Isolation and Characterization of a Novel 5-Oxoprolinase (without ATP-hydrolyzing) from Alcaligenes faecalis N-38A

A screening test was undertaken to isolate a microorganism that produced 5-oxoprolinase (without ATP-hydrolyzing). The 5-oxoprolinase (without ATP-hydrolyzing) activity (decyclization activity toward L-pyroglutamate) was found in a cell-free extract of Alcaligenes faecalis N-38A, newly isolated from a soil sample. The enzyme was purified as a homogeneous preparation. The molecular weight of the enzyme was estimated to be 47,000. The decyclization activity was specific for L-pyroglutamate, and independent of ATP and metal ions. The reaction was a reversible one, i.e., cyclization reaction of L-glutamate to yield pyroglutamate was identified.     

Dipeptides delay the onset of morphine withdrawal in the mouse

The effect of dipeptides was studied on naloxone-precipitated morphine withdrawal in the mouse. In accordance with previous data, s.c. treatment with Z-prolyl-D-leucine delayed the onset of withdrawal jumpings . Replacement of L-proline by L-glutamate or L-pyroglutamate resulted in dipeptides which were more potent in morphine withdrawal than was Z-prolyl-D-leucine.     

Influence of dipeptides on precipitated morphine withdrawal in the mouse

Effects of various dipeptides on naloxone-precipitated morphine withdrawal were studied in the mouse. Mice were rendered dependent on morphine by implantation of morphine pellets and the withdrawal syndrome was measured by the latency of the onset of stereotyped jumpings. In accordance with previous data, subcutaneous injection of Z-prolyl-D-leucine significantly delayed the onset of morphine withdrawal. The all-L enantiomer of the dipeptide (Z-L-prolyl-L-leucine) did not affect morphine withdrawal in the dose studied. Replacement of L-proline by L-glutamate or L-pyroglutamate (Z-L-glutamyl-L-leucine and L-pyroglutamyl-L-leucine) resulted in dipeptides which were more potent towards morphine withdrawal than Z-prolyl-D-leucine. Z-L-glycyl-L-proline attenuated the morphine withdrawal syndrome more effectively than Z-L-prolyl-D-leucine, but Z-L-leucyl-L-glycine was ineffective in this respect. The data reveal that certain dipeptides—which in their nonprotected forms are normal sequences of endogenous peptides—affect morphine withdrawal more potently than Z-prolyl-D-leucine, a synthetic dipeptide known to attenuate morphine dependence.     

Dependence of energy coupling on nucleotide base structure in the reaction catalyzed by 5-oxo-L-prolinase

5-Oxo-L-prolinase catalyzes the virtually complete hydrolysis of 5-oxo-L-proline (L-pyroglutamate) to L-glutamate. The thermodynamic driving force for this endergonic amide hydrolysis is supplied by the coupled stoichiometric hydrolysis of ATP to ADP and Pi. We report here that the efficiency of the coupling between nucleotide and amide hydrolysis is dependent on the nucleotide base. Thus, with both ATP and dATP there is one to one stoichiometry between nucleotide cleavage and 5-oxoproline hydrolysis. With ITP, GTP, or UTP, however, the hydrolysis of NTP exceeds amide hydrolysis by 6 to 50-fold. In the absence of 5- oxoproline, the enzyme catalyzes a slow ATPase reaction, but it catalyzes very rapid ITPase, GTPase and UTPase reactions. These NTPase reactions, which under some conditions are faster than the ATP-mediated overall coupled reaction, are inhibited by 5-oxoproline and by analogs of 5-oxoproline that bind to the enzyme.     

Use of a bioreactor consisting of sequentially aligned L-glutamate dehydrogenase and L-glutamate oxidase for the determination of ammonia by chemiluminescence

A chemiluminometric method for the automated flow injection analysis of ammonia is described. The essence of the invention is the use of a bioreactor consisting of both immobilized L-glutamate dehydrogenase (GLDH) and L-glutamate oxidase (GLXD), which are sequentially aligned in this order in a minicolumn measuring 2.0 X 20 mm. The unidirectional constant flow of liquid through the column reactor minimizes the reversed diffusion of the solutes so that the following sequence of reactions is ensured. Thus, ammonia to be determined is first transformed by GLDH into L-glutamate, which then produces hydrogen peroxide by GLXD. Hydrogen peroxide in the effluent from the column is then determined by its chemiluminescence upon admixing with luminol and potassium ferricyanide. The present method gives linearity of the standard curve for ammonia up to 1.0 mM. It is at least 100 times more sensitive than the conventional method for ammonia assay using ultraviolet absorption measurement.     

