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Isolation and identification of ??-citryl-L-glutamic acid from newborn rat brain
Abstract
Ab unknwon compound containing glutamic acid residue was found in newborn rat brain. The compound occurred predominantly in brain. Its concentration was approx. 1 μmol/g tissue at birth and decreased to one-tenth 24 days after birth.The compound was isolated from newborn rat brains, and subjected to elementary analysis and to infrared and mass spectrometric analysis. Glutamic acid and citric acid were formed from the compound on acid hydrolysis. The compound was presumed to be a citryglutamic acid.Two isomers, α- and β-citrylglutamic acid, were sunthesized. The unknown compound was identified as β-citryl-L-glutamic acid. The occurrence of this compound has not been reported in nature.
... After e~aporation of water, the resulhng residue was dissolved in a minimal amount of methanol and subjecte0 to preparative TLC on pre-coated cellulose plates (Merck) with a solvent system of n-butanol-formic acid-H20 (20: 20:1). The active spot at Rf of 0.36, which gave an orange leaction with p-dimethylaminobenzaldehyde-acetic anhydride (Altman) reagent [14], was scraped off and extracted with methanol, Evaporation of the solvent gave a pale yellow oil. The overall yield was approx. ...
... The crude extract prepared from 10 I of the supernatant was further purified by column chromatography on Bio-gel P2 and subsequently on TSK-gel. TLC of the active concentrate from the TSK-gel column gave two major colored spots at Rf 0.36 (orange) and 0.54 (pink) after spraying with Altman reagent which visualizes acidic compounds [14]. Only the compound at Rf 0.36 was active in reversing growth inhibition by EDDA, as shown in Fig. 1. ...
A compound with siderophore activity was purified by successive column and thin layer chromatographic procedures from Dowex 1 x 8 extracts of culture supernatants of Vibrio parahaemolyticus AQ 3354. The strain synthesized the compound in culture media containing less than 2 microM added FeCl3. Hydrolysis of the compound yielded alanine, ethanolamine, citric acid and 2-ketoglutaric acid. The 1H-NMR spectrum exhibited the presence of a residue from each of these components in the intact molecule. The fast-atom bombardment mass spectrum of the methyl ester derivative indicated a prominent ion at m/z 477, probably corresponding to [M + 1] ion. Other strains of V. parahaemolyticus were also found to produce this compound when grown in an iron-limited medium.
... β-citryl-L-glutamate (bCG) was first identified in rat brain by [14]. In the rat nervous system, bCG level is highest at birth and then continuously decreases during postnatal development [15]. ...
Chromatin remodelling in spermatids is an essential step in spermiogenesis and involves the exchange of most histones by protamines, which drives chromatin condensation in late spermatids. The gene Rimklb encodes a citrylglutamate synthase highly expressed in testes of vertebrates and the increase of its reaction product, β-citrylglutamate, correlates in time with the appearance of spermatids. Here we show that deficiency in a functional Rimklb gene leads to male subfertility, which could be partially rescued by in vitro fertilization. Rimklb-deficient mice are impaired in a late step of spermiogenesis and produce spermatozoa with abnormally shaped heads and nuclei. Sperm chromatin in Rimklb-deficient mice was less condensed and showed impaired histone to protamine exchange and retained transition protein 2. These observations suggest that citrylglutamate synthase, probably via its reaction product β-citrylglutamate, is essential for efficient chromatin remodelling during spermiogenesis and may be a possible candidate gene for male subfertility or infertility in humans.
... L-acetylcarnitine (LAC) has a known biological function of improving the efficiency of mitochondrial function by facilitating the movement of acetyl CoA into the matrices (Wutzke and Lorenz 2004 . These features both correlated to beta-citryl-Lglutamic acid (BCG), which is a derivative of glutamate found in the developing brain of rats (Miyake, Kakimoto, and Sorimachi 1978). This metabolite has been shown to be an iron carrier that is used to serotonin (Weissbach, Redfield, and Axelrod 1960), and has been shown to act as a classic antioxidant through hydrogen donation (Barsacchi et al. 1998). ...
Acute social defeat represents a naturalistic form of conditioned fear and is an excellent model in which to investigate the biological basis of stress resilience. While there is growing interest in identifying biomarkers of stress resilience, until recently, it has not been feasible to associate levels of large numbers of neurochemicals and metabolites to stress-related phenotypes. The objective of the present study was to use an untargeted metabolomics approach to identify known and unknown neurochemicals in select brain regions that distinguish susceptible and resistant individuals in two rodent models of acute social defeat. In the first experiment, male mice were first phenotyped as resistant or susceptible. Then, mice were subjected to acute social defeat, and tissues were immediately collected from the ventral medial prefrontal cortex (vmPFC), basolateral/central amygdala (BLA/CeA), nucleus accumbens (NAc), and dorsal hippocampus (dHPC). Ultra-high performance liquid chromatography coupled with high resolution mass spectrometry (UPLC-HRMS) was used for the detection of water-soluble neurochemicals. In the second experiment, male Syrian hamsters were paired in daily agonistic encounters for 2 weeks, during which they formed stable dominant-subordinate relationships. Then, 24 h after the last dominance encounter, animals were exposed to acute social defeat stress. Immediately after social defeat, tissue was collected from the vmPFC, BLA/CeA, NAc, and dHPC for analysis using UPLC-HRMS. Although no single biomarker characterized stress-related phenotypes in both species, commonalities were found. For instance, in both model systems, animals resistant to social defeat stress also show increased concentration of molecules to protect against oxidative stress in the NAc and vmPFC. Additionally, in both mice and hamsters, unidentified spectral features were preliminarily annotated as potential targets for future experiments. Overall, these findings suggest that a metabolomics approach can identify functional groups of neurochemicals that may serve as novel targets for the diagnosis, treatment, or prevention of stress-related mental illness.
