Formation Of Aspartate Research Articles

#5797 AMINOPEPTIDASE A REGULATES ACUTE ANGIOTENSIN DEPENDENT HYPERTENSION

Abstract Background and Aims Aminopeptidase A (APA) is an enzyme that cleaves a single aspartate residue from the amino-terminus of several renin-angiotensin systems (RAS) peptides. We have shown that genetic deficiency of APA was associated with increased blood pressure and ultrastructural glomerular pathology in mice. Here we studied the effect of this Aminopeptidase on different peptides of the RAS and its effect on acute hypertension. Method We developed an in vitro assay based on Aspartate formation by APA to detect which Angiotensin (Ang) peptides are the substrate of this enzyme. The identified Ang peptides were injected to determine their relative effects on systolic blood pressure (SBP) in anesthetized WT mice. The effect of recombinant Aminopeptidase A on Ang I, Ang II and Ang 1-12 mediated SBP increase was further assessed in APA deficiency using global knockout mice (KO), and after systemic injection of recombinant APA. Results In the in vitro assay, the rAPA showed significant cleavage activities for Ang II (22427±3353, n = 12), Ang I (21883±1692, n = 3), Ang1-7 (15833±3395, n = 3), Ang1-9 (15773±3395, n = 3) and Ang1-12 (13170±220, n = 3) as evidenced by the formation of aspartate measured as a readout of enzymatic cleavage. When the five angiotensin peptides were injected into WT mice, only Ang I, Ang II and Ang 1-12 caused a substantial increase in SBP (Figure 1, left). In APA KO mice the bolus administration of Ang II showed an exaggerated increase in SBP compared to WT mice (Figure 1, right). The effect of Ang I and Ang 1-12 in APA KO appeared similar (not shown). When Ang I, Ang II and Ang 1-12 were administrated in WT mice preinjected with APA the increase in SBP was markedly reduced (p<0,001, p = 0,0024 and p = 0,0063, respectively). Conclusion Aminopeptidase A can cleave the N-terminal aspartate from Ang II as well as from other RAS peptides upstream [Ang 1-12, Ang I (1-10) and Ang 1-9] and downstream of Ang II (Ang 1-7). Aminopeptidase A deficiency is associated with exaggerated SBP response to Ang I, Ang II and Ang 1-12 whereas the recombinant enzyme restores the effect on acute hypertension caused by infusion of these peptides. This suggests that APA could be used to treat hypertension due to RAS overactivity.

Brain CoA and Acetyl CoA Metabolism in Mechanisms of Neurodegeneration

The processes of biotransformation of pantothenic acid (Pan) in the biosynthesis and hydrolysis of CoA, key role of pantothenate kinase (PANK) and CoA synthetase (CoASY) in the formation of the priority mitochondrial pool of CoA, with a high metabolic turnover of the coenzyme and limited transport of Pan across the blood-brain barrier are considered. The system of acetyl-CoA, a secondary messenger, which is the main substrate of acetylation processes including formation of N-acetyl aspartate and acetylcholine, post-translational modification of histones, predetermines protection of the neurons against degenerative signals and cholinergic neurotransmission. Biochemical mechanisms of neurodegenerative syndromes in the cases of PANK and CoASY defects, and the possibility of correcting of CoA biosynthesis in the models with knockouts of these enzymes have been described. The data of a post-mortem study of the brains from the patients with Huntington's and Alzheimer's diseases are presented, proving Pan deficiency in the CNS, which is especially pronounced in the pathognomonic neurostructures. In the frontal cortex of the patients with Parkinson's disease, combined immunofluorescence of anti-CoA- and anti-tau protein was detected, reflecting CoAlation during dimerization of the tau protein and its redox sensitivity. Redox activity and antioxidant properties of the precursors of CoA biosynthesis were confirmed in vitro with synaptosomal membranes and mitochondria during modeling of aluminum neurotoxicity accompanied by the decrease in the level of CoA in CNS. The ability of CoA biosynthesis precursors to stabilize glutathione pool in neurostructures, in particular, in the hippocampus, is considered as a pathogenetic protection mechanism during exposure to neurotoxins, development of neuroinflammation and neurodegeneration, and justifies the combined use of Pan derivatives (for example, D-panthenol) and glutathione precursors (N-acetylcysteine). Taking into account the discovery of new functions of CoA (redox-dependent processes of CoAlation of proteins, possible association of oxidative stress and deficiency of Pan (CoA) in neurodegenerative pathology), it seems promising to study bioavailability and biotransformation of Pan derivatives, in particular of D-panthenol, 4'-phospho-pantetheine, its acylated derivatives, and compositions with redox pharmacological compounds, are promising for their potential use as etiopathogenetic agents.

