N-acetyl-l-glutamate in brain: Assay,levels, and regional and subcellular distribution

N-Acetyl-L-glutamate (NAG), the activator of mitochondrial carbamoyl phosphate synthetase (CPS), is demonstrated by several methods, including a new HPLC assay, in the brain of mammals and of chicken. The brain levels of NAG are 200–300 times lower than the levels of N-acetyl-l-aspartate (NAA), and are similar to the levels of NAG in rat liver. The NAG levels in chicken liver are very low. Although NAG is mitochondrial in the liver, it is cytosolic in brain. Using enzyme activity and immuno assays we did not detect CPS in brain (detection limit, 12.5 g/g brain), excluding that brain NAG is involved in citrullinogenesis. The regional distribution of brain NAG differs from that of NAA and resembles that of N-acetyl-l-aspartyl-l-glutamate (NAAG), suggesting that NAG and NAAG are related. NAG might be involved in the modulation of NAAG degradation.Special issue dedicated to Dr. Santiago Grisolía     

An expedient synthesis of l-ribulose and derivatives

A significant improvement in the production of l-ribulose from inexpensive and commercially available starting materials, l-arabinose and sodium aluminate, is demonstrated. This has facilitated expeditious access to gram-scale quantities of l-ribulofuranoside derivatives.     

Astrocytes as potential modulators of mercuric chloride neurotoxicity

Summary 1. MC has been shown to inhibit the uptake ofl-glutamate and increased-aspartate release from preloaded astrocytes in a dose-dependent fashion.2. Two sulfhydryl (SH-)-protecting agents; reduced glutathione (GSH), a cell membrane-nonpenetrating compound, and the membrane permeable dithiothreitol (DTT), have been shown consistently to reverse the above effects. MC-inducedd-aspartate release is completely inhibited by the addition of 1 mM DTT or GSH during the actual 5-min perfusion period with MC (5µM); when added after MC treatment, DTT fully inhibits the MC-inducedd-aspartate release, while GSH does not.3. Neither DTT nor GSH, in the absence of MC, have any effect on the rate of astrocyticd-aspartate release. Other studies demonstrate that although MC treatment (5µM) does not induce astrocytic swelling, its addition to astrocytes swollen by exposure to hypotonic medium leads to their failure to volume regulate.4. Omission of calcium from the medium greatly potentiates the effect of MC on astrocyticd-aspartate release, an effect which can be reversed by cotreatment of astrocytes with the dihydropyridine Ca²⁺-channel antagonist nimodipine (10µM), indicating that one possible route of MC entry into the cells is through voltage-gated L-type channels.     

Origin ofd-serine present in urine of mutant mice lackingd-amino-acid oxidase activity

Summary Urine of ddY/DAO mice lackingd-amino-acid oxidase contained 5.7 times more serine than that of normal ddY/DAO⁺ mice. Most of the serine wasd-isomer. The origin of this^d-serine was examined. Oral administration of 0.02% amoxicillin and 0.004% minocycline to the ddY/ DAO^- mice for 7 days did not reduce the urinaryd-serine, indicating that thed-serine was not of intestinal bacterial origin. When the mouse diet was changed to one with different compositions, the urinaryd-serine was considerably reduced. Furthermore, starvation of the ddY/DAO^- mice for 24 hours reduced the urinaryd-serine to 33% of the original level. These results indicate that most of the urinaryd-serine comes from the diet. However, the urine of the starved ddY/DAO^- mice still contained 4.6 times mored-serine than that of the ddY/DAO⁺ mice, suggesting a part of the D-serine have an endogenous origin.     

d-Aspartyl residue in a peptide can be liberated and metabolized by pig kidney enzymes

Summary The presence of an enzyme activity which hydrolyzes glycyl-d-aspartate was found in the homogenates of pig kidney cortex. The activity was inhibited by metal chelating agents and cilastatin, suggesting that the enzyme was a cilastatin-sensitive metallo-peptidase. Of the two hydrolysis products,d-aspartate was found to be less accumulated than glycine. The fate ofd-aspartate was, therefore, examined and the amino acid was found to be converted tol-aspartate,l-alanine and pyruvate, in the presence ofl-glutamate. Experiments with enzyme inhibitors suggested that the conversion involvedd-aspartate oxidase, aspartate aminotransferase and alanine aminotransferase as well as decarboxylation of oxaloacetate produced fromd-aspartate. All the results indicate that the enzymes in the pig kidney can liberate thed-aspartyl residue in the peptide and convert it to the compounds readily utilizable. The finding suggests a probable metabolic pathway of thed-aspartate-containing peptide.     

