Concentrations Of L-aspartate Research Articles

The effect of receptor interaction on the bacterial chemotactic adaptation rate

Different receptors have evolved in organisms to sense different stimuli in their surroundings. The interaction among the receptors can significantly increase sensory sensitivity and adaptation precision. To study the influence of interaction among different types of chemoreceptors on the adaptation rate in the bacterial chemotaxis signaling network, we systematically compared the adaptation time between the wild-type strain expressing mixed types of receptors and the mutant strain expressing only Tar receptors (namely, the Tar-only strain) under stepwise addition of different concentrations of L-aspartate using FRET (Förster resonance energy transfer) and bead assays. We find that the wild type exhibits faster adaptation than the mutant under the same concentration of saturated stimulus. In contrast, the wild type exhibits slower adaptation than the mutant under unsaturated stimuli that induce the same magnitude of response, and this is independent of the level of receptor expression. The same result is obtained for the network relaxation time by monitoring the steady-state rotational signal of the flagellar motors. By simulating bacterial chemotaxis with different adaptation rates in a stable gradient of chemoattractants, we confirm that the interaction of different types of receptors can effectively promote chemotaxis of <i>Escherichia coli</i> under a stable spatial gradient of attractants while ensuring minimum noise in the cell position distribution.

Aspartate aminotransferase--key enzyme in the human systemic metabolism

Aspartate aminotransferase is an organ-nonspecific enzyme located in many tissues of the human body where it catalyzes reversible reaction of transamination. There are two aspartate aminotransferase isoforms--cytoplasmic (AST1) and mitochondrial (AST2), that usually occur together and interact with each other metabolically. Both isoforms are homodimers containing highly conservative regions responsible for catalytic properties of enzyme. The common feature of all aspartate aminotransfeses is Lys - 259 residue covalent binding with prosthetic group - pyridoxal phosphate. The differences in the primary structure of AST isoforms determine their physico-chemical, kinetic and immunological properties. Because of the low concentration of L-aspartate (L-Asp) in the blood, AST is the only enzyme, which supply of this amino acid as a substrate for many metabolic processes, such as urea cycle or purine and pyrimidine nucleotides in the liver, synthesis of L-arginine in the kidney and purine nucleotide cycle in the brain and the skeletal muscle. AST is also involved in D-aspartate production that regulates the metabolic activity at the auto-, para- and endocrine level. Aspartate aminotransferase is a part of the malate-aspartate shuttle in the myocardium, is involved in gluconeogenesis in the liver and kidney, glyceroneogenesis in the adipose tissue, and synthesis of neurotransmitters and neuro-glial pathway in the brain. Recently, the significant role of AST in glutaminolysis - normal metabolic pathway in tumor cells, was demonstrated. The article is devoted the role of AST, known primarily as a diagnostic liver enzyme, in metabolism of various human tissues and organs.

Mechanisms of inhibition of Rhizobium etli pyruvate carboxylase by L-aspartate.

l-Aspartate is a regulatory feedback inhibitor of the biotin-dependent enzyme pyruvate carboxylase in response to increased levels of tricarboxylic acid cycle intermediates. Detailed studies of l-aspartate inhibition of pyruvate carboxylase have been mainly confined to eukaryotic microbial enzymes, and aspects of its mode of action remain unclear. Here we examine its inhibition of the bacterial enzyme Rhizobium etli pyruvate carboxylase. Kinetic studies demonstrated that l-aspartate binds to the enzyme cooperatively and inhibits the enzyme competitively with respect to acetyl-CoA. l-Aspartate also inhibits activation of the enzyme by MgTNP-ATP. The action of l-aspartate was not confined to inhibition of acetyl-CoA binding, because the acetyl-CoA-independent activity of the enzyme was also inhibited by increasing concentrations of l-aspartate. This inhibition of acetyl-CoA-independent activity was demonstrated to be focused in the biotin carboxylation domain of the enzyme, and it had no effect on the oxamate-induced oxaloacetate decarboxylation reaction that occurs in the carboxyl transferase domain. l-Aspartate was shown to competitively inhibit bicarbonate-dependent MgATP cleavage with respect to MgATP but also probably inhibits carboxybiotin formation and/or translocation of the carboxybiotin to the site of pyruvate carboxylation. Unlike acetyl-CoA, l-aspartate has no effect on the coupling between MgATP cleavage and oxaloacetate formation. The results suggest that the three allosteric effector sites (acetyl-CoA, MgTNP-ATP, and l-aspartate) are spatially distinct but connected by a network of allosteric interactions.