Differentiation of fresh and thawed Atlantic salmon using NMR metabolomics

NMR metabolomics approach was used to distinguish fresh and thawed Atlantic salmon. Statistical analysis revealed significant differences in the concentration of some metabolites in reference and frozen-thawed fish during its storage. It was found that salmon freezing/thawing caused a significant increase in the concentration of fumarate and phenylalanine in stored salmon muscle. The concentration of fumarate increased until the 3rd–5th day after thawing and then gradually decreased, reaching zero after two weeks of storage. The concentration of phenylalanine was constantly increased during the storage time. Furthermore, it was detected that aspartate was formed in the flesh of only thawed fish after the second day of storage. Its concentration followed the same trend as fumarate reaching its maximal concentration on the 3rd–5th day after thawing (up to 3.8 mg in 100 g of muscle) and gradually decreased to zero. Aspartate formation was influenced by storage time after thawing and not by the time after slaughter. We propose to use the formation of aspartate in stored salmon flesh as a marker of salmon freezing/thawing.

Obtaining and Properties of L-Aspartic Acid Solutions of Chitosan

Conditions for obtaining L-aspartic acid solutions of chitosan were considered: dissolution of the polymer with the formation of chitosan aspartate occurs in pH range of 3.6–3.8 at a minimum stoichiometric ratio of [acid]/[chitosan (−NH2)] ∼ 0.43. The hydrodynamic, electrochemical, optical and biological properties of aqueous solutions of chitosan aspartic acid salt are investigated. It has been established that macromolecules of polymeric salt exhibit properties of a polyelectrolyte with a partially compensated charge in an aqueous medium. Huggins constants and temperature viscosity coefficients were calculated, which indicate an increased rigidity of the chitosan macrochain in the studied solutions and deterioration in the polymer—solvent interaction with increasing temperature. Chitosan aspartate powders in the form of lamellar microparticles with fractal ordering were isolated. Low biocompatibility and antibacterial activity of the polymeric salt are demonstrated, which allows recommending the production of biomedical products on its basis.

Active Role of the Substrate During Catalysis by the Therapeutic Enzyme L‐Asparaginase II

Bacterial type II L‐Asparaginases (ASPII) have been used for over four decades to treat acute lymphoblastic leukemia, yet a full reaction mechanism remains unknown. ASPII enzymes catalyze the deamidation of both asparagine (Asn) and glutamine (Gln), which results in the formation of aspartate (Asp) and glutamate (Glu) respectively, and the by‐product ammonia. Proposed ASPII mechanisms to date have yet to explain the absolute requirement of a substrate α‐carboxyl group, and clearly identify the role of the catalytic threonines T12 and T89. Here, we study the reaction mechanism of asparagine degradation by ASPII through ab initio molecular dynamics (MD) simulations. We select a reduced system from the substrate‐bound enzyme obtained by classical MD, and explore different initial reaction pathways by driving the system in steered simulations. Our results show that direct nucleophilic attack by T12 produces a highly unstable substrate‐enzyme intermediate, as the stabilization provided by the nearby protons (i.e., the “oxyanion hole”) is insufficient to sustain the high energy state. We find that the substrate‐enzyme intermediate can be stabilized by first protonating the substrate's amide oxygen through the K162‐T89 proton bridge. Furthermore, the α‐carboxyl of the substrate acts as a proton acceptor for the hydroxyl sidechain of T12 during nucleophilic attack. We conclude by showing that a complete deamidation mechanism may require a sequence of several nucleophilic attacks by both T12 and T89, with K162 playing a critical role as a proton buffer during the course of the reaction.