A Metal Ion as a Cofactor Attenuates Substrate Inhibition in the Enzymatic Production of a High Concentration of <Emphasis Type="SmallCaps">D</Emphasis>-glutamate Using <Emphasis Type="Italic">N</Emphasis>-acyl-<Emphasis Type="SmallCaps">D</Emphasis>-glutamate Amidohydrolase

N-Acyl-D-glutamate amidohydrolase (D-AGase) was inhibited by 94 % when 1 mol/l N-acetyl-DL- glutamate was used as a substrate. The addition of 1 mM Co²⁺ stabilized D-AGase. Moreover, the substrate inhibition was weakened to 88% with the addition of 0.4 mM Co²⁺ to the reaction mixture. Although D-AGase is a zinc-metalloenzyme, the addition of Zn²⁺ from 0.01 to 10 mM did not increase the D-glutamic acid production in the saturated substrate. Under optimal conditions, 0.38 M D-glutamic acid was obtained from N-acyl-DL-glutamate with 100% of the theoretical yield after 48 h.     

The VANA glycopeptide resistance protein is related to d-alanyl-d-alanine ligase cell wall biosynthesis enzymes

Summary Inducible resistance to the glycopeptide antibiotics vancomycin and teicoplanin is mediated by plasmid pIP816 in Enterococcus faecium strain BM4147. Vancomycin induced the synthesis of a ca. 40 kDa membrane-associated protein designated VANA. The resistance protein was partially purified and its N-terminal sequence was determined. A 1761 by DNA restriction fragment of pIP816 was cloned into Escherichia coli and sequenced. When expressed in E. coli, this fragment encoded a ca. 40 kDa protein that comigrated with VANA from enterococcal membrane fractions. The ATG translation initiation codon for VANA specified the methionine present at the N-terminus of the protein indicating the absence of signal peptide processing. The amino acid sequence deduced from the sequence of the vanA gene consisted of 343 amino acids giving a protein with a calculated Mr of 37400. VANA was structurally related to the d-alanyl-d-alanine (d-ala-d-ala) ligases of Salmonella typhimurium (36% amino acid identity) and of E. coli (28%). The vanA gene was able to transcomplement an E. coli mutant with thermosensitive d-ala-d-ala ligase activity. Thus, the inducible resistance protein VANA was structurally and functionally related to cytoplasmic enzymes that synthesize the target of glycopeptide antibiotics. Based on these observations we discuss the possibility that resistance is due to modification of the glycopeptide target.     

d-Aminoaciduria in mutant mice lackingd-amino-acid oxidase activity

Summary Urine of mutant ddY/DAO^– mice lackingd-amino-acid oxidase activity contained more serine and proline than that of normal ddY/DAO⁺ mice.d-Amino-acid oxidase treatment of urinary amino acids decreased the serine and proline, suggesting that they containedd-isomers. An HPLC analysis confirmed the presence ofd-serine. Urinary serine and proline contents were not decreased when the ddY/DAO^– mice were fed a diet which did not contain supplementaryd-methionine or when they were given water containing antibiotics. These results suggest that thed-serine andd-proline do not derive from thed-methionine supplemented in the diet or from intestinal bacteria. In urine of the ddY/DAO^– mice, a substance which seemed to bed-methionine sulfoxide and/ord-methionine sulfone was present. It is probably a metabolite of thed-methionine supplemented in the diet. Thed-aminoaciduria in the mutant mice lackingd-amino-acid oxidase activity indicates that this enzyme is involved in the metabolism of thed-amino acids in normal mice.     