The impact of chronic imipramine treatment on amino acid concentrations in the hippocampus of mice

The relationship between antidepressants and monoamine concentrations in the brain has been well investigated, but few studies have investigated the relationship between antidepressants and amino acid concentrations in the brain. The purpose of the present study was therefore to investigate the effect of the chronic antidepressant imipramine on amino acid and monoamine concentrations in the mouse brain and plasma. Chronic imipramine treatment decreased the concentration of 5-hydroxyindoleaceticacid/5-hydroxytryptamine in the cerebral cortex and increased that of norepinephrine (NE) in the hippocampus. Since these changes were conspicuous effects of the antidepressant, we concluded that imipramine acts on the central nervous system. No change in amino acid concentrations in plasma was induced by chronic imipramine treatment, but several changes were confirmed in the cerebral cortex, the hypothalamus and the hippocampus. Chronic imipramine treatment caused increases in l-methionine, l-tyrosine, and l-lysine in the cerebral cortex, and an increase in l-aspartate in the hypothalamus. Contrary to this, the concentrations of l-aspartate, l-serine, l-asparagine, glycine, l-glutamine, gamma-aminobutyric acid, l-threonine, l-arginine, l-proline, l-valine, and l-methionine in the hippocampus were decreased by chronic imipramine treatment. The present results demonstrate that the metabolism of several amino acids in the brain, but not of those in plasma, was altered by chronic imipramine treatment. The findings in the present study may help to further elucidate the relationship between amino acids and the effects and side effects of antidepressants.

The use of nucleotide analogs to evaluate the mechanism of the heterotropic response of Escherichia coli aspartate transcarbamoylase.

As an alternative method to study the heterotropic mechanism of Escherichia coli aspartate transcarbamoylase, a series of nucleotide analogs were used. These nucleotide analogs have the advantage over site-specific mutagenesis experiments in that interactions between the backbone of the protein and the nucleotide could be evaluated in terms of their importance for function. The ATP analogs purine 5'-triphosphate (PTP), 6-chloropurine 5'-triphosphate (Cl-PTP), 6-mercaptopurine 5'-triphosphate (SH-PTP), 6-methylpurine 5'-triphosphate (Me-PTP), and 1-methyladenosine 5'-triphosphate (Me-ATP) were partially synthesized from their corresponding nucleosides. Kinetic analysis was performed on the wild-type enzyme in the presence of these ATP analogs along with GTP, ITP, and XTP. PTP, Cl-PTP, and SH-PTP each activate the enzyme at subsaturating concentrations of L-aspartate and saturating concentrations of carbamoyl phosphate, but not to the same extent as does ATP. These experiments suggest that the interaction between N6-amino group of ATP and the backbone of the regulatory chain is important for orienting the nucleotide and inducing the displacements of the regulatory chain backbone necessary for initiation of the regulatory response. Me-PTP and Me-ATP also activate the enzyme, but in a more complex fashion, which suggests differential binding at the two sites within each regulatory dimer. The purine nucleotides GTP, ITP, and XTP each inhibit the enzyme but to a lesser extent than CTP. The influence of deoxy and dideoxynucleotides on the activity of the enzyme was also investigated. These experiments suggest that the 2' and 3' ribose hydroxyl groups are not of significant importance for binding and orientation of the nucleotide in the regulatory binding site. 2'-dCTP inhibits the enzyme to the same extent as CTP, indicating that the interactions of the enzyme to the O2-carbonyl of CTP are critical for CTP binding, inhibition, and the ability of the enzyme to discriminate between ATP and CTP. Examination of the electrostatic surface potential of the nucleotides and the regulatory chain suggest that the complimentary electrostatic interactions between the nucleotides and the regulatory chain are important for binding and orientation of the nucleotide necessary to induce the local conformational changes that propagate the heterotropic effect.