Uptake and metabolism of fructose by rat neocortical cells in vivo and by isolated nerve terminals in vitro

Fructose reacts spontaneously with proteins in the brain to form advanced glycation end products (AGE) that may elicit neuroinflammation and cause brain pathology, including Alzheimer's disease. We investigated whether fructose is eliminated by oxidative metabolism in neocortex. Injection of [(14) C]fructose or its AGE-prone metabolite [(14) C]glyceraldehyde into rat neocortex in vivo led to formation of (14) C-labeled alanine, glutamate, aspartate, GABA, and glutamine. In isolated neocortical nerve terminals, [(14) C]fructose-labeled glutamate, GABA, and aspartate, indicating uptake of fructose into nerve terminals and oxidative fructose metabolism in these structures. This was supported by high expression of hexokinase 1, which channels fructose into glycolysis, and whose activity was similar with fructose or glucose as substrates. By contrast, the fructose-specific ketohexokinase was weakly expressed. The fructose transporter Glut5 was expressed at only 4% of the level of neuronal glucose transporter Glut3, suggesting transport across plasma membranes of brain cells as the limiting factor in removal of extracellular fructose. The genes encoding aldose reductase and sorbitol dehydrogenase, enzymes of the polyol pathway that forms glucose from fructose, were expressed in rat neocortex. These results point to fructose being transported into neocortical cells, including nerve terminals, and that it is metabolized and thereby detoxified primarily through hexokinase activity. We asked how the brain handles fructose, which may react spontaneously with proteins to form 'advanced glycation end products' and trigger inflammation. Neocortical cells took up and metabolized extracellular fructose oxidatively in vivo, and isolated nerve terminals did so in vitro. The low expression of fructose transporter Glut5 limited uptake of extracellular fructose. Hexokinase was a main pathway for fructose metabolism, but ketohexokinase (which leads to glyceraldehyde formation) was expressed too. Neocortical cells also took up and metabolized glyceraldehyde oxidatively.

Active Role of the Substrate during Catalysis by the Therapeutic Enzyme L-Asparaginase II

Bacterial type II L-Asparaginases (ASPII) have been used for over four decades to treat acute lymphoblastic leukemia, yet a full reaction mechanism remains unknown. ASPII enzymes catalyze the deamidation of both asparagine (Asn) and glutamine (Gln), which results in the formation of aspartate (Asp) and glutamate (Glu) respectively, and the by-product ammonia. Proposed ASPII mechanisms to date have yet to explain the absolute requirement of a substrate α-carboxyl group, and clearly identify the role of the catalytic threonines T12 and T89. Here, we study the reaction mechanism of asparagine degradation by ASPII through ab initio molecular dynamics (MD) simulations. We selected a reduced system from the substrate-bound enzyme obtained by classical MD, and explore different initial reaction pathways by driving the system in steered simulations. Our results show that direct nucleophilic attack by T12 produces a highly unstable substrate-enzyme intermediate, as the stabilization provided by the nearby protons (i.e., the “oxyanion hole”) is insufficient to sustain the high energy state. We find that the substrate-enzyme intermediate can be stabilized by first protonating the substrate's amide oxygen through the K162-T89 proton bridge. Furthermore, the α-carboxyl of the substrate acts as a proton acceptor for the hydroxyl sidechain of T12 during nucleophilic attack. We conclude by showing that a complete deamidation mechanism may require a sequence of several nucleophilic attacks by both T12 and T89, with K162 playing a critical role as a proton buffer during the course of the reaction.