... We now show that RIMKLA can also ligate a second glutamate to BCG, forming the previously unknown BCG 2 . The intriguing metabolite BCG was first identified in newborn rat brain, where it is present up to low millimolar levels (60). It is present at particularly high levels in the developing brain of many species but decreases to high micromolar levels with age (61). ...
The ubiquitous efflux transporter ABCC5 (ATP-binding cassette subfamily C member 5) is present at high levels in the blood-brain
barrier, neurons, and glia, but its in vivo substrates and function are not known. Using untargeted metabolomic screens, we show that Abcc5−/− mice accumulate endogenous glutamate conjugates in several tissues, but brain in particular. The abundant neurotransmitter
N-acetylaspartylglutamate was 2.4-fold higher in Abcc5−/− brain. The metabolites that accumulated in Abcc5−/− tissues were depleted in cultured cells that overexpressed human ABCC5. In a vesicular membrane transport assay, ABCC5 also
transported exogenous glutamate analogs, like the classic excitotoxic neurotoxins kainic acid, domoic acid, and NMDA; the
therapeutic glutamate analog ZJ43; and, as previously shown, the anti-cancer drug methotrexate. Glutamate conjugates and analogs
are of physiological relevance because they can affect the function of glutamate, the principal excitatory neurotransmitter
in the brain. After CO2 asphyxiation, several immediate early genes were expressed at lower levels in Abcc5−/− brains than in wild type brains, suggesting altered glutamate signaling. Our results show that ABCC5 is a general glutamate
conjugate and analog transporter that affects the disposition of endogenous metabolites, toxins, and drugs.
... BCG is also detected in kidneys, heart, and to a much lower extent in intestine, spinal cord, and lungs of young rats. The content of BCG in all organs decreases rapidly after birth to the noticeable exception of testes, where its concentration increases during sexual maturation and remains constant during adulthood (1)(2)(3). Although the exact physiological function of BCG is presently unknown, different observations suggest that it may play an important role during brain development and spermatogenesis (4,5). ...
β-Citrylglutamate (BCG), a compound present in adult testis and in the CNS during the pre- and perinatal periods is synthesized by an intracellular enzyme encoded by the RIMKLB gene and hydrolyzed by an as yet unidentified ectoenzyme. To identify β-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca(2+), the purified enzyme specifically hydrolyzed β-citrylglutamate and did not act on N-acetyl-aspartylglutamate (NAAG). However, both compounds were hydrolyzed in the presence of Mn(2+). This behavior and the fact that the enzyme was glycosylated and membrane-bound suggested that β-citrylglutamate hydrolase belonged to the same family of protein as glutamate carboxypeptidase 2 (GCP2), the enzyme that catalyzes the hydrolysis of N-acetyl-aspartylglutamate. The mouse tissue distribution of β-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to β-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed β-citrylglutamate in the presence of Ca(2+), and acted on both N-acetyl-aspartylglutamate and β-citrylglutamate in the presence of Mn(2+), whereas recombinant GCP2 only hydrolyzed N-acetyl-aspartylglutamate and this, in a metal-independent manner. A comparison of the structures of the catalytic sites of GCP2 and GCP3, as well as mutagenesis experiments revealed that a single amino acid substitution (Asn-519 in GCP2, Ser-509 in GCP3) is largely responsible for GCP3 being able to hydrolyze β-citrylglutamate. Based on the crystal structure of GCP3 and kinetic analysis, we propose that GCP3 forms a labile catalytic Zn-Ca cluster that is critical for its β-citrylglutamate hydrolase activity.