Multiple Consequences of Mutating Two Conserved β-Bridge Forming Residues in the Translocation Cycle of a Neuronal Glutamate Transporter

Glutamate transporters remove this neurotransmitter from the synapse in an electrogenic process. After sodium-coupled glutamate translocation, the cycle is completed by obligatory outward translocation of potassium. In the crystal structure of an archaeal homologue, two conserved residues form a beta-bridge, which points away from the binding pocket. In the neuronal glutamate transporter EAAC1, the equivalent residues are asparagine 366 and aspartate 368. Substitution mutants N366Q and D368E, but not N366D and D368N, show glutamate-induced inwardly rectifying steady-state currents, but their apparent substrate affinity is dramatically decreased. Such currents, which reflect electrogenic net uptake of substrate are not observed with the reciprocal double mutant N366D/D368N. Remarkably, the double mutant exhibits slow substrate-induced voltage-dependent capacitative transient currents. These currents apparently reflect the reversible sodium-coupled glutamate translocation step, because the interaction of the double mutant with potassium is largely impaired. Moreover, when the analogous double mutant in the glutamate transporter GLT-1 is reconstituted into liposomes, a slow exchange of radioactive and unlabeled acidic amino acids is observed. Our results suggest that it is the interaction of asparagine 366 and aspartate 368 that is important during the glutamate translocation step. On the other hand, the side chains of these residues themselves are required for the subsequent potassium relocation step.

Amino acids and transaminases activity in ventricular CSF and in brain of normal and Alzheimer patients

The present study was conducted to determine the concentration of amino acids in the cerebrospinal spinal fluid (CSF) and the activities of two tramsaminases: glutamic oxaloacetate transaminase (GOT) and glutamic pyruvate transaminase (GPT) in human Alzheimer disease (AD) and normal brain. l-Glutamic acid, l-glutamine and l-alanine are the most abundant amino acids in the CSF (50–55% of total amino acids). l-Glutamine occurs at much higher levels in Alzheimer CSF compared to the normal CSF (229 ± 91.8 nmol/ml in AD versus 107 ± 47.2 nmol/ml in normal; P = 0.0041). In contrast, l-aspartate occurs at significantly lower concentrations in Alzheimer CSF than normal CSF (46.1 ± 25.7 nmol/ml in Alzheimer versus 95.2 ± 52.6 nmol/ml in normal; P = 0.020). In Alzheimer brain (frontal, parietal and occipital cortices) GOT is present at significantly higher activities than in normal brain cortices (about 1.5 times higher; P < 0.01). No significant differences for GPT activity occurred between normal and AD brain. Since CSF receives amino acids from brain tissues, and since GOT catalyzes the conversion of l-aspartate to l-glutamate, the higher concentrations of l-glutamine (which is derived from l-glutamate), and the lower concentrations of l-aspartate found in Alzheimer CSF could be considered as a consequence of the higher activity of GOT that occurs in Alzheimer brain.

Occurrence of free D-aspartate and aspartate racemase in the blood shell Scapharca broughtonii.

Substantial concentrations of D-aspartate were found in several tissues of Scapharca broughtonii together with approximately equal concentrations of L-aspartate. The foot and mantle extracts also contained an aspartate racemase activity. The formation of L-aspartate from the D-enantiomer by the foot extract was apparently slower than the reverse reaction, and this unbalance seemed to be due to the presence of an enzyme activity which rapidly converted L-aspartate to L-alanine. The possible role of D-aspartate in the anaerobiosis was discussed.

Utilization of Aspartate as a Nitrogen Source in Escherichia coli: ANALYSIS OF NITROGEN FLOW AND CHARACTERIZATION OF THE PRODUCTS OF ASPARTATE CATABOLISM

15N NMR, reverse-phase high performance liquid chromatography and gas chromatography/mass spectrometry have been used to follow nitrogen metabolism in Escherichia coli labeled on medium containing L-[15N]aspartate. The flow of 15N through various nitrogen-containing metabolites was followed over the course of the labeling period. For wild-type E. coli labeled on L-[15N]aspartate as sole source of nitrogen, significant 15N labeling was detected only in the intracellular L-glutamate, L-alanine, L-aspartate, and putrescine pools. Intracellular concentrations of L-aspartate and L-glutamate differed significantly in extracts of an arginine auxotroph (argG-), which is deficient in a potential aspartate-assimilating reaction. When the L-[15N]aspartate containing labeling medium was supplemented with unlabeled arginine, extracts of wild-type E. coli were shown to contain significant amounts of unlabeled ammonia and putrescine. There was substantial dilution of 15N in the glutamate pool. The observation that glutamate, aspartate, and alanine are 15N-labeled, but ammonia is not, suggests that these amino acids are not the immediate source of ammonia. Our results suggest that arginine may be an intermediate for the degradation of some of the aspartate and that arginine may be an intermediate for ammonia production during nitrogen-limited growth. Our results also strongly suggest the presence of a previously uncharacterized pathway of arginine degradation.