Dipeptidyl peptidase IV activity in commercial solutions of human serum albumin

Due to the heterogeneous nature of commercial human serum albumin (cHSA), other components, such as the protease dipeptidyl peptidase IV (DPP-IV), possibly contribute to the therapeutic effect of cHSA. Here, we provide evidence for the first time that DPP-IV activity contributes to the formation of aspartate–alanine diketopiperazine (DA-DKP), a known immunomodulatory molecule from the N terminus of human albumin. cHSA was assayed for DPP-IV activity using a specific DPP-IV substrate and inhibitor. DPP-IV activity was assayed at 37 and 60°C because cHSA solutions are pasteurized at 60°C. DPP-IV activity in cHSA was compared with other sources of albumin such as a recombinant albumin (rHSA). In addition, the production of DA-DKP was measured by negative electrospray ionization/liquid chromatography mass spectrometry (ESI−/LCMS). Significant levels of DPP-IV activity were present in cHSA. This activity was abolished using a specific DPP-IV inhibitor. Fully 70 to 80% DPP-IV activity remained at 60°C compared with the 37°C incubate. No DPP-IV activity was present in rHSA, suggesting that DPP-IV activity is present only in HSA produced using the Cohn fractionation process. The formation of DA-DKP at 60°C was observed with the DPP-IV inhibitor significantly decreasing this formation. DPP-IV activity in cHSA results in the production of DA-DKP, which could account for some of the clinical effects of cHSA.

Rerouting citrate metabolism in Lactococcus lactis to citrate-driven transamination.

Oxaloacetate is an intermediate of the citrate fermentation pathway that accumulates in the cytoplasm of Lactococcus lactis ILCitM(pFL3) at a high concentration due to the inactivation of oxaloacetate decarboxylase. An excess of toxic oxaloacetate is excreted into the medium in exchange for citrate by the citrate transporter CitP (A. M. Pudlik and J. S. Lolkema, J. Bacteriol. 193:4049-4056, 2011). In this study, transamination of amino acids with oxaloacetate as the keto donor is described as an additional mechanism to relieve toxic stress. Redirection of the citrate metabolic pathway into the transamination route in the presence of the branched-chain amino acids Ile, Leu, and Val; the aromatic amino acids Phe, Trp, and Tyr; and Met resulted in the formation of aspartate and the corresponding α-keto acids. Cells grown in the presence of citrate showed 3.5 to 7 times higher transaminase activity in the cytoplasm than cells grown in the absence of citrate. The study demonstrates that transaminases of L. lactis accept oxaloacetate as a keto donor. A significant fraction of 2-keto-4-methylthiobutyrate formed from methionine by citrate-driven transamination in vivo was further metabolized, yielding the cheese aroma compounds 2-hydroxy-4-methylthiobutyrate and methyl-3-methylthiopropionate. Reducing equivalents required for the former compound were produced in the citrate fermentation pathway as NADH. Similarly, phenylpyruvate, the transamination product of phenylalanine, was reduced to phenyllactate, while the dehydrogenase activity was not observed for the branched-chain keto acids. Both α-keto acids and α-hydroxy acids are known substrates of CitP and may be excreted from the cell in exchange for citrate or oxaloacetate.

Regulation of glutamate metabolism by hydrocortisone and branched chain keto acids in cultured rat retinal Müller cells (TR-MUL)

Glutamate released from retinal neurons during neurotransmission is taken up by retinal Müller cells, where much of the amino acid is subsequently amidated to glutamine or transaminated to α-ketoglutarate for oxidation. Müller cell glutamate levels may have to be carefully maintained at fairly low concentrations to avoid excesses of glutamate in extracellular spaces of the retina that would otherwise cause excitotoxicity. We employed a cultured rat retinal Müller cell line in order to study the metabolism and the role of Müller cell specific enzymes on the glutamate disposal pathways. We found that the TR-MUL cells express the glial specific enzymes, glutamine synthetase, the mitochondrial isoform of branched chain aminotransferase (BCATm) and pyruvate carboxylase, all of which are involved in glutamate metabolism and homeostasis in the retina. Hydrocortisone treatment of TR-MUL cells increased glutamine synthetase expression and the rate of glutamate amidation to glutamine. Addition of branched chain keto acids (BCKAs) increased lactate and aspartate formation from glutamate and also oxidation of glutamate to CO2 and H2O. The two glutamate disposal pathways (amidation and oxidation) did not influence each other. When glutamate levels were independently depleted within TR-MUL cells, the uptake of glutamate from the extracellular fluid increased compared to uptake from control (undepleted) cells suggesting that the level of intracellular glutamate may influence clearing of extracellular glutamate.

