L-glutamate Complex Research Articles

Crystallization and preliminary X-ray crystallographic analysis of the GluR0 ligand-binding core fromNostoc punctiforme

GluR0 from Nostoc punctiforme (NpGluR0) is a bacterial homologue of the ionotropic glutamate receptor. The ligand-binding core of NpGluR0 was crystallized at 294 K using the hanging-drop vapour-diffusion method. The L-glutamate-complexed crystal belongs to space group C222(1), with unit-cell parameters a = 78.0, b = 145.1, c = 132.1 A. The crystals contain three subunits in the asymmetric unit, with a VM value of 2.49 A3 Da(-1). The diffraction limit of the L-glutamate complex data set was 2.1 A using synchrotron X-ray radiation at beamline BL-4A of the Pohang Accelerator Laboratory (Pohang, Korea).

Aluminum l-glutamate complex in rat brain cortex: in vivo prevention of aluminum deposit by magnesium d-aspartate

Our previous experiments in the rat showed that aluminum l-glutamate complex (Al l-Glu) crosses the blood–brain barrier and accumulates in selective brain areas and that Al salts may increase d-aspartic acid forms in living brain proteins, probably by inducing more thermodynamically stable structures than L isomers. As magnesium blocks NMDA receptors, d-aspartic acid was used in the present study in the form of magnesium salt to prevent the excitotoxicity of dicarboxylic amino acids. Effects on brain amino acids and Al cortex levels in mature rats were studied after chronic treatment with Al l-Glu or Na l-Glu alone or in association with magnesium d-aspartate (Mg d-Asp). Results demonstrate that treatment with Mg d-Asp induces a decrease in the Al concentration in brain cortex of Al l-Glu-treated rats. In aluminum-free treated controls, treatment with Mg d-Asp in association with Na l-Glu also induces a decrease in Al concentration in brain cortex. These data indicate that Mg d-Asp administration protects rat brain cortex from Al accumulation and suggest that this treatment may be useful in preventing brain Al intoxication.

Chronic administration of aluminium l-glutamate in young mature rats: effects on iron levels and lipid peroxidation in selected brain areas

Clinical and experimental studies have demonstrated the neurotoxicity of aluminium (Al), notably as a result of lipid peroxidation in vitro. We previously showed that Al is able to cross the blood–brain barrier as an l-glutamate complex and be deposited in rat brain. The present work in young mature rats investigated the in vivo effects of chronic Al– l-glutamate treatment on Al and iron movement in plasma and selected brain regions. Brain lipid peroxidation was determined by evaluating the production of thiobarbituric acid reactive substances (TBARS) and analysing polyunsaturated fatty acids (PUFAs) such as C20:4 n-6 and C22:6 n-3. Our results indicate that iron concentration was decreased in plasma and that Al accumulated especially in striatum where iron levels were decreased and in the hippocampus where TBARS were increased without PUFA modifications. These data show that Al administered chronically as an l-glutamate complex is neurotoxic in vivo and thus provides a good model for studying Al toxic mechanisms.

Effect of cryosolvent on transient kinetics of the glutamate dehydrogenase reaction.

The transient and steady-state kinetics of the oxidative deamination of L-glutamate by glutamate dehydrogenase and NADP in both aqueous solution and 30% methanol are compared. Methanol causes an approximately 5-fold tightening of the enzyme--L-glutamate binary complex and an approximately 2-fold reduction of the interaction parameter for the ternary enzyme--NADP--L-glutamate complex. The most dramatic effect of methanol on the time course of the reaction is what appears to be a conversion of the enzyme at substoichiometric initial levels of reactant NADP to a form from which product alpha-ketoglutarate does not readily dissociate. This conversion appears only at NADP concentrations over one-third of the enzyme active site concentration.

Reaction mechanism of L-glutamate dehydrogenase. Transient complexes in the oxidative deamination of L-glutamate catalyzed by NAD(P)-dependent L-glutamate dehydrogenase.

Characteristic optical properties of the reduced nicotinamide group of the coenzyme NADPH (absorbance, fluorescence, and circular dichroism) have enabled us to describe the sequential formation of two transients during the oxidative deamination of l-glutamste catalyzed by l-glutamate dehydrogenase in the presence of NADP. The first transient detected, called complex I, is characterized by a reduced nicotinamide absorption maximum at 333 nm and a very low specific NADPH fluorescence (Fs∼ 0.2). This complex is identified as the E · NADPH · 2-oxoglutarate complex which is known to possess very similar optical properties. The second transient, called complex II, is characterized and identified as the E · NADPH ·l-glutamate complex by the detection of an unique negative ellipticity during the steady-state phases. Using certain kinetic arguments, the formation of complex II (i.e., binding of l-glutamate to E · NADPH) is shown to be rate-limited by the dissociation of 2-oxoglutarate from complex II (i.e., E · NADPH · 2-oxo-glutarate). The conclusion is also drawn that the complex II accumulates prior to the liberation of the free product NADPH during the presteady-states phases. The rate-limiting step of the overall reaction is the dissociation of NADPH from the complex II which attains a, steady-state concentration level corresponding to 80% of the total enzyme concentration under our experimental conditions. The influence of the known catalytic effectors GTP and ADP on the accumulation of these transients has also been studied. The inhibitor GTP, under these same conditions, allows the accumulation of complex I, although its rate of formation is slower (2-fold), but thereafter blocks the reaction (>90%) by inhibiting the liberation of the products, especially that of 2-oxoglutarate. There is no formation of complex II which confirms the fact that 2-oxoglutarate is retained under the complex E · NADPH · 2-oxoglutarate form. The complex I accumulates to the same maximal concentration (∼ 50%, total available enzyme sites) as the reaction in the absence of effector. It is not known whether the extremely slow increase in absorbance which follows is due to a slow binding of the substrate (2-oxoglutarate) to the remaining sites and/or to a very slow liberation of NADPH permitting the enzyme turnover. The activator ADP modulates the accumulation of these two transients in a different fashion. The complex I is also formed more slowly (2-fold) but it then decomposes more quickly (2-oxoglutarate liberated twice as fast). Its maximal accumulation is thus lower than in the reaction in the absence of ADP (∼ 30%). The formation of complex II however does occur and accumulates to about 80% of the enzyme concentration. Here again the enzyme turnover is thought to be governed by the release of NADPH from complex II, which is about 2–3 times faster than the reaction without effector. This fact suffices to explain the activation (2–3 times) observed by classical steady-state kinetics.

Spectrophotometric observation of a glutamate dehydrogenase- l-glutamate complex

Ultraviolet differential spectroscopic measurements show the existence of a glutamate dehydrogenase ( l-glutamate:NAD(P) + oxidoreductase (deaminating), EC 1.4.1.3)— l-glutamate complex. The spectral features resemble perturbation difference spectra of enzyme aromatic amino acid chromophores and allow the determination of the l-glutamate concentration dependence. The dissociation constant for this enzyme- l-glutamate complex was approximately 48 mM and was independent of enzyme concentration. The lack of interaction between the binding of l-glutamate and the activating monocarboxylic amino acids indicates that they bind at totally separate sites.