In an era when population aging is increasing the burden of neurodegenerative conditions, deciphering the mechanisms underlying brain senescence is more important than ever. Here, we present a spatial metabolomics analysis of age-induced neurochemical alterations in the mouse brain using negative ionization mode mass spectrometry imaging. The age-dependent effects of the acetylcholinesterase inhibitor tacrine were simultaneously examined. For ultrahigh mass resolution analysis, we utilized a Fourier-transform ion cyclotron resonance spectrometer. To complement this, a trapped ion mobility spectrometry time-of-flight analyzer provided high speed and lateral resolution. The chosen approach facilitated the detection and identification of a wide range of metabolites, from amino acids to sphingolipids. We reported significant, age-dependent alterations in brain lipids which were most evident for sulfatides and lysophosphatidic acids. Sulfatide species, which are mainly localized to white matter, either increased or decreased with age, depending on the carbon chain length and hydroxylation stage. Lysophosphatidic acids were found to decrease with age in the detailed cortical and hippocampal subregions. An age-dependent increase in the glutamine/glutamate ratio, an indicator of glia-neuron interconnection and neurotoxicity, was detected after tacrine administration. The presented metabolic mapping approach was able to provide visualizations of the lipid signaling and neurotransmission alterations induced by early aging and can thus be beneficial to further elucidating age-related neurochemical pathways.
Resistance training promotes metabolic health and stimulates muscle hypertrophy, but the precise routes by which resistance exercise (RE) conveys these health benefits are largely unknown.
Aim:
To investigate how acute RE affects human skeletal muscle metabolism.
Methods:
We collected vastus lateralis biopsies from six healthy male untrained volunteers at rest, before the first of 13 RE training sessions, and 45 min after the first and last bouts of RE. Biopsies were analysed using untargeted mass spectrometry-based metabolomics.
Results:
We measured 617 metabolites covering a broad range of metabolic pathways. In the untrained state RE altered 33 metabolites, including increased 3-methylhistidine and N-lactoylvaline, suggesting increased protein breakdown, as well as metabolites linked to ATP (xanthosine) and NAD (N1-methyl-2-pyridone-5-carboxamide) metabolism; the bile acid chenodeoxycholate also increased in response to RE in muscle opposing previous findings in blood. Resistance training led to muscle hypertrophy, with slow type I and fast/intermediate type II muscle fibre diameter increasing by 10.7% and 10.4%, respectively. Comparison of post-exercise metabolite levels between trained and untrained state revealed alterations of 46 metabolites, including decreased N-acetylated ketogenic amino acids and increased beta-citrylglutamate which might support growth. Only five of the metabolites that changed after acute exercise in the untrained state were altered after chronic training, indicating that training induces multiple metabolic changes not directly related to the acute exercise response.
Conclusion:
The human skeletal muscle metabolome is sensitive towards acute RE in the trained and untrained states and reflects a broad range of adaptive processes in response to repeated stimulation.
Sleep has evolved as a universal core function to allow for restorative biological processes. Detailed knowledge of metabolic changes necessary for the sleep state in the brain is missing. Herein, we have performed an in-depth metabolic analysis of four mouse brain regions and uncovered region-specific circadian variations. Metabolites linked to oxidative stress were altered during sleep including acylcarnitines, hydroxylated fatty acids, phenolic compounds, and thiol-containing metabolites. These findings provide molecular evidence of a significant metabolic shift of the brain energy metabolism. Specific alterations were observed for brain metabolites that have previously not been associated with a circadian function including the microbiome-derived metabolite ergothioneine that suggests a regulatory function. The pseudopeptide β-citryl-glutamate has been linked to brain development and we have now discovered a previously unknown regioisomer. These metabolites altered by the circadian rhythm represent the foundation for hypothesis-driven studies of the underlying metabolic processes and their function.
E-mail: dulee@dongguk.ac.krReceived July 1, 2008Key Words : Trimethylcitryl-β-D-galactopyranoside, Practical synthesis, Glycosylation, DeacetylationThe rhizome of Gastrodia elata Blume (GastrodiaeRhizoma, Orchidaceae) has been used in traditional medi-cine as an anticonvulsant and sedatives in Korea, Japan andChina.
Unlabelled:
Glutamate carboxypeptidase III (GCPIII) is best known as a homologue of glutamate carboxypeptidase II [GCPII; also known as prostate-specific membrane antigen (PSMA)], a protease involved in neurological disorders and overexpressed in a number of solid cancers. However, mouse GCPIII was recently shown to cleave β-citrylglutamate (BCG), suggesting that these two closely related enzymes have distinct functions. To develop a tool to dissect, evaluate and quantify the activities of human GCPII and GCPIII, we analysed the catalytic efficiencies of these enzymes towards three physiological substrates. We observed a high efficiency of BCG cleavage by GCPIII but not GCPII. We also identified a strong modulation of GCPIII enzymatic activity by divalent cations, while we did not observe this effect for GCPII. Additionally, we used X-ray crystallography and computational modelling (quantum and molecular mechanical calculations) to describe the mechanism of BCG binding to the active sites of GCPII and GCPIII, respectively. Finally, we took advantage of the substantial differences in the enzymatic efficiencies of GCPII and GCPIII towards their substrates, using enzymatic assays for specific detection of these proteins in human tissues. Our findings suggest that GCPIII may not act merely as a complementary enzyme to GCPII, and it more likely possesses a specific physiological function related to BCG metabolism in the human body.
Database:
The X-ray structure of GCPII Glu424Ala in complex with BCG has been deposited in the RCSB Protein Data Bank under accession code 5F09.