Postnatal Deamidation of 4E-BP2 in Brain Enhances Its Association with Raptor and Alters Kinetics of Excitatory Synaptic Transmission

The eIF4E-binding proteins (4E-BPs) repress translation initiation by preventing eIF4F complex formation. Of the three mammalian 4E-BPs, only 4E-BP2 is enriched in the mammalian brain and plays an important role in synaptic plasticity and learning and memory formation. Here we describe asparagine deamidation as a brain-specific posttranslational modification of 4E-BP2. Deamidation is the spontaneous conversion of asparagines to aspartates. Two deamidation sites were mapped to an asparagine-rich sequence unique to 4E-BP2. Deamidated 4E-BP2 exhibits increased binding to the mammalian target of rapamycin (mTOR)-binding protein raptor, which effects its reduced association with eIF4E. 4E-BP2 deamidation occurs during postnatal development, concomitant with the attenuation of the activity of the PI3K-Akt-mTOR signaling pathway. Expression of deamidated 4E-BP2 in 4E-BP2(-/-) neurons yielded mEPSCs exhibiting increased charge transfer with slower rise and decay kinetics relative to the wild-type form. 4E-BP2 deamidation may represent a compensatory mechanism for the developmental reduction of PI3K-Akt-mTOR signaling.

Structural investigation of a phosphorylation-catalyzed, isoaspartate-free, protein succinimide: crystallographic structure of post-succinimide His15Asp histidine-containing protein.

Aspartates and asparagines can spontaneously cyclize with neighboring main-chain amides to form succinimides. These succinimides hydrolyze to a mixture of isoaspartate and aspartate products. Phosphorylation of aspartates is a common mechanism of protein regulation and increases the propensity for succinimide formation. Although typically regarded as a form of protein damage, we hypothesize succinimides could represent an effective mechanism of phosphoaspartate autophosphatase activity, provided hydrolysis is limited to aspartate products. We previously reported the serendipitous creation of a protein, His15Asp histidine-containing protein (HPr), which undergoes phosphorylation-catalyzed formation of a succinimide whose hydrolysis is seemingly exclusive for aspartate formation. Here, through the high-resolution structure of postsuccinimide His15Asp HPr, we confirm the absence of isoaspartate residues and propose mechanisms for phosphorylation-catalyzed succinimide formation and its directed hydrolysis to aspartate. His15Asp HPr represents the first characterized protein example of an isoaspartate-free succinimide and lends credence to the hypothesis that intramolecular cyclization could represent a physiological mechanism of autophosphatase activity. Furthermore, this indicates that current strategies for succinimide evaluation, based on isoaspartate detection, underestimate the frequencies of these reactions. This is considerably significant for evaluation of protein stability and integrity.

Truncation of the Krebs Cycle During Hypoglycemic Coma

There is a misconception that hypoglycemic nerve cell death occurs easily, and can happen in the absence of coma. In fact, coma is the prerequisite for neuronal death, which occurs via metabolic excitatory amino acid release. The focus on nerve cell death does not explain how most brain neurons and all glia survive. Brain metabolism was interrogated in rats during and following recovery from 40 min of profound hypoglycemia using ex vivo (1)H MR spectroscopy to determine alterations accounting for survival of brain tissue. As previously shown, a time-dependent increase in aspartate was equaled by a reciprocal decrease in glutamate/glutamine. We here show that the kinetics of aspartate formation during the first 30 min (0.36 +/- 0.03 micromol g(-1) min(-1)) are altered such that glutamate, via aspartate aminotransferase, becomes the primary source of carbon when glucose-derived pyruvate is unavailable. Oxaloacetate is produced directly from alpha-ketoglutarate, so that reactions involving the six-carbon intermediates of the tricarboxylic acid cycle are bypassed. These fundamental observations in basic metabolic pathways in effect redraw the tricarboxylic acid cycle from a tricarboxylic to a dicarboxylic acid cycle during hypoglycemia. The basic neurochemical alterations according to the chemical equilibrium of mass action augments flux through a truncated Krebs cycle that continues to turn during hypoglycemic coma. This explains the partial preservation of energy charge and brain cell survival during periods of glucose deficiency.

