Isolation and identification of β-citryl-L-glutamic acid from newborn rat brain

Miyake, Masakazu; Kawaguchi, Yasuo; Sorimachi, Masaru

doi:10.1016/0304-4165(78)90340-9

Cited by 30 publications

(27 citation statements)

References 13 publications

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“…It was first identified in newborn rat brain, where its concentration reaches 0.5-1 mol/g at birth and then decreases with age (7). BCG also was detected in kidneys and heart and to a much lower extent in intestine, spinal cord, and lung of newborn rats.…”

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confidence: 99%

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

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Stroobant

Lamosa

et al. 2010

Journal of Biological Chemistry

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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 ␤-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 ␤-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 ␤-citrylglutamate. N-Acetylaspartylglutamate (NAAG),3 the most abundant dipeptide in the CNS, is present in brain and in the spinal cord, most particularly in neurons of the anterior horn (1). NAAG can be released from neurons upon calcium depolarization (reviewed in Ref. 1) and is a substrate for two glial peptidases, glutamate carboxypeptidase-II (2) and, to a lower extent, glutamate carboxypeptidase-III (3), which are anchored to the plasma membrane with their catalytic site oriented toward the outside of the cell. NAAG long has been thought to be a neurotransmitter able to bind to the metabotropic glutamate receptors mGluR3 (4). Two recent reports suggest, however, that the effects of NAAG as a neurotransmitter are due to a 0.5% glutamate contamination present in commercial NAAG (5, 6). The function of NAAG is, therefore, presently unknown.␤-Citrylglutamate (BCG), which is structurally close to NAAG, is less well characterized. It was first identified in newborn rat brain, where its concentration reaches 0.5-1 mol/g at birth and then decreases with age (7). BCG also was detected in kidneys and heart and to a much lower extent in intestine, spinal cord, and lung of newborn rats. The content of BCG in all organs decreased with age to the noticeable exception of testes, where its concentration increases during sexual maturation and remains constant in adulthood (8,9). BCG is present not only in the testes of mammals but also in those of amphibians and fishes. There is evidence in germinal cells for a role in spermatogenesis (9). BCG rec...

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confidence: 99%

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

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Stroobant

Lamosa

et al. 2010

Journal of Biological Chemistry

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“…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).…”

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confidence: 99%

Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3

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Vertommen

Constantinescu

et al. 2011

Journal of Biological Chemistry

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␤-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 ␤-citrylglutamate hydrolase, this enzyme was partially purified from mouse testis and characterized. Interestingly, in the presence of Ca 2؉ , the purified enzyme specifically hydrolyzed ␤-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 ␤-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 ␤-citrylglutamate hydrolase was strikingly similar to that of the glutamate carboxypeptidase 3 (GCP3) mRNA, but not that of the GCP2 mRNA. Furthermore, similarly to ␤-citrylglutamate hydrolase purified from testis, recombinant GCP3 specifically hydrolyzed ␤-citrylglutamate in the presence of Ca 2؉ , and acted on both N-acetyl-aspartylglutamate and ␤-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 ␤-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 ␤-citrylglutamate hydrolase activity. ␤-Citrylglutamate (BCG)3 is a pseudodipeptide first identified in newborn rat brain at a concentration of 0.5 to 1 mol/g. 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 (1-3). 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). Recently, BCG has been proposed to be an iron and copper chelator (6, 7).BCG is structurally close to N-acetyl-aspartylglutamate (NAAG), the most abundant dipeptide in the adult brain. NAAG is secreted by neurons upon calcium-dependent depolarization and has long been thought to bind to the glutamate metabotropic receptor mGluR3, possibly attenuating the glutamate-induced excitotoxicity (8). However, this neurotransmitter function of NAAG is still debated (9 -11). NAAG is hydrolyzed into N-acetyl-aspartate (NAA) and glutamate by glutamate carboxypeptidase 2 (GCP2), a membrane-bound, glycosylat...

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“…β-Citryl-l-glutamate (β-CG) was synthesized as previously described. 15) [Fe(II)(Citrate)] complex were prepared from Fe(II) O and trisodium citrate as previously described. 16) All other reagents were purchased from commercial sources at the highest grade available.…”

Section: Methodsmentioning

confidence: 99%

Nitric Oxide Promotes Survival of Cerebral Cortex Neurons with Simultaneous Addition of [Fe(II)(β-Citryl-L-glutamate)] Complex in Primary Culture

Hamada-Kanazawa

Narahara

Takano

et al. 2013

Biological & Pharmaceutical Bulletin

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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).NO is a free radical produced by a wide variety of cell types, that reacts predominantly with Fe(III) or Fe(II) ions of heme proteins and Fe-S centers, as well as molecular oxygen, and superoxide anion. 4,5) Moreover, NO easily reaches mitochondria from cytosolic or extracellular sources, due to its low molecular radius and hydrophobic nature.6) It has also been reported that induction of NO synthesis or exposure of different cell types to NO-donors resulted in early loss of mitochondrial aconitase (m-aconitase) activity. 1)NO-mediated inactivation of m-aconitase has been reported in a variety of cells including macrophages, 7) fibroblasts, 8) tumor cells, 9,10) and Escherichia (E.) coli, 11) though results from in vitro studies are somewhat contradictory. Indeed, in vitro studies that used porcine heart m-aconitase found that low concentrations of NO did not inactivate aconitase, whereas high concentrations led to moderate inhibition.12) Resistance of purified E. coli aconitase and human recombinant cytosolic aconitase (c-aconitase) to NO-dependent inactivation has also been reported. 13) On the contrary, inactivation by either NO or a NO-donor was reported for both m-aconitase and c-aconitase in the presence and absence of a substrate. 14)In agreement with those findings, it was shown that the active form [4Fe-4S] of m-aconitase is rapidly and directly oxidized by ONOO − , to [3Fe-4S], causing a loss of catalytic activity. 12)Another report 11) also noted that E. coli aconitase is highly sensitive to NO-mediated inactivation apparently independent of ONOO − formation. Recently, it was also reported that NO binds to Fe a in the [4Fe-4S] cluster of recombinant porcine maconitase and slowly promotes complete cluster disassembly. 6)The present findings demonstrate that NO-donors promote Fe-dependent activation of m-aconitase ([3Fe-4S] form) in vitro. In intact mitochondria, the NO-donors also promoted Fe-dependent reactivation of the enzyme disassembled by ammonium peroxodisulfate (APS). In mixed neuronal and

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Isolation and identification of β-citryl-L-glutamic acid from newborn rat brain

Cited by 30 publications

References 13 publications

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

Molecular Identification of N-Acetylaspartylglutamate Synthase and β-Citrylglutamate Synthase

Molecular Identification of β-Citrylglutamate Hydrolase as Glutamate Carboxypeptidase 3

Nitric Oxide Promotes Survival of Cerebral Cortex Neurons with Simultaneous Addition of [Fe(II)(β-Citryl-L-glutamate)] Complex in Primary Culture

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