Defects in the gene encoding the persulfide dioxygenase ETHE1 are known to cause the severe inherited metabolic disorder ethylmalonic encephalopathy (EE). In spite of known clinical characteristics, the molecular mechanisms underlying the ETHE1 deficiency are still obscure. Herein, to further analyze the molecular phenotype of the disease, we applied an untargeted metabolomics approach on cultivated fibroblasts of EE patients for pinpointing alterations in metabolite levels. Metabolites, as direct signatures of biochemical functions, can decipher biochemical pathways involved in the cellular phenotype of patient cells. Using liquid chromatography-mass spectrometry-based untargeted metabolomics, we identified 18 metabolites that have altered levels in fibroblasts from EE patients. Our data demonstrate disrupted redox state in EE patient cells, which is reflected by significantly decreased level of reduced glutathione. Furthermore, the down-regulation of several intermediate metabolites such as the redox cofactors NAD(+) and NADH as well as Krebs cycle intermediates revealed clear alteration in metabolic regulation. Pantothenic acid and several amino acids exhibited decreased levels, whereas the β-citrylglutamate with a putative role in brain development had an increased level in the EE patient cells. These observations indicate the severe impact of ETHE1 deficiency on cellular physiology and redox state, meanwhile suggesting targets for experimental studies on novel treatment options for the devastating metabolic disorder.
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Developmental changes in the concentration of β‐citryl‐L‐glutamate (β‐CG) have been examined in the cerebrum and optic lobe of the developing chick brain and in primary cultured neuronal cells from the chick embryo optic lobes with gas chromatographic and HPLC methods originated in our studies. A sharp peak was shown by β‐CG, with a maximal concentration at 13 days of incubation in the optic lobe of the developing chick brain but decreasing markedly to adult levels. The developmental change in primary cultured neurons was similar to that in the optic lobe of the developing chick brain. Changes in synthetic and hydrolytic activities of β‐CG were studied during growth of primary cultured neurons. Incorporation of radioactivities from radiolabeled pyruvate and alanine into β‐CG increased significantly on day 3 of culture, reaching a plateau on day 6, whereas that from radioactive glutamine and glutamate increased gradually from day 3 to day 12 of culture. The hydrolyzing enzyme activity of β‐CG during neuron growth was low until day 3 of culture, when it increased significantly until day 12. Similar developmental changes were observed in the developing chick embryo optic lobes.
It has been reported that the activity of mitochondrial aconitase (m-aconitase) is rapidly inhibited in a variety of cells when exposed to nitric oxide (NO). In present study, we found that NO significantly increased the number of surviving neurons via enhanced mitochondrial functions with simultaneous addition of the [Fe(II)(β-citryl-L-glutamate; β-CG)] complex. In vitro, a variety of aconitase-inhibitors, such as fluorocitrate, cyanide ion, ferricyanide ([Fe(CN)6]), and various oxidants including superoxide anion, inhibited the activity of m-aconitase even in the presence of Fe(II), whereas a NO-donor, nitroprusside (SNP) ([Fe(CN)5NO]), was the only agent that significantly increased activity of that enzyme. Therefore, it is reasonable to assume that NO released from SNP promotes Fe-dependent activation of aconitase. All other tested NO-donors, including 3-morpholino-sydnonimine (SIN), Deta NONOate (NOC18), and NaNO2, also promoted activation of m-aconitase in time- and dose-dependent manners in the presence of Fe(II). The promoting effects of the NO-donors on activation disappeared with the addition of NO-scavengers. In intact mitochondria, all tested NO-donors promoted reactivation of aconitase in a dose-dependent manner in the presence of Fe(II), whereas that was not seen in its absence. These findings suggest that NO released from NO-donors promotes Fe-dependent activation of aconitase. In mixed neuronal and glial cultures, NO-donors except for SNP enhanced mitochondrial activity at low concentrations. Furthermore, simultaneous addition of the [Fe(II)(β-CG)] complex significantly enhanced those activities and greatly increased the number of surviving neurons. Thus, NO can carry Fe ions into m-aconitase via the guide of the tag of β-CG addressed to the enzyme.
A convenient method for the synthesis of symmetric and asymmetric diamides of amino acids including DOPA and citric acid from 2-tert-butyl-1,3-di(N-hydroxysuccinimidyl)citrate and 1-tert-butyl-2,3-di(N-hydroxysuccinimidyl)citrate is described.
Asym-monomethyl citrate was prepared by hydrolysis of sym-dimethyl citrate. Sym-monomethyl and asym-dimethyl citrate were prepared by a selective hydrolysis of trimethyl citrate.