Altered metabolic flux due to deletion of odhA causes L-glutamate overproduction in Corynebacterium glutamicum

L-glutamate overproduction in Corynebacterium glutamicum, a biotin auxotroph, is induced by biotin limitation or by treatment with certain fatty acid ester surfactants or with penicillin. We have analyzed the relationship between the inductions, 2-oxoglutarate dehydrogenase complex (ODHC) activity, and L-glutamate production. Here we show that a strain deleted for odhA and completely lacking ODHC activity produces L-glutamate as efficiently as the induced wild type (27.8 mmol/g [dry weight] of cells for the ohdA deletion strain compared with only 1.0 mmol/g [dry weight] of cells for the uninduced wild type). This level of production is achieved without any induction or alteration in the fatty acid composition of the cells, showing that L-glutamate overproduction can be caused by the change in metabolic flux alone. Interestingly, the L-glutamate productivity of the odhA-deleted strain is increased about 10% by each of the L-glutamate-producing inductions, showing that the change in metabolic flux resulting from the odhA deletion and the inductions have additive effects on L-glutamate overproduction. Tween 40 was indicated to induce drastic metabolic change leading to L-glutamate overproduction in the odhA-deleted strain. Furthermore, optimizing the metabolic flux from 2-oxoglutarate to L-glutamate by tuning glutamate dehydrogenase activity increased the l-glutamate production of the odhA-deleted strain.     

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Studies of the chemical mechanism

Rat kidney 5-oxo-L-prolinase catalyzes the endergonic hydrolysis of 5-oxo-L-proline (L-pyroglutamate, L-2-pyrrolidone-5-carboxylate) to form L-glutamate; the reaction is driven by and dependent on the stoichiometric concomitant hydrolysis of ATP to ADP and inorganic phosphate. The present studies are concerned with the mechanism by which the free energy of ATP hydrolysis is conserved and made available for 5-oxoproline hydrolysis. Studies with 18O-labeled substrates showed that (a) all three oxygen atoms of 5-oxoproline are recovered in the product glutamate, and (b) the two water molecules consumed in the reaction contribute one oxygen atom to inorganic phosphate and one oxygen atom to the gamma-carboxyl group of glutamate. It was shown that the enzyme also catalyzes the intrinsically exergonic hydrolysis of alpha-hydroxyglutarate lactone, a reaction that is ATP-dependent. Intermediates in the 5-oxoprolinase reaction were not detected by exchange experiments with radioactive ADP and phosphate, nor were they trapped by adding hydroxylamine. In the presence of very high glutamate concentrations, a slow reversal of the 5-oxoprolinase reaction was demonstrated by measuring ATP formation. The findings are consistent with a mechanism in which 5-oxo-L-proline is phosphorylated by ATP on the amide carbonyl oxygen and the resulting intermediate is subsequently hydrolyzed to yield gamma-glutamyl phosphate; the latter is hydrolyzed to glutamate and inorganic phosphate.     

5-Oxo-L-prolinase (L-pyroglutamate hydrolase). Purification and catalytic properties.