Increased transmitter amino acid concentration in human ventricular CSF after brain trauma.

The concentrations of L-aspartate, L-glutamate, L-serine, glycine and gamma-aminobutyric acid (GABA) were determined in repeated samples of ventricular CSF from five patients with severe closed head injury. The values were compared with those obtained from five subjects undergoing surgical treatment for intractable depression. In the head-injured patients, the concentrations of aspartate, glutamate and glycine were 2- to 8-fold higher and the concentration of GABA 56- to 317-fold higher than control values; the concentration of serine was unaffected. Spearman correlation analysis indicated that the concentration of glutamate significantly increased after injury (Rs = 0.60, p < 0.0001, n = 42), reaching an average concentration of about 7 microM 3 days after the injury. This is probably sufficient to cause further excitotoxicity, which suggests the use of excitatory amino acid receptor antagonists as a treatment following severe head injury may require prolonged administration for maximum therapeutic benefit.

Pharmacological characterization of the quisqualate receptor coupled to phospholipase C (Qp) in striatal neurons

A detailed pharmacological characterization of the quisqualate (QA) receptor coupled to phospholipase C (Qp) was performed in striatal neurons. The experiments were carried out in the presence of the ionotropic antagonists MK-801 (1 μM) and 6-cyano-7-nitroquinoxaline-2.3-dione (30 μM), concentrations that block N-methyl-D-aspartate (NMDA) or α-amino-hydroxy-5-methyl-4-isoxazolepropionic acid (AMPA) receptors in these cells. QA, ibotenate and trans-1-aminocyclopentyl-1,3-dicarboxylate (ACPD) evoked dose-dependent inositol phosphate formations with EC 50 values of 0.3, 6.7 and 29 μM, respectively. QA and ibotenate had the same maximal effect (295.7 ± 17.9% of basal, n = 6) whereas the efficacy of ACPD was somewhat lower (70.2 ± 8.9% of the maximal quisqualate effect, n = 4). The QA-, ibotenate- and ACPD-induced maximal effects were not additive, and the inositol phosphate formations induced by high concentrations of L-aspartate (L-ASP), AMPA, kainate (KA) and domoate (DO) (100 μM or higher) were also not additive. The inositol phosphate responses induced by all these agonists were totally blocked by the phorbol ester phorbol 12,13-dibutyrate (PdBu), but not by atropine or prazosin suggesting that all these substances were able to stimulate the Qp excitatory amino acid receptor in striatal neurons. Of the excitatory amino acid receptor antagonists tested, only D,L-2-amino-3-phosphonoproprionate (D,L-AP3) inhibited QA-induced InsP formation in a competitive manner (mean pKi = 4.45 ± 0.43, n = 4). However, this durg was also a partial agonist of the Qp receptor since it stimulated the inositol phosphate formation. We found that D,L-AP3 also inhibited NMDA-induced calcium increase, in a competitive manner (mean pIC 50 = 4.34 ± 0.22, n = 8, and mean pKi = 3.7 ± 0.11, n = 5). The Qp excitatory amino acid receptor in striatal neurons therefore closely resembles Qp receptors with high potency for agonists as described in striatal and retinal slices and synaptoneurosomes, and has several pharmacological differences compared to the Qp receptors which have low potency for agonists described in hippocampal and cortical slices, cerebellar granule cells, astrocytes and rat brain mRNA-injected oocytes.

Structural transitions in crystals of native aspartate carbamoyltransferase.