ß-Citryl-l-glutamate-hydrolysing enzyme (ß-CGHE) was purified from rat testis particulate fraction 13 000-fold, at a yield of 7%. The enzyme was purified by ammonium sulfate fractionation, hydroxyapatite, chelating Sepharose, ß-CG-Sepharose affinity chromatography and Sephacryl S-300 gel filtration. The purified enzyme usually migrated as two periodic acid Schiff's-stained bands on native polyacrylamide gel-electrophoresis (PAGE) with molecular weights of 350 and 420 kDa. Both bands hydrolyzed ß-citryl-l-glutamate (ß-CG) to citrate and glutamate. The 420 kDa band was changed by digestion with N-glycosidase F, into a 350 kDa band on native PAGE. The purified enzyme was composed of 90, 100, 115 and 130 kDa subunits on SDS-PAGE under non-reduced conditions. The purified enzyme was pharmacologically similar to the ß-CGHE activity partially purified from rat testis. This enzyme required manganese ions for full activity and it was strongly inhibited by nucleotides such as ATP or GTP and phosphate ions. ß-CGHE was also potently inhibited by an excitatory amino acid agonist, l-quisqualate, but not by another agonists, and kinate. It had high substrate specificity for ß-CG. The antibodies against the purified enzyme reacted mainly to the 115 kDa band on the SDS-PAGE and precipitated the enzyme activity from the crude and purified enzyme solution.
Physico-chemical and chemical properties of citric acid and methods of synthesis of its derivatives, namely esters, amides, 1,3-dioxolan-4-ones etc. are presented. The important group of natural derivatives of this acid belonging to the siderophore family, i.e. compounds fulfilling in nature the function of biocarriers of ferric ions, is described in detail. The recent advances in the total synthesis of natural siderophores — citric acid derivatives, its homologues and analogues of the “retro” type are summarized.
Abstract The developmental changes of N-acetylaspartic acid (NA-Asp), N-acetyl--aspartylglutamic acid (NA-Asp-Glu), and β-citryl-L-glutamic acid (β-CG) have been examined in the cerebrum, cerebellum, brain stem and spinal cord of both rat and guinea pig by the gas chromatographic method developed in our studies. A rapid increase in the concentration of NA-Asp was observed postnatally in every region of the rat brain. On the other hand, all regions of guinea pig brain showed the prenatal increases. NA-Asp-Glu showed a different developmental profile, depending on region of the brain, in the two species. The concentration of NA-Asp-Glu remained constantly low during brain maturation in the rostral regions. In the caudal portions it showed a marked increase during maturation and reached a high level in the adult brain. The concentration of β-CG was highest at birth in all regions of rat brain and rapidly decreased by 20 days after birth and remained low thereafter. The rapid decrease occurred in the guinea pig during the foetal period, and β-CG content decreased to an adult level at birth.
A simple and sensitive gas-chromatographic method for the determination of N-acetyl-l-aspartic acid (NA-Asp), N-acetyl-α-aspartylglutamic acid (NA-Asp-Glu) and β-citryl-l-glutamic acid (β-CG) was developed. The organ, regional and phylogenetic distributions of these compounds were studied. NA-Asp and NA-Asp-Glu were highly concentrated in nervous tissue, and less than 1% of the amounts in the nervous tissues were found in nonnervous organs. These two compounds showed a reciprocal relationship in their regional distribution in mature brains, but such a relationship was not evident or was even reversed in immature brains. The two compounds also showed different developmental changes in different regions of the brain. Fish brain contained a relatively high concentration of NA-Asp, but only a trace amount of NA-Asp-Glu. By contrast, a 10 times higher concentration of NA-Asp-Glu than NA-Asp was found in frog brain. Reptilian brain contained similar amounts of each compound. Avian and mammalian brain had NA-Asp at a roughly 10 times higher concentration than NA-Asp-Glu. β-CG occurred at the highest concentration in the immature brain of rat and guinea pig, but disappeared in the mature brains. The adult frog brain, however, contained a large amount of β-CG. In the adult rat, testis contained the highest concentration of β-CG.
The compound β-citryl-L-glutamate (β-CG) was initially isolated from developing brains, though its functional roles remain unclear. In in vitro experiments, the [Fe(II)(β-CG)] complex activated aconitase in the presence of reducing reagents, whereas no Fe complex with citrate, glutamate, or deferoxamine displayed such an effect. β-CG and [Fe(II)(β-CG)] both bound to the fourth labile Fe atom (Fe(a)) in the [4Fe-4S] cluster of aconitase. Furthermore, [Fe(II)(β-CG)] reactivated aconitase damaged by ammonium peroxodisulfate (APS), while β-CG and citrate had no effect. These findings suggest that [Fe(II)(β-CG)] can transfer Fe to aconitase disassembled by APS. In intact mitochondria, both β-CG and [Fe(II)(β-CG)] bound to Fe(a) of aconitase, whereas only [Fe(II)(β-CG)] reactivated the enzyme disassembled by APS. In cultured neuronal cells, β-CG significantly enhanced cell viability by accelerating mitochondrial activity in primary cultures of neurons from newborn mouse cerebrum tissues. Thus, the β-CG plays a role as an Fe-carrier for mitochondrial aconitase, and then activates it. Taken together, these findings suggest that β-CG is an endogenous low molecular weight Fe chaperone for aconitase.