Reversible Denaturation and Reactivation of Aspartase from Hafnia alvei

Aspartase (L-aspartate ammonia-lyase, EC 4.3.1.1) catalyzes the reversible conversion of L-aspasteate to fumarate and ammonium ion. Aspartase is a catabolic enzyme in both bacteria and plants, but the reaction is reversible and favors aspartate formation with ΔG = 3.2 kcal/mol for aspartate deamination. The aspartase from Hafnia alvei, that is important for aspartate synthesis in industrial purpose, is a tetramer composed of four apparently identical subunits of molecular weight 55,000 Da. Although many aspects of H. alvei aspatase reactions are well studied, the role of subunit-subunit interaction in this oligomeric protein is not known yet. As the relationship between subunit assembly and function are very important for elucidation of the protein functions at molecular basis, we have studied the activitysubunit interaction relationship and effect of environmental factors, such as temperature and pH, on reversible denaturation and reactivation of the H. alvei aspartase. As shown in Figure 1(a) at 1 M Gu-HCl was 40%. On a temperature scale (0-40 C), the optimum temperature for reactivation was attained at 25 C (Figure 2a). There was no reactivation between 0-10 C, progressed with linearly increase at 10-25 C, with a level off between 25-35 C and completely stopped at 35 C. The optimum pH for reactivation was 7.0 and little degree of reactivation was attained at the acidic or alkaline pH, presumably due to charge interaction of polypeptide chains (Figure 2b). When the temperature of renaturation was shifted from 4 to 25 C, a rapid increase in the rate of reactivation was observed with ~45 min lag phase (Figure 3), reasoning need of time for enzyme to remove the Gu-HCl by dilution. These results attributed a temperature-dependent process, as a rate-determining step in the reactivation of H. alvei aspartase. This indicates that the enzyme reached to restructured form during renaturation process and reactivated at the suitable temperature. Many cases are known, actually in which enzymes are not able to be readily reactivated simply by removal of the causes due to partial or serious denaturation of the polypeptides. However, the fact that the native confirmation of most proteins is uniquely determined by the amino acid sequence in the protein, any protein to which this generalization applies should always be able to recover its native conformation, provided that its primary sequence remains intact. Sachiko et al. identified an active dimeric form of aspartase as a denaturation intermediate of E. coli. We have proposed the intermediate forms of H. alvei aspartase during reversible denaturation (Scheme 1). As at ≥ 1 M GuHCl, aspartase dissociates to monomers and largely unfolds, in the present investigation, the denaturation process at < 1 M Gu-HCl can be explained in terms of a two-state transition. The 100% renaturation below 1 M Gu-HCl knowed out

Glycogen is a preferred glutamate precursor during learning in 1‐day‐old chick: Biochemical and behavioral evidence

Bead discrimination training in chicks sets in motion a tightly timed series of biochemical events, including glutamate release, increase in forebrain level of glutamate and utilization of glycogen and glucose. Inhibition of glycogen breakdown by the glycogen phosphorylase inhibitor 1,4-dideoxy-1,4-imino-D-arabinitol (DAB) around the time of training abolishes the increase in glutamate 5 min posttraining in the left hemisphere, in spite of uninhibited glucose metabolism. It also reduces the contents of glutamate, glutamine, and aspartate in the right hemisphere. Behavioral evidence supports the conclusion that glucose breakdown serves to provide energy, whereas glycogen acts as a substrate for glutamate, glutamine, and aspartate formation, requiring both pyruvate dehydrogenation to acetyl coenzyme A and pyruvate carboxylation in astrocytes. Inhibition of memory consolidation caused by DAB or 2-deoxyglucose (2-DG), an inhibitor of glucose phosphorylation without effect on glycogen metabolism, was challenged by intracerebral administration of acetate, aspartate, glutamine, lactate or glucose. DAB-mediated memory inhibition was successfully challenged by administration at 0 or 20 min posttraining of acetate (an astrocyte-specific acetyl CoA precursor) together with aspartate, substituting for pyruvate carboxylation, or of glutamine at 0-2.5 or 30 min posttraining. 2-DG-mediated memory impairment was not challenged by acetate with or without aspartate at 0 time but was challenged by acetate without aspartate at 20 min. Lactate, a substrate for both dehydrogenation and pyruvate carboxylation challenged both DAB and 2-DG. Doses of DAB and 2-DG which, on their own were subeffective, were not additive, further supporting the existence of one pathway using glucose and another using glycogen.