5-Oxo-L-prolinase, an enzyme that catalyzes the conversion of 5-oxo-L-proline (L-pyroglutamate; L-2-pyrrolidone-5-carboxylate) to L-glutamate coupled with the cleavage of ATP to ADP and Pi, has been purified about 1600-fold from rat kidney. Purification was carried out in the presence of 5-oxo-L-proline which protects the enzyme under a variety of conditions. An estimate of the molecular weight (about 325,000) was made by gel filtration on Sephadex G-200. K+ (or NH4+) and Mg2+ were required for activity. GTP, ITP, CTP, and UTP were much less active than ATP; dATP was 43% as active as ATP. ADP inhibited and addition of pyruvate kinase and phosphoenolpyruvate activated the reaction. The enzyme, which is protected during storage by dithiothreitol, is inhibited by p-hydroxymercuribenzoate, N-ethylmaleimide, and iodoacetamide. The apparent Km values for 5-oxo-L-proline and ATP are, respectively, 0.05 and 0.17 mM. The pH profile indicates a broad range of activity from about pH 5.5 to pH 11.2 with apparent maxima at about pH 7 and pH 9.7. The formation of Pi and glutamate was equimolar over a wide pH range. When the enzyme was incubated with ATP, Mg2+, K+, and L-2-imidazolidone-4-carboxylate or L-dihydroorotate, cleavage of ATP to ADP and Pi occurred, but no cleavage of the imino acid substrates was observed; when the enzyme was incubated under these conditions with 2-piperidone-6-carboxylate, 4-oxy-5-oxoproline, and 3-oxy-5-oxoproline, the corresponding dicarboxylic amino acids were formed, but the molar ratio of Pi to amino acid formation was significantly greater than unity.     

Development and characterization of a polymer gel with an immobilized enzyme to measure L-glutamate

Glutamate dehydrogenase (GDH) is used in an enzyme electrode to measure L-glutamate. GDH is covalently immobilized in a hydrophilic, permeable, and semirigid gel produced by the copolymerization of polyacrylamide and N-acryloxysuccinimide. Experimental conditions necessary to retain GDH in the gel with high efficiency and minimum denaturation are optimized. The abilities of enzymatic cofactors and coenzymes, NADH, NAD, ATP, ADP, GTP, and ZnCl2, to protect the enzyme during immobilization are explored. Under optimum experimental procedures an enzyme-containing gel is produced that is reproducible and long lasting in its functional behavior. The gel responds to the presence of L-glutamate with high velocity, the delay being less than 500 ms; high specificity, being 1000-fold more responsive to L-glutamate than D-glutamate, D- or L-aspartate, and N-acetylhistidine; and high sensitivity, a concentration of about 3 microM can be measured.     

The transient-state kinetics of L-glutamate dehydrogenase. pH-dependence of the burst rate parameters.

The pH dependence of the initial transient velocity of NADPH production during the burst phase of the oxidative deamination of L-glutamate by L-glutamate dehydrogenase (L-glutamate : NAD(P)+ oxidoreductase (deaminating), EC 1.4.1.3) and NADP+ has been measured by stopped-flow spectrophotometry. These studies provide evidence that the entire pH dependence below pH 8.26 arises from reaction steps contributing to V of the burst with an apparent pKa of 8.1 +/- 0.1. The data are consistent with a model in which the formation of the first enzyme-coenzyme-substrate ternary complex on the reaction path equilibrates rapidly and in which the pH-dependent steps are mechanistically close to and may include the catalytic hydrogen transfer itself. At pH 8.87, there is evidence that L-glutamate binds less tightly to the enzyme and to the enzyme-NADP+ complex than at lower pH values.     

Reconstitution and partial purification of several Na+ cotransport systems from renal brush-border membranes. Properties of the L-glutamate transporter in proteoliposomes

Brush-border membranes of renal proximal tubules were solubilized with deoxycholate and some proteins were separated and incorporated into proteoliposomes by a reconstitution procedure which was analyzed in detail. The proteoliposomes contained mainly polypeptides with molecular weights of 152,000, 94,000, and 52,000, each of which could be separated further into homologous polypeptides with different isoelectric points. In the proteoliposomes, Na+ cotransport systems for D-glucose, acidic and neutral amino acids, and mono- and dicarboxylic acids were demonstrated by showing that due to an inwardly directed Na+ gradient the substrate concentrations in the proteoliposomes increased significantly over their respective equilibrium values. Using inhibition experiments, selectivity of the different transporters could be demonstrated. Studying the reconstituted L-glutamate transporter in detail, countertransport of L-glutamate and K+ was shown (i) at Na+ equilibrium the intraliposomal L-glutamate concentration increased significantly over the equilibrium value if an outside-directed K+ gradient was applied; (ii) Rb+ influx was significantly stimulated by the outflux of L-glutamate. By applying a K+ diffusion potential across the liposomal membrane by addition of valinomycin it could be shown that during L-glutamate transport in the presence of Na+ and K+ positive charge is transferred together with L-glutamate and Na+. The apparent Km value of L-glutamate uptake driven by concentration differences of 89 mM Na+ (out greater than in) and 89 mM K+ (in greater than out) was 26.3 +/- 1.3 microM. The Vmax value of 70.2 +/- 2.3 pmol X mg of protein-1 X S-1 was half the value measured in intact membranes.     