β-Citryl-L-glutamate (β-CG) is a unique compound initially isolated from developing brains, which also appears in high concentrations during the period characterized by growth and differentiation of neurons in developing animals, and then decreases with maturation. However, its functional roles remain unclear. The stability constant obtained in our previous pH titration studies showed that β-CG forms relatively strong complexes with copper. Reactive oxygen species (ROS) and nitric oxide (NO) have been suggested to act as mediators of the cell death that occurs in neurons during development of the nervous system. However, regulation of ROS and NO formation by Cu in the developing brain remains poorly understood. The activity of superoxide dismutase (SOD), a key superoxide scavenging enzyme, is low in the developing brain. Furthermore, xanthine oxidase (XO) has been implicated in diverse pathological situations due to its capability of generating both ROS and NO. Therefore, we examined the effects of β-CG and its Cu-complex on SOD and XO activities. We found that the [Cu(II)(β-CG)] complex had SOD activity and a strong competitive inhibition of XO, while reduced glutathione caused concentration-dependent decreases of the XO inhibitory activities in the [Cu(II)(β-CG)] complex.
The purpose of the present work was to determine the identity of the enzymes that synthesize N-acetylaspartylglutamate (NAAG), the most abundant dipeptide present in vertebrate central nervous system (CNS), and β-citrylglutamate,
a structural analogue of NAAG present in testis and immature brain. Previous evidence suggests that NAAG is not synthesized
on ribosomes but presumably is synthesized by a ligase. As attempts to detect this ligase in brain extracts failed, we searched
the mammalian genomes for putative enzymes that could catalyze this type of reaction. Mammalian genomes were found to encode
two putative ligases homologous to Escherichia coli RIMK, which ligates glutamates to the C terminus of ribosomal protein S6. One of them, named RIMKLA, is almost exclusively
expressed in the CNS, whereas RIMKLB, which shares 65% sequence identity with RIMKLA, is expressed in CNS and testis. Both
proteins were expressed in bacteria or HEK293T cells and purified. RIMKLA catalyzed the ATP-dependent synthesis of N-acetylaspartylglutamate from N-acetylaspartate and l-glutamate. RIMKLB catalyzed this reaction as well as the synthesis of β-citrylglutamate. The nature of the reaction products
was confirmed by mass spectrometry and NMR. RIMKLA was shown to produce stoichiometric amounts of NAAG and ADP, in agreement
with its belonging to the ATP-grasp family of ligases. The molecular identification of these two enzymes will facilitate progress
in the understanding of the function of NAAG and β-citrylglutamate.
The compound beta-citryl-L-glutamate (beta-CG) was initially isolated from developing brains, while it has also been found in high concentrations in testes and eyes. However, its functional roles are unclear. To evaluate its coordination with metal ions, we performed pH titration experiments. The stability constant, logbeta(pqr) for M(p)(beta-CG)(q)H(r) was calculated from pH titration data, which showed that beta-CG forms relatively strong complexes with Fe(III), Cu(II), Fe(II) and Zn(II). beta-CG was also found able to solubilize Fe more effectively from Fe(OH)(2) than from Fe(OH)(3). Therefore, we examined the effects of beta-CG on Fe-dependent reactive oxygen species (ROS)-generating systems, as well as the potential ROS-scavenging activities of beta-CG and metal ion-(beta-CG) complexes. beta-CG inhibited the Fe-dependent degradation of deoxyribose and Fe-dependent damage to DNA or plasmid DNA in a dose-dependent manner, whereas it had no effect on Cu-mediated DNA damage. In addition, thermodynamic data showed that beta-CG in a physiological pH solution is an Fe(II) chelator rather than an Fe(III) chelator. Taken together, these findings suggest that beta-CG is an endogenous low molecular weight Fe chelator.
An enzyme responsible for the deacylation of beta-citryl-L-glutamate to citrate and glutamate has been characterized in rat testis. The enzyme required manganese ion for full activity and was strongly inhibited by nucleotides such as ATP or GTP. The activity was localized in the particulate fractions. The enzyme favored N-formyl-L-glutamate greater than beta-citrly-L-glutamate greater than beta-citryl-L-glutamine in a decreasing order. The amidohydrolyase activity was highest in the testis and lung, a moderate activity was detected in heart, kidney and intestine, and low in brain, thymus, stomach, skeletal muscle, spleen and liver. These findings suggest that the amidohydrolase is different from any of amidohydrolases reported so far, amidohydrolase I (EC 3.5.1.14), II (EC 3.5.1.15), III, N-acetyl-lysine deacylase (EC 3.5.1.17) and N-acetyl-beta-alanine deacetylase (EC 3.5.1.21), and various peptidases.
beta-Citryl-L-glutamic acid, which is known to be highly concentrated in the brains of immature animals, is preferentially localized in the testes of various adult animals, including mammals, amphibians and fish, mainly in the germinal cells. In young rats, the citrylglutamate concentration increases with age and coincides with the development of late spermatocytes into early spermatids. Rats with seminiferous tubule failure induced by ductuli efferentes ligation and experimental cryptorchidism are infertile as a result of germ cell depletion, especially spermatocytes and early spermatids. In these animals, the testicular citrylglutamate content was much lower than in normal testes.
