazole from 4-(2-benzothiazole)-4-thiabutanoyl-CoA (BTTB-CoA). As expected, reactivity toward BTTB-CoA is substantially reduced (690-fold) for the E164Q enzyme compared to wild-type. However, E144Q is also less active than wild-type (180-fold) even though elimination of 2-mercaptobenzothiazole (pK(a) 6.8) should require no assistance from an acid catalyst. Clearly, the ability of E164 to function as an acid-base in the active site is affected by mutation of E144 and it is concluded that the two glutamates act in concert to effect catalysis.