The effect of the carbon source on ammonium regulation in Aspergillus nidulans

Summary Ammonium regulation of L-glutamate transport is annuled when L-glutamate is the sole or main carbon source. This shows that ammonium regulation mechanisms are not operative where the substrate of the ammonium repressible system can be used as a carbon as well as a nitrogen source. There is rapid loss of NADP L-glutamate dehydrogenase when glucose is replaced by L-glutamate as a carbon source. This suggests that NADP L-glutamate dehydrogenase may be subject to carbon as well as nitrogen control.     

Mechanism of L-glutamate transport in membrane vesicles from Bacillus stearothermophilus.

In the presence of electrochemical energy, several branched-chain neutral and acidic amino acids were found to accumulate in membrane vesicles of Bacillus stearothermophilus. The membrane vesicles contained a stereo-specific transport system for the acidic amino acids L-glutamate and L-aspartate, which could not translocate their respective amines, L-glutamine and L-asparagine. The transport system was thermostable (Ti = 70 degrees C) and showed highest activities at elevated temperatures (60 to 65 degrees C). The membrane potential or pH gradient could act as the driving force for L-glutamate uptake, which indicated that the transport process of L-glutamate is electrogenic and that protons are involved in the translocation process. The electrogenic character implies that the anionic L-glutamate is cotransported with at least two monovalent cations. To determine the mechanistic stoichiometry of L-glutamate transport and the nature of the cotranslocated cations, the relationship between the components of the proton motive force and the chemical gradient of L-glutamate was investigated at different external pH values in the absence and presence of ionophores. In the presence of either a membrane potential or a pH gradient, the chemical gradient of L-glutamate was equivalent to that specific gradient at different pH values. These results cannot be explained by cotransport of L-glutamate with two protons, assuming thermodynamic equilibrium between the driving force for uptake and the chemical gradient of the substrate. To determine the character of the cotranslocated cations, L-glutamate uptake was monitored with artificial gradients. It was established that either the membrane potential, pH gradient, or chemical gradient of sodium ions could act as the driving force for L-glutamate uptake, which indicated that L-glutamate most likely is cotranslocated in symport with one proton and on sodium ion.     

Thermodynamics of active-site ligand binding to Escherichia coli glutamine synthetase

Active-site ligand interactions with dodecameric glutamine synthetase from Escherichia coli have been studied by calorimetry and fluorometry using the nonhydrolyzable ATP analogue 5'-adenylyl imidodiphosphate (AMP-PNP), L-glutamate, L-Met-(S)-sulfoximine, and the transition-state analogue L-Met-(S)-sulfoximine phosphate. Measurements were made with the unadenylylated enzyme at pH 7.1 in the presence of 100 mM KCl and 1.0 mM MnCl2, under which conditions the two catalytically essential metal ion sites per subunit are occupied and the stoichiometry of active-site ligand binding is equal to 1.0 equiv/subunit. Thermodynamic linkage functions indicate that there is strong synergism between the binding of AMP-PNP and L-Met-(S)-sulfoximine (delta delta G' = -6.4 kJ/mol). In contrast, there is a small antagonistic effect between the binding of AMP-PNP and L-glutamate (delta delta G' = +1.4 kJ/mol). Proton effects were negligible (less than or equal to 0.2 equiv of H+ release or uptake/mol) for the different binding reactions. The binding of AMP-PNP (or ATP) to the enzyme is entropically controlled at 303 K with delta H = +5.4 kJ/mol and delta S = +150 J/(K.mol). At 303 K, the binding of L-glutamate (delta H = -22.2 kJ/mol) or L-Met-(S)-sulfoximine [delta H = -45.6 kJ/mol with delta Cp approximately equal to -670 +/- 420 J/(K.mol)] to the AMP-PNP.Mn.enzyme complex is enthalpically controlled with opposing delta S values of -29 or -46 J/(K.mol), respectively. The overall enthalpy change is negative and the overall entropy change is positive for the simultaneous binding of AMP-PNP and L-glutamate or of AMP-PNP and L-Met-(S)-sulfoximine to the enzyme. For the binding of the transition-state analogue L-Met-(S)-sulfoximine phosphate (which inactivates the enzyme by blocking active sites), both enthalpic and entropic contributions also are favorable at 303 K [delta G' approximately equal to -109 and delta H = -54.8 kJ/mol of subunit and delta S approximately equal to +180 J/(K.mol)].     