Screened precession x-ray photographs of crystals of native aspartate carbamoyltransferase (EC 2.1.3.2, from Escherichia coli) ligated with L-aspartate and phosphate reveal the presence of a crystal unit-cell dimension that is intermediate between the T (tense) and R (relaxed) states. Characterizing the intermediate (I) crystal is a c-axis unit-cell dimension of 149 A, halfway between the c-axis length of the T (c = 142 A) and R (c = 156 A) states, in the space group P321. Preservation of the P321 space group indicates that the intermediate crystal form retains a threefold axis of symmetry, and therefore the enzyme has at minimum a threefold axis; however, we do not know whether the molecular twofold axis is conserved. The I crystals are formed by soaking T-state crystals with L-aspartate and phosphate. By raising the concentration of L-aspartate we can further transform the I crystals, without fragmentation, to a form that has the same unit-cell dimensions as R-state crystals grown in the presence of N-(phosphonoacetyl)-L-aspartate.

Regulation of reductive production of succinate under anaerobic conditions in baker's yeast.

When baker's yeast grown aerobically on ethanol as a carbon source was anaerobically cultured in a medium containing glucose, the activity of a cytoplasmic fumarate reductase irreversibly catalyzing the conversion of fumarate to succinate increased, reaching about 3 times the original activity after 12 h, while the activity of succinate dehydrogenase was almost lost after 10 h. These results indicate that the citrate cycle is partially modified to become a reductive pathway leading to succinate during the anaerobic cultivation. In non-proliferating cells grown anaerobically on glucose, the rates of accumulating succinate and pyruvate were decreased and increased, respectively, with increasing concentrations of L-aspartate or NH4Cl in the medium containing glucose as a substrate. These changes were accompanied with increase in the cellular content of aspartate, an inhibitor of pyruvate carboxylase that is involved in supplying the intermediates of the citrate cycle, and pyruvate, a substrate of the enzyme. The aminotransferase inhibitor, aminooxyacetate, prevented the changes in succinate accumulation and cellular aspartate following the addition of NH4Cl. The addition of L-glutamate caused a marked increase in the rate of succinate accumulation without changing the cellular content of aspartate. Neither L-glutamate nor L-aspartate had the ability to produce succinate. The rate of glucose consumption was not changed upon adding these nitrogen compounds. Similar findings were also observed in experiments using proliferating cells. This report presents evidence that in cells containing a large amount of the fumarate reductase, the production of succinate from glucose is regulated by the cellular level of aspartate through the pyruvate carboxylase reaction and that glutamate regulates the succinate production by a mechanism distinct from that involved in the regulation by L-aspartate.

L-Aspartate-induced activation of aspartase.

During the catalysis of the fumarate amination reaction, aspartase was markedly activated by the product, L-aspartate, as shown by a steep increase in the reaction rate. When NH4+ was replaced by NH2OH, the hydroxylamination reaction proceeded without any acceleration, and was activated upon addition of L-aspartate. The activation required the Mg2+ ion and the alkaline pH, and the half-saturation concentration of L-aspartate for activation was as low as 0.07 mM, which was far lower than the Km value for catalysis. Fumarate showed no activating effect in contrast to L-aspartate, and L-aspartate lowered the Km value for fumarate instead of acting as a competitive inhibitor. Besides L-aspartate, alpha-methyl-DL-aspartate exhibited an activating effect without serving as a substrate. These results suggest that the activation is mediated by an indirect action of L-aspartate which is bound to a site distinct from the catalytic site.

Excitotoxin lesions suggest an aspartatergic projection from rat medial prefrontal cortex to ventral tegmental area

High affinity D-[ 3H]aspartate uptake and amino acid concentrations were examined in synaptosome-enriched preparations of microdissected rat ventral tegmental area 6–7 days following N-methyl- D-aspartate lesions confined to medial prefrontal cortex. Specific reductions were observed in the high affinity uptake of D-[ 3H]aspartate (59% of control, P< 0.005) and concentrations of L-aspartate (79% of control, P < 0.05) in the ventral tegmental area suggesting the presence of an aspartatergic projection from medial prefrontal cortex to this area.

The sub-cellular localisation and regulatory properties of pyruvate carboxylase from Rhizopus arrhizus.