N-Acetyl-L-aspartic acid (NA-Asp), N-acetyl-alpha-L-aspartyl-L-glutamic acid (NA-Asp-Glu) and beta-citryl-L-glutamic acid (beta-CG), which are known to occur in the brain, have been isolated from human urine. Their identities were proved by comparing them with synthetic NA-Asp, NA-Asp-Glu and beta-CG using electrophoretic and chromatographic methods and by acid hydrolysis. A method was developed for the quantitation of NA-Asp, NA-Asp-Glu and beta-CG in human urine. It consists of ion-exchange chromatography followed by gas-chromatographic analysis. The amounts of urinary excretion of NA-Asp, NA-Asp-Glu and beta-CG were 41.2 +/- 10.1 (n = 27), 20.8 +/- 9.6 (n = 27) and 30.2 +/- 13.2 (n = 21) mumol/g creatinine in adult males, and 62.2 +/- 16.3 (n = 27), 24.0 +/- 8.2 (n = 27) and 40.5 +/- 21.1 (n = 24) mumol/g creatinine in adult females, respectively.
The beta-CG concentration in the chicken brain was high during embryonic development and decreased rapidly to a lower level close to hatching, while the concentration in the eyeball which was also high during the embryonic life retained a fairly high level after hatching. The distribution of beta-CG in the bovine eye was determined. About 95% of total beta-CG content in the whole eye was localized in the lens. However, the distribution of beta-CG in the eye varied depending on species. beta-CG was exclusively localized in the lens in the eyes of fish and mammals, but distributed in both lens and retina in frogs. The molecule was localized in the retina rather than the lens in the chicken eye, although the concentrations was extremely low compared to those in the mammalian, amphibian and fish eyes. It was found that beta-CG is present ubiquitously in the lens or retina in various species. The distribution of beta-CG in the bovine lens was determined in the three cortex regions and nucleus. beta-CG was present at the highest concentration in the equatorial cortex, at a moderate concentration in the posterior and anterior cortex, and at the lowest concentration in the nucleus. Similar distribution patterns were also found in the rabbit and rat lens. When embryonic chick lens epithelial cells were cultured in the presence of fetal calf serum, the cells elongated, differentiated into fiber cells and formed lentoid bodies. The cells of lentoid bodies were stained strongly by the anti-beta-CG antibody, while cells around the structures were not. In addition, the beta-CG content in the lenses from the galactose cataractous rat decreased to about 20-30% of that in the normal lens. These findings suggest that beta-CG may play a role in the differentiation of epithelial cells into fiber cells.
Beta-citryl-L-glutamate (beta-CG) concentration was determined by HPLC during the differentiation of bovine lens epithelial cells into lens fiber cells in culture. beta-CG increased from 1 to 4 weeks of culture and then decreased slightly, while alpha-crystallin, a marker of lens cell differentiation, increased rapidly 4 weeks after the culture and continued to increase gradually until week 11. In addition, the localization of beta-CG was immunohistochemically examined using anti-beta-CG antibody. Cells around lentoid bodies were stained with anti-beta-CG antibody, whereas cells in the bodies were stained strongly with anti-gamma-crystallin antibody. These findings suggest that beta-CG accumulated immediately before the differentiation of the bovine lens epithelial cells into lens fiber cells and may play a role in regulating the differentiation of lens cells.
The immunocytochemical localization of beta-citryl-L-glutamate (beta-CG) in primary neuronal cells and in the differentiation of P19 cells was examined. 1: Cells with the morphological features of neurons in the primary culture were specifically stained with the anti-beta-CG antibody both in neurites and in the cell body. 2: The neuronal cells differentiated from P19 cells were distinctly stained with the anti-beta-CG antibody both in neurites and in the cell body, while the non-neuronal cells were not. 3: The concentration of beta-CG was low in the P19 cells, but increased significantly with the differentiation of P19 cells into neurons. It was shown that beta-CG was localized exclusively in neurons. These findings suggest that beta-CG plays functional roles in the differentiation and growth of neuron.
A cDNA encoding rat homologue of the previously characterized mouse Sox6 was isolated by a polymerase chain reaction (PCR) cloning strategy. Comparison of this eDNA with homologous mouse, human and rainbow trout cDNA exhibited an overall amino acid sequence identity of 99.6, 89.3 and 76.3% respectively. The leucine-zipper and HMG-box motif were almost completely conserved between these homologues. The expression of Sox6 was determined in rat by Northern hybridization and Real-time quantitative reverse transcription (RT)-PCR. rSox6 (rat Sox6) was specifically expressed in the neonatal brain and adult testis with Northern blotting. Real-time quantitative RT-PCR for the determination of Sox6 mRNA was examined. The rSox6 was expressed in the neonatal brain and adult testis as well as by Northern blotting and also expressed in the adult eyeball and slightly in the ovary.