Glutamine as an energy substrate in cultured neurons during glucose deprivation

During glucose deprivation an increase in aspartate formation from glutamine has been observed in different brain preparations, including synaptosomes and cultured astrocytes. To what extent this reaction, which provides a substantial amount of energy, occurs in different types of neurons is unknown. The present study shows that (14)CO(2) formation from [U-(14)C]glutamine in cerebellar granule neurons, a glutamatergic preparation, increased by 60% during glucose deprivation, indicating enhanced aspartate formation or increased complete oxidative degradation of glutamine. In primary cultures of cerebrocortical interneurons, a GABAergic preparation, the rate of (14)CO(2) production from [U-(14) C] glutamine was four times lower and not stimulated by glucose deprivation. During incubation with glutamine (0.8 mM) as the only metabolic substrate, cerebellar granule cells maintained an oxygen consumption rate of 12 nmol/min/mg protein, corresponding to an aspartate formation of 8 nmol/min/mg protein (three oxidations occur between glutamine and aspartate) or to a total oxidative degradation of 3 nmol/min/mg protein. During glucose deprivation, the rate of aspartate formation increased, and during a 20-min incubation in phosphate-buffered saline it amounted to 3.3 nmol/min/mg protein at 0.2 mM glutamine, which might have been more if measured at 0.8 mM glutamine. These values are consistent with the rate of glutamine utilization calculated based on oxygen consumption and leaves open the possibility that some glutamine is completely degraded oxidatively, as has been shown by other authors based on pyruvate recycling and labeling of lactate from aspartate in cerebellar granule neurons.

Evidence for a higher glycolytic than oxidative metabolic activity in white matter of rat brain

Different values exist for glucose metabolism in white matter; it appears higher when measured as accumulation of 2-deoxyglucose than when measured as formation of glutamate from isotopically labeled glucose, possibly because the two methods reflect glycolytic and tricarboxylic acid (TCA) cycle activities, respectively. We compared glycolytic and TCA cycle activity in rat white structures (corpus callosum, fimbria, and optic nerve) to activities in parietal cortex, which has a tight glycolytic-oxidative coupling. White structures had an uptake of [ 3H]2-deoxyglucose in vivo and activities of hexokinase, glucose-6-phosphate isomerase, and lactate dehydrogenase that were 40–50% of values in parietal cortex. In contrast, formation of aspartate from [U- 14C]glucose in awake rats (which reflects the passage of 14C through the whole TCA cycle) and activities of pyruvate dehydrogenase, citrate synthase, α-ketoglutarate dehydrogenase, and fumarase in white structures were 10–23% of cortical values, optic nerve showing the lowest values. The data suggest a higher glycolytic than oxidative metabolism in white matter, possibly leading to surplus formation of pyruvate or lactate. Phosphoglucomutase activity, which interconverts glucose-6-phosphate and glucose-1-phosphate, was similar in white structures and parietal cortex (∼3 nmol/mg tissue/min), in spite of the lower glucose uptake in the former, suggesting that a larger fraction of glucose is converted into glucose-1-phosphate in white than in gray matter. However, the white matter glycogen synthase level was only 20–40% of that in cortex, suggesting that not all glucose-1-phosphate is destined for glycogen formation.

Reduced N-Acetylaspartate Levels in Mice Lacking Aralar, a Brain- and Muscle-type Mitochondrial Aspartate-glutamate Carrier

Aralar is a mitochondrial calcium-regulated aspartate-glutamate carrier mainly distributed in brain and skeletal muscle, involved in the transport of aspartate from mitochondria to cytosol, and in the transfer of cytosolic reducing equivalents into mitochondria as a member of the malate-aspartate NADH shuttle. In the present study, we describe the characteristics of aralar-deficient (Aralar-/-) mice, generated by a gene-trap method, showing no aralar mRNA and protein, and no detectable malate-aspartate shuttle activity in skeletal muscle and brain mitochondria. Aralar-/- mice were growth-retarded, exhibited generalized tremoring, and had pronounced motor coordination defects along with an impaired myelination in the central nervous system. Analysis of lipid components showed a marked decrease in the myelin lipid galactosyl cerebroside. The content of the myelin lipid precursor, N-acetylaspartate, and that of aspartate are drastically decreased in the brain of Aralar-/- mice. The defect in N-acetylaspartate production was also observed in cell extracts from primary neuronal cultures derived from Aralar-/- mouse embryos. These results show that aralar plays an important role in myelin formation by providing aspartate for the synthesis of N-acetylaspartate in neuronal cells.