Importance of the l-galactonolactone pool for enhancing the ascorbate content revealed by l -galactonolactone dehydrogenase-overexpressing tobacco plants

l-Galactono-1,4-lactone (GalL) dehydrogenase (GLDH) is an enzyme that catalyzes the last step of l-ascorbate (AsA) biosynthesis in plants. To re-evaluate the importance of the enzyme and the possibility of manipulating the AsA content in plants, a cDNA encoding GLDH from sweet potato was introduced into tobacco plants by Agrobacterium-mediated transformation under the control of a CaMV 35S promoter. Protein blot analysis revealed the elevation of GLDH protein contents in three GLDH-transformed lines. Furthermore, these transgenic lines showed 6- to 10-fold higher GLDH activities in the roots than the non-transformed plants, SR1. Despite the elevated GLDH activity, the AsA content in the leaves did not change in all lines; i.e., the AsA content in GLDH-transformed lines was 3–7 μmol g⁻¹ FW, comparable to that in the non-transformed plants. Incubation of leaf discs in a GalL solution led to a rapid 2- to 3-fold increase in the AsA content in both GLDH-transformed and non-transformed plants in the same manner. These results suggest that the supply of GalL is a crucial factor for determining the AsA pool size and that the upstream genes in the AsA biosynthetic pathway are responsible for enhancing the AsA content in plants.     

Biochemical and genetic characterization of l-glutamate transport and utilization in Salmonella typhimurium LT-2 mutants

Two systems for l-glutamate transport were found in Salmonella typhimurium LT-2 GltU⁺ (glutamate utilization) mutants. The first one is similar to the glt system previously described in Escherichia coli; by transductional analysis the structural gene, gltS, coding for the transport protein was located at minute 80 of the chromosome as part of the operon gltC-gltS, and its regulator, the gltR gene, near minute 90; the gltS gene product transports both l-glutamate and l-aspartate, is sodium independent, and is -hydroxyaspartate sensitive. The second transport system, whose structural gene was called gltF and is located at minute 0, was l-glutamate specific, sodium independent, and -methylglutamate sensitive. Two aspartase activities occurred in S. typhimurium LT-2: the first one was present only in the GltU⁺ mutants, had a pH 6.4 optimum, was essential for both l-glutamate and l-aspartate metabolism, and mapped at minute 94, close to the ampC gene. The second one had a pH 7.2 optimum, could be induced by several amino acids, and thus may have a general role in nitrogen metabolism.     

Crystal structures of rare disaccharides, α-d-glucopyranosyl β-d-psicofuranoside, and α-d-galactopyranosyl β-d-psicofuranoside

The crystal structures of α-d-glucopyranosyl β-d-psicofuranoside and α-d-galactopyranosyl β-d-psicofuranoside were determined by a single-crystal X-ray diffraction analysis, refined to R₁ = 0.0307 and 0.0438, respectively. Both disaccharides have a similar molecular structure, in which psicofuranose rings adopt an intermediate form between ⁴E and ⁴T₃. Unique molecular packing of the disaccharides was found in crystals, with the molecules forming a layered structure stacked along the y-axis.     

Development of a high-throughput method for the determination of pharmacological levels of plasma d-serine

d-Serine administration has been shown to be effective for the treatment of schizophrenia symptoms. However, d-serine must be administered at high doses to observe clinical effects. This is due in large part to d-serine undergoing oxidation by d-amino acid oxidase (DAAO) before it reaches the brain. Consequently, coadministration of d-serine with a DAAO inhibitor has been suggested as a way to lower the dose of d-serine required to treat schizophrenia. During the characterization of DAAO inhibitors as potential drugs, inhibitors are evaluated in rodents for their ability to increase plasma d-serine levels after oral coadministration. Current high-performance liquid chromatography (HPLC)-based methodologies to measure d-serine in plasma are time-consuming and are not amenable to concomitant analysis of multiple samples. We report the characterization of a 96-well format assay to monitor d-serine in plasma that greatly expedites analysis time. The assay involves the use of strong cation exchange solid phase extraction (SPE) to isolate d-serine from plasma followed by quantitation of d-serine using the DAAO-catalyzed reaction. Plasma d-serine determination using this assay could also be used as pharmacodynamic marker and as biomarker.     