The microacidity constants for citric acid have been redetermined from pH titrations of citric acid and selected methyl esters in aqueous solution at 25°C and at two ionic strengths. The results obtained differ from those previously published. Calculated acidity constants, derived from a careful consideration of the empirical substituent effect, are also given and are in reasonable agreement with the experimental acidity constants.
N-Acetylaspartic acid (NAA) occurs at relatively high concentrations exclusively in the mammalian and avian brain and undergoes rapid rise in level soon after birth (Tallan, 1957).
The amount of NAA in brains of mentally abnormal human beings and of young human beings was measured. The route by which NAA is synthesized was shown to involve a direct acetylation of aspartic acid. The degradative activity of the brain toward NAA is slight. Some experiments indicate that NAA in the brain is a physiologically and metabolically active compound.
The acetylated amino acid N-acetyl-l-aspartic acid (NA-Asp)* was first isolated from mammalian tissue by Tallan et al. (1956). It is present in adult mammalian brain at a concentration of 5–6 μmol/g tissue, but the values in nonmammalian species are variable. Values range from 1.1 μmol/g in reptilian brain to 5–8 μmol/g in fish brain and 6 μmol/g in avian brain. It is undetectable in frog brain and in the central ganglia of invertebrates such as the lobster and horseshoe crab (Tallan, 1957; Tsukada et al., 1964). The concentration of NA-Asp in the brain of the newborn rat or rabbit is one-fifth that of the adult—adult levels are reached in 20 days (Tallan, 1957; Jacobson, 1959). In other tissues, the level of NA-Asp is significantly lower; e.g., in the liver and kidney of the cat, values range from only 0.06 to 0.17 μmol/g tissue (Tallan, 1957).
This chapter discusses anatomical, biochemical, and neurophysiological changes with the development of functional and behavioral patterns of brain that indicate the necessary degree of maturation required for various functions. The neurological maturation of the brain assessed with the concomitant chemical and anatomical development considers various factors in the total development of the animal. These factors include the rate of prenatal development, a correlary of which is the maturity of the animal at birth, and the rate of postnatal development. Other important points are the complexity of the brain organization and the total metabolic rate of the animal as a whole as well as that of the brain itself. The data used in the chapter were obtained on whole brain and reported on a wet weight basis. The human infant is born more mature than that of the rat or rabbit, and based on the visual evoked potential, more mature than the cat. After birth, development in man appears to proceed more slowly than in the other species examined.
A number of N-hydroxysuccinimide esters of acylamino acids have been synthesized. These compounds are crystalline solids which react readily with amino acids or peptides or their esters. Peptide formation under aqueous conditions goes well. Because of the water solubility of N-hydroxysuccinimide, these esters appear to be more generally useful than the analogous esters of N-hydroxyphthalimide.
N-acetyl-l-glutamine, pyroglutamic acid, and the butyl ester of pyroglutamic acid were isolated in pure form from an aqueous extract of human brain. These compounds were isolated by combination of paper and ion exchange chromatography. The isolated substance identified as N-acetyl-l-glutamine did not react with the ninhydrin reagent but yielded glutamic acid and ammonia upon acid hydrolysis. An acetyl hydrazide was identified by paper chromatography from hydrazinolysates of the isolated substance. The glutamic acid liberated by hydrolysis had the l-configuration. The results of elementary analysis of the isolated compound were in full accord with the analysis calculated for synthetic N-acetyl-l-glutamine. A large amount of pyroglutamic acid and a substance identical with the butyl ester of pyroglutamic acid were isolated in pure form. The results of our studies suggest that pyroglutamic and the butyl ester derivative were artifacts formed during the isolation and purification procedures.
An acetylated dipeptide composed of equimolar quantities of aspartic acid and glutamic acid has been isolated in pure form from human brain by a combination of ion exchange and paper chromatographic techniques. Hydrazinolysis and acid hydrolysis studies indicate that the peptide is N-acetyl aspartyl glutamate. Preliminary synthesis studies suggest that the peptide may be of the alpha rather than the beta structural configuration.
An unidentified substance with acetylcholine-like activity has been isolated from human brain by Chromatographie procedures. The substance is more active on the frog reclus preparation than on the guinea pig ileum. It has an Ritf value of 0.65 ± 0.05 in BuOH: EtOH:Ac:H2O. An amino acid analysis of material isolated from this region of the chromatogram revealed two major ninhydrin peaks corresponding to leucine (0.112 μmoles) and isoleucine (0.052 μmoles). Two additional prominent ninhydrin peaks appeared in an equivalent amount of an acid hydrolyzate which corresponded to aspartic acid (1.925 μmoles) and glutamic acid (1.709) μmoles). The amounts of leucine and isoleucine were of the same order as those in the unhydrolyzed sample. Only trace amounts of other amino acids were found. Dinitrophenylation experiments did not give rise to either DNP-glutamic acid or DNP-aspartic acid on hydrolysis. Hydrozinolysis indicated glutamic to be a C-terminal amino acid. The hydrolyzed material possessed no pharmacological activity. The active substance was temporarily presumed to be an acetylated dipeptide of aspartic and glutamic acid.