Aquifex aeolicus Aspartate Transcarbamoylase, an Enzyme Specialized for the Efficient Utilization of Unstable Carbamoyl Phosphate at Elevated Temperature

Aquifex aeolicus, an organism that flourishes at 95 degrees C, is one of the most thermophilic eubacteria thus far described. The A. aeolicus pyrB gene encoding aspartate transcarbamoylase (ATCase) was cloned, overexpressed in Escherichia coli, and purified by affinity chromatography to a homogeneous form that could be crystallized. Chemical cross-linking and size exclusion chromatography showed that the protein was a homotrimer of 34-kDa catalytic chains. The activity of A. aeolicus ATCase increased dramatically with increasing temperature due to an increase in kcat with little change in the Km for the substrates, carbamoyl phosphate and aspartate. The Km for both substrates was 30-40-fold lower than the corresponding values for the homologous E. coli ATCase catalytic subunit. Although rapidly degraded at high temperature, the carbamoyl phosphate generated in situ by A. aeolicus carbamoyl phosphate synthetase (CPSase) was channeled to ATCase. The transient time for carbamoyl aspartate formation was 26 s, compared with the much longer transient times observed when A. aeolicus CPSase was coupled to E. coli ATCase. Several other approaches provided strong evidence for channeling and transient complex formation between A. aeolicus ATCase and CPSase. The high affinity for substrates combined with channeling ensures the efficient transfer of carbamoyl phosphate from the active site of CPSase to that of ATCase, thus preserving it from degradation and preventing the formation of toxic cyanate.

Role of the glutamate dehydrogenase reaction in furnishing aspartate nitrogen for urea synthesis: studies in perfused rat liver with 15N.

The present study was designed to determine: (i) the role of the reductive amination of alpha-ketoglutarate via the glutamate dehydrogenase reaction in furnishing mitochondrial glutamate and its transamination into aspartate; (ii) the relative incorporation of perfusate 15NH4Cl, [2-15N]glutamine or [5-15N]glutamine into carbamoyl phosphate and aspartate-N and, thereby, [15N]urea isotopomers; and (iii) the extent to which perfusate [15N]aspartate is taken up by the liver and incorporated into [15N]urea. We used a liver-perfusion system containing a physiological mixture of amino acids and ammonia similar to concentrations in vivo, with 15N label only in glutamine, ammonia or aspartate. The results demonstrate that in perfusions with a physiological mixture of amino acids, approx. 45 and 30% of total urea-N output was derived from perfusate ammonia and glutamine-N respectively. Approximately two-thirds of the ammonia utilized for carbamoyl phosphate synthesis was derived from perfusate ammonia and one-third from glutamine. Perfusate [2-15N]glutamine, [5-15N]glutamine or [15N]aspartate provided 24, 10 and 10% respectively of the hepatic aspartate-N pool, whereas perfusate 15NH4Cl provided approx. 37% of aspartate-N utilized for urea synthesis, secondary to the net formation of [15N]glutamate via the glutamate dehydrogenase reaction. The results suggest that the mitochondrial glutamate formed via the reductive amination of alpha-ketoglutarate may have a key role in ammonia detoxification by the following processes: (i) furnishing aspartate-N for ureagenesis; (ii) serving as a scavenger for excess ammonia; and (iii) improving the availability of the mitochondrial [glutamate] for synthesis of N -acetylglutamate. In addition, the current findings suggest that the formation of aspartate via the mitochondrial aspartate aminotransferase reaction may play an important role in the synthesis of cytosolic argininosuccinate.