Solubilisation of a Glutamate Binding Protein from Rat Brain

Rat brain synaptic plasma membranes were solubilised in either 1% Triton X-100 or potassium cholate and subjected to batch affinity adsorption on L-glutamate/bovine serum albumin reticulated glass fibre. The fibre was extensively washed, and bound proteins eluted with 0.1 mM L-glutamate in 0.1% detergent, followed by repeated dialysis to remove the glutamate from the eluted proteins. Aliquots of the dialysed extracts were assayed for L-[3H]glutamate binding activity in the presence or absence of 0.1 mM unlabelled L-glutamate (to define displaceable binding). Incubations were conducted at room temperature and terminated by rapid filtration through nitrocellulose membranes. Binding to solubilised fractions could be detected only following affinity chromatography. Binding was saturable and of relatively low affinity: KD = 1.0 and 1.8 microM for Triton X-100 and cholate extracts, respectively. The density of binding sites was remarkably high: approximately 18 nmol/mg protein for Triton X-100-solubilised preparations, and usually double this when cholate was employed. Analysis of structural requirements for inhibition of binding revealed that only a very restricted number of compounds were effective, i.e., L-glutamate, L-aspartate, and sulphur-containing amino acids. Binding was not inhibited significantly by any of the selective excitatory amino acid receptor agonists--quisqualate, N-methyl-D-aspartate, or kainate. The implication from this study is that the glutamate binding protein is similar if not identical to one previously isolated and probably is not related to the pharmacologically defined postsynaptic receptor subtypes, unless solubilisation of synaptic membranes resulted in major alterations to binding site characteristics. Since solubilisation with Triton X-100 is known to preserve synaptic junctional complexes, it seems likely that the origin of the glutamate binding protein may be extrajunctional, although its functional role is unknown.     

Actions of the insecticide 2(nitromethylene)tetrahydro-1,3-thiazine on insect and vertebrate nicotinic acetylcholine receptors

The nitromethylene heterocyclic compound 2(nitromethylene)tetrahydro)1,3-thiazine (NMTHT) inhibits the binding of [125I]alpha-bungarotoxin to membranes prepared from cockroach (Periplaneta americana) nerve cord and fish (Torpedo californica) electric organ. Electrophysiological studies on the cockroach fast coxal depressor motorneuron (Df) reveal a dose-dependent depolarization in response to bath-applied NMTHT. Responses to ionophoretic application of NMTHT onto the cell-body membrane of motorneuron Df are suppressed by bath-applied mecamylamine (1.0 x 10(-4) M) and alpha-bungarotoxin (1.0 x 10(-7) M). These findings, together with the detection of a reversal potential close to that estimated for acetylcholine, provide evidence for an agonist action of this nitromethylene on an insect neuronal nicotinic acetylcholine receptor. The binding of [3H]H12-histrionicotoxin to Torpedo membranes was enhanced in the presence of NMTHT indicating an agonist action at this vertebrate peripheral nicotinic acetylcholine receptor. NMTHT is ineffective in radioligand binding assays for rat brain GABAA receptors, rat brain L-glutamate receptors and insect (Musca domestica) L-glutamate receptors. Partial block of rat brain muscarinic acetylcholine receptors is detected at millimolar concentrations of NMTHT. Thus nitromethylenes appear to exhibit selectivity for acetylcholine receptors and exhibit an agonist action at nicotinic acetylcholine receptors.     

Sodium ion-dependent amino acid transport in membrane vesicles of Bacillus stearothermophilus.