Cell-free extracts of Rhizopus arrhizus contain exclusively cytosolic pyruvate carboxylase and NAD-glutamate dehydrogenase, a single mitochondrial isoenzyme of NADP-isocitrate dehydrogenase, and both mitochondrial and cytosolic isoenzymes of NADP-malate dehydrogenase (decarboxylating). Other enzymes examined have sub-cellular localisations similar to those characteristic of mammalian liver. Purified preparations of R. arrhizus pyruvate carboxylase are subject to partial regulatory inhibition by L-aspartate and 2-oxoadipate. L-Glutamate acts as a less effective analogue of L-aspartate while 2-oxoglutarate is ineffective. Competition studies indicate the presence of separate inhibitory sites for L-aspartate and 2-oxoadipate. Under routine assay conditions R. arrhizus pyruvate carboxylase shows significant activation by acyl derivatives of coenzyme A with long chain acyl CoA being more effective than acetyl-CoA. This activation is no longer observed in the presence of high concentrations of pyruvate, MgATP2- and HCO-3. The concentrations of L-aspartate and 2-oxoadipate required to give 50% inhibition ([I]0.5), and the maximal extents of inhibition, are increased by addition of acetyl-CoA. Acetyl-CoA increases the sigmoidal character of the relationship: initial rate/[L-aspartate], but decreases this parameter for the relationship: initial rate/[2-oxoadipate]. The studies indicate that R. arrhizus possesses an entirely cytosolic pathway for the conversion of glucose to fumaric acid and that both the organisation of pyruvate metabolism and the regulation of pyruvate carboxylase differ significantly in this organism as compared to that proposed previously for Aspergillus nidulans.

Pyruvate carboxylase from Aspergillus nidulans. Effects of regulatory modifiers on the structure of the enzyme.

A method is described for purification of pyruvate carboxylase from Aspergillus nidulans by affinity chromatography on monomeric avidin-Sepharose. The purified enzyme is homogeneous as judged by electrophoretic and immunochemical analysis. The sub-unit Mr determined by electrophoresis in the presence of sodium dodecyl sulphate is 133000 +/- 5000. Electron microscopic analysis of purified A. nidulans pyruvate carboxylase after negative staining with uranyl acetate reveals the presence of molecules showing rhomboid and triangular projections as well as a projection showing two intensity maxima. A cleft running along the longitudinal axis of the sub-unit is observed in the rhomboid and triangular projections. Interconversion between all three projections can be obtained in a tilt series. Significantly better preservation of molecular structure is obtained if A. nidulans pyruvate carboxylase is prepared for electron microscopy in the presence of acetyl-CoA, 2-oxoglutarate or as the enzyme-avidin complex. L-Aspartate has no significant effect when added alone but markedly decreases the enhanced preservation given by acetyl-CoA. No marked alterations in molecular dimensions are caused by any of these additions. L-Aspartate, but not 2-oxoglutarate, enhances the rate of inactivation observed on incubation of A. nidulans pyruvate carboxylase at 4 degrees C in the presence of 0.5 M KCl. Addition of L-aspartate in low concentrations enhances the effectiveness of inhibition by 2-oxoglutarate by causing a decrease in the value of [I]0.5 without affecting the Hill coefficient h or the extent of activity observed at saturating 2-oxoglutarate concentrations. Conversely addition of low concentrations of 2-oxoglutarate decreases the concentration of L-aspartate required to give 50% inhibition but also causes a fall in h and an absolute increase in the extent of activity observed in the presence of saturating L-aspartate concentrations. The data are consistent with the proposal that A. nidulans pyruvate carboxylase is a tetrameric molecule in which the four sub-units are located at the corners of a tetrahedron. Metabolites which regulate the activity of the enzyme do not cause major alterations in this molecular structure but may alter its stability.(ABSTRACT TRUNCATED AT 400 WORDS)

Pyruvate carboxylase from Aspergillus nidulans. Regulatory properties.