Characterization of <Emphasis Type="SmallCaps">d</Emphasis>-tagatose-3-epimerase from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis> that converts <Emphasis Type="SmallCaps">d</Emphasis>-fructose into <Emphasis Type="SmallCaps">d-</Emphasis>psicose

A non-characterized gene, previously proposed as the d-tagatose-3-epimerase gene from Rhodobacter sphaeroides, was cloned and expressed in Escherichia coli. Its molecular mass was estimated to be 64 kDa with two identical subunits. The enzyme specificity was highest with d-fructose and decreased for other substrates in the order: d-tagatose, d-psicose, d-ribulose, d-xylulose and d-sorbose. Its activity was maximal at pH 9 and 40°C while being enhanced by Mn²⁺. At pH 9 and 40°C, 118 g d-psicose l⁻¹ was produced from 700 g d-fructose l⁻¹ after 3 h. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Physiological comparison of d-cysteine desulfhydrase of Escherichia coli with 3-chloro-d-alanine dehydrochlorinase of Pseudomonas putida CR 1-1

d-Cysteine desulfhydrase of Escherichia coli W3110 trpED102/F trpED102 was physiologically characterized. It was found to be located in the cytosolic fraction, as 3-chloro-d-alanine dehydrochlorinase is. d-Cysteine desulfhydrase catalyzed not only the ,-elimination reaction of O-acetyl-d-serine to form pyruvate, acetic acid and ammonia, but also the -replacement reaction of O-acetyl-d-serine with sulfide to form d-cysteine. However, these reactions appeared not to proceed in vivo. No other activity of d-cysteine synthesis from O-acetyl-d-serine and sulfide was detected in a crude cell extract of E. coli which was immunotitrated with antibodies raised against the purified d-cysteine desulfhydrase. Although d-cysteine desulfhydrase catalyzes the degradation (,-elimination reaction) of 3-chloro-d-alanine, which is an effective antibacterial agent, E. coli W3110 trpED102/F trpED102 did not show resistance against 3-chloro-d-alanine. Therefore, d-cysteine desulfhydrase does not contribute to 3-chloro-d-alanine detoxification in vivo.     

Organization and characterization of three genes involved in d-xylose catabolism in Lactobacillus pentosus

Summary A cluster of three genes involved in d-xylose catabolism (viz. xylose genes) in Lactobacillus pentosus has been cloned in Escherichia coli and characterized by nucleotide sequence analysis. The deduced gene products show considerable sequence similarity to a repressor protein involved in the regulation of expression of xylose genes in Bacillus subtilis (58%), to E. coli and B. subtilis d-xylose isomerase (68% and 77%, respectively), and to E. coli d-xylulose kinase (58%). The cloned genes represent functional xylose genes since they are able to complement the inability of a L. casei strain to ferment d-xylose. NMR analysis confirmed that ¹³C-xylose was converted into ¹³C-acetate in L. casei cells transformed with L. pentosus xylose genes but not in untransformed L. casei cells. Comparison with the aligned amino acid sequences of d-xylose isomerases of different bacteria suggests that L. pentosus d-xylose isomerase belongs to the same similarity group as B. subtilis and E. coli d-xylose isomerase and not to a second similarity group comprising d-xylose isomerases of Streptomyces violaceoniger, Ampullariella sp. and Actinoplanes. The organization of the L. pentosus xylose genes, 5-xylR (1167 bp, repressor) — xylA (1350 bp, D-xylose isomerase) — xylB (1506 bp, d-xylulose kinase) — 3 is similar to that in B. subtilis. In contrast to B. subtilis xylR, L. pentosus xylR is transcribed in the same direction as xylA and xylB.     

Release of arachidonic acid by NMDA-receptor activation in the rat hippocampus

In hippocampal slices arachidonic acid released after NMDA post-synaptic receptor activation is thought to act as a retrograde trans-synaptic messenger which facilitates the pre-synaptic release of L-glutamate to be involved in the expression of long-term synaptic potentiation (LTP). We measured the mass amount of arachidonic acid released from hippocampal slices incubated under conditions which maintain the electrophysiological responsiveness of the slice. Melittin released arachidonic, oleic and docosahexaenoic acids by phospholipase A₂ activation but not palmitic or stearic acids. Of greater interestl-glutamate, N-methyl-d-aspartate and incubation conditions known to induce LTP selectively and rapidly increased the release of archidonic acid in amounts over basal levels of 200–300 ng/mg protein. This is the first direct determination of the mass amount of arachidonic acid released following NMDA receptor activation in the hippocampus.Special issue dedicated to Dr. Louis Sokoloff.     