Amino acid transport in membrane vesicles of Bacillus stearothermophilus was studied. A relatively high concentration of sodium ions is needed for uptake of L-alanine (Kt = 1.0 mM) and L-leucine (Kt = 0.4 mM). In contrast, the Na(+)-H(+)-L-glutamate transport system has a high affinity for sodium ions (Kt less than 5.5 microM). Lithium ions, but no other cations tested, can replace sodium ions in neutral amino acid transport. The stimulatory effect of monensin on the steady-state accumulation level of these amino acids and the absence of transport in the presence of nonactin indicate that these amino acids are translocated by a Na+ symport mechanism. This is confirmed by the observation that an artificial delta psi and delta mu Na+/F but not a delta pH can act as a driving force for uptake. The transport system for L-alanine is rather specific. L-Serine, but not L-glycine or other amino acids tested, was found to be a competitive inhibitor of L-alanine uptake. On the other hand, the transport carrier for L-leucine also translocates the amino acids L-isoleucine and L-valine. The initial rates of L-glutamate and L-alanine uptake are strongly dependent on the medium pH. The uptake rates of both amino acids are highest at low external pH (5.5 to 6.0) and decline with increasing pH. The pH allosterically affects the L-glutamate and L-alanine transport systems. The maximal rate of L-glutamate uptake (Vmax) is independent of the external pH between pH 5.5 and 8.5, whereas the affinity constant (Kt) increases with increasing pH. A specific transport system for the basic amino acids L-lysine and L-arginine in the membrane vesicles has also been observed. Transport of these amino acids occurs most likely by a uniport mechanism.     

In vivo demonstration of the enhancement of MK-801 by L-glutamate

MK-801 infused bilaterally into the nucleus accumbens of rats produced a dose-related increase in locomotor activity that was not blocked by intra-accumbens infusion of haloperidol (2.5 micrograms). Intra-accumbens infusion of L-glutamate (1.0 microgram) was without effect when administered alone, but significantly enhanced the increase in locomotor activity produced by MK-801 (5.0 micrograms). The inactive isomer of MK-801 did not produce hypermotility and L-glutamate did not enhance amphetamine(intra-accumbens)-induced hypermotility. These results extend to an in vivo model electrophysiological and radioligand binding studies demonstrating an enhancement of MK-801 activity by L-glutamate. The results also reinforce the concept that such an enhancement would occur during an escalation of excitatory amino acid levels accompanying pathological overload and, thus, would trigger a compensatory neuronal protection by MK-801.     

Glutamate inhibits protein phosphatases and promotes insulin exocytosis in pancreatic beta-cells

In human type 2 diabetes mellitus, loss of glucose-sensitive insulin secretion from the pancreatic beta-cell is an early pathogenetic event, but the mechanisms involved in glucose sensing are poorly understood. A messenger role has been postulated for L-glutamate in linking glucose stimulation to sustained insulin exocytosis in the beta-cell, but the precise nature by which L-glutamate controls insulin secretion remains elusive. Effects of L-glutamate on the activities of ser/thr protein phosphatases (PPase) and Ca(2+)-regulated insulin exocytosis in INS-1E cells were investigated. Glucose increases L-glutamate contents and promotes insulin secretion from INS-1E cells. L-glutamate also dose-dependently inhibits PPase enzyme activities analogous to the specific PPase inhibitor, okadaic acid. L-glutamate and okadaic acid directly and non-additively promote insulin exocytosis from permeabilized INS-1E cells in a Ca(2+)-independent manner. Thus, an increase in phosphorylation state, through inhibition of protein dephosphorylation by glucose-derived L-glutamate, may be a novel regulatory mechanism linking glucose sensing to sustained insulin exocytosis.     

Radioisotopic measurement of femtomolar amounts of NAD(P)H in the assay of enzymatic activity at a single cell level

A radioisotopic method for the assay of reduced or oxidized pyridine nucleotides, based on the interconversion of 2-[U-14C]ketoglutarate or 2-keto[3,4-3H]glutarate and labelled L-glutamate in the reaction catalyzed by glutamate dehydrogenase, was applied to the measurement of lactate dehydrogenase activity in rat pancreatic islet homogenates. Using the tritiated tracer, the limit of sensitivity of the procedure for NAD(P)H assay was close to 1.0 fmol/sample, and lactate dehydrogenase activity could be measured in as little as 0.0005 islet/sample i.e., at a single cell level. This radioisotopic procedure, which can be used for the assay of various metabolites and enzymic activities, thus provides a tool for investigating the heterogeneity in metabolic behaviour of individual cells.