Partially purified preparations of pyruvate carboxylase from Aspergillus nidulans are inhibited significantly only by l‐aspartate and 2‐oxoglutarate out of nine dicarboxylic and tricarboxylic acids tested. Initial velocity/inhibitor concentration relationships are sigmoid for both dicarboxylic acids and, for l‐aspartate, inhibition is incomplete even at saturating concentrations. l‐Aspartate acts as a competitive inhibitor with respect to HCO3‐ and a non‐competitive inhibitor with respect to pyruvate and MgATP2−. Secondary slope and intercept plots are non‐linear functions of l‐aspartate concentration. 2‐Oxoglutarate induces complex kinetic behaviour for all three substrates. Inhibition appears competitive with respect to pyruvate and at lower substrate concentrations uncompetitive with respect to HCO3−. Both the sigmoid character of the initial velocity/[2‐oxoglutarate] relationship and the concentration of 2‐oxoglutarate required for 50% inhibition ([I]0.5) are markedly and specifically increased by an increase in pyruvate concentration. Addition of acetyl‐CoA causes activation in the presence of either l‐aspartate or 2‐oxoglutarate but does not activate the enzyme in the absence of these dicarboxylic acids. In the presence of 2‐oxoglutarate this effect is less marked at more alkaline pH. Longer‐chain homologues of acetyl‐CoA also activate under these conditions with oleoyl‐CoA as the most effective activator. An apparently competitive relationship exists between the effects of acetyl‐CoA and l‐aspartate on catalysis by pyruvate carboxylase from A. nidulans.l‐Aspartate increases the apparent KA for acetyl‐CoA but does not change the hyperbolic character of the initial velocity/[acetyl‐CoA] relationship. Acetyl‐CoA has no effect on the maximal extent of inhibition by l‐aspartate but increases both the sigmoidicity of the initial velocity [l‐aspartate] relationship and the concentration of l‐aspartate giving 50% inhibition. A non‐competitive relationship exists between the effects of acetyl‐CoA and 2‐oxoglutarate. The relationship between initial velocity and [acetyl‐CoA] remains hyperbolic in the presence of 2‐oxoglutarate but the apparent KA is increased. Acetyl‐CoA causes a slight increase in the concentration of 2 required for 50% inhibition but decreases the sigmoid character of the initial velocity/[2‐oxoglutarate] relationship. Incubation of pyruvate carboxylase from A. nidulans with trinitrobenzenesulphonate causes selective loss of the capacity for activation by acetyl‐CoA with no significant effect on the extent of inhibition by l‐aspartate or 2‐oxoglutarate. Catalytic activity in the absence of these effectors decreases less markedly. These effects are largely prevented if the enzyme is incubated with trinitrobenzenesulphonate in the presence of acetyl‐CoA. We propose that pyruvate carboxylase from A. nidulans carries an allosteric acyl‐CoA activator site but that under the conditions examined occupancy of this site affects catalysis only if either l‐aspartate or 2‐oxoglutarate is present. We further propose that the enzyme carries distinct regulatory sites for l‐aspartate and 2‐oxoglutarate.

Studies on Rat Liver Argininosuccinate Synthetase Inhibition by Various Amino Acids

Inhibition studies of crystallized rat liver argininosuccinate synthetase [EC 6.3.4.5] are described. 1. L-Argininosuccinate, L-histidine, and L-tryptophan inhibited the enzyme activity at saturating amounts of the substrates. 2. L-Norvaline, L-argininosuccinate, L-arginine, L-isoleucine, and L-valine competitively inhibited the enzyme activity at a low concentration of L-citrulline, with Ki values of 1.3 x 10(4) M, 2.5 X 10(-4) M, 6.7 X 10(-4) M, 6.3 X 10(-4) M, and 6.0 x 10(-4) M, respectively. 3. L-Argininosuccinate and L-arginine competitively inhibited the enzyme activity at a low concentration of L-aspartate, with Ki values of 9.5 x 10(-4) M and 1.2 x 10(-3) M, respectively. 4. The modes of inhibition by L-histidine were mixed-noncompetitive, uncompetitive, and noncompetitive types with respect to L-citrulline, L-aspartate, and ATP, respectively. 5. When the enzyme was preincubated with L-citrulline, the enzyme activity was slightly increased in the presence of a low concentration of L-histidine in the assay mixture. 6. The conformation of the enzyme was markedly changed by the addition of L-histidine as judged from the CD spectrum. This change was partially reversed by incubation with L-citrulline.

Activation of aspartate transaminase by pyridoxal 5′-phosphate in the serum of normal subjects

Preincubation of 16 normal sera with pyridoxal 5′-phosphate resulted in a 56% increase of aspartate transminase activity. The magnitude of aspartate transaminase activation by PP is dependent on the concentration of L-aspartate and 2-oxoglutarate in the final reaction mixture.