Automethylation of protein (d -aspartyl/l -isoaspartyl) carboxyl methyltransferase, a response to enzyme aging

A question that is central to understanding the mechanisms of aging and cellular deterioration is whether enzymes involved in recognition and metabolism of spontaneously damaged proteins are themselves damaged, either becoming substrates for their own activity; or being unable to act upon themselves, initiating cascades of cellular damage. We show here byin vitro experiments that protein (d-aspartyl/l-isoaspartyl) carboxyl methyltransferase (PCM) from bovine erythrocytes does methylate age-dependent amino acid damage in its own sequence. The subpopulation that is methylated, termed thePCM fraction, appears to be formed through age-dependent deamidation of an asparaginyl site to either anl-isoaspartyl ord-aspartyl site because (a) the stoichiometry of automethylation of purified PCM is less than 1%, a value typical of the substoichiometric methylation of many other aged protein substrates, (b)PCM is slightly more acidic than the bulk of PCM, and (c) the methyl esterified site inPCM has the characteristic base-lability of this type of methyl ester. Also, the methyl group is not incorporated into the enzyme as an active site intermediate because the incorporated methyl group is not chased onto substrate protein. The effect of enzyme dilution on the rate of the automethylation reaction is consistent with methylation occurring between protein molecules, showing that the pool of PCM is autocatalytic even though individual molecules may not be. The automethylation and possible self-repair of the PCM pool has implications for maintaining thein vivo efficiency of methylation-dependent protein repair.     

Determination of [d-Ala, d-Leu]enkephalin and the metabolites containing C-terminal d-leucine by high-performance liquid chromatography

A high-performance liquid chromatographic procedure has been developed for the determination of [d-Ala², d-Leu⁵]enkephalin (DADLE) and the fragments containing d-leucine in rat blood. The procedure was applied to the determination of blood levels of [³H-d-Leu⁵]DADLE and the C-terminal fragments after intravenous administration of [³H-d-Leu⁵]DADLE to a rat. Unlabelled DADLE and the C-terminal fragments were spiked as carriers to rat blood samples and the blood samples were extracted with 1% trifluoroacetic acid in methanol. The recoveries from rat blood were quantitative for all compounds. DADLE and the C-terminal four fragments were well separated on a reversed-phase column with gradient elution using a mobile phase composed of 0.14% HClO₄ and acetonitrile.     

Fermentation of <Emphasis Type="SmallCaps">d</Emphasis>-glucose and <Emphasis Type="SmallCaps">d</Emphasis>-xylose mixtures by <Emphasis Type="Italic">Candida tropicalis</Emphasis> NBRC 0618 for xylitol production

The fermentation of d-glucose and d-xylose mixtures by the yeast Candida tropicalis NBRC 0618 has been studied under the most favourable operation conditions for the culture, determining the most adequate initial proportion in these sugars for xylitol production. In all the experiments a synthetic culture medium was used, with an initial total substrate concentration of 25 g L⁻¹, a constant pH of 5.0 and a temperature of 30 °C. From the experimental results, it was deduced that the highest values of specific rates of production and of overall yield in xylitol were achieved for the mixtures with the highest percentage of d-xylose, specifically in the culture with the initial d-glucose and d-xylose concentrations of 1 and 24 g L⁻¹, respectively, with an overall xylitol yield of 0.28 g g⁻¹. In addition, the specific rates of xylitol production declined over the time course of the culture and the formation of this bioproduct was favoured by the presence of small quantities of d-glucose. The sum of the overall yield values in xylitol and ethanol for all the experiments ranged from 0.26 to 0.56 g bioproduct/g total substrate.     

Improving the thermostability of <Emphasis Type="Italic">N</Emphasis>-carbamyl-<Emphasis Type="SmallCaps">d</Emphasis>-amino acid amidohydrolase by error-prone PCR

To facilitate the easier production of d-amino acids using N-carbamyl-d-amino acid amidohydrolase (DCase) in an immobilized form, we improved the enzymatic thermostability of highly soluble DCase-M3 of Ralstonia pickettii using directed mutagenesis. Six novel mutation sites were identified in this study, apart from several thermostability-related amino acid sites reported previously. The most thermostable mutant, in which the 12th amino acid had been changed from glutamine to leucine, showed a 7 °C increase in thermostability. Comparative characterization of the parental and mutant DCases showed that although there was a slight reduction in the oxidative stability of the mutants, their kinetic properties and high solubility were not affected. The mutated enzymes are expected to be applied to the development of a fully enzymatic process for the industrial production of d-amino acids.