Presence Of Alanine Research Articles

The Nature and Thiknesses of Passive Films on Interface Cu/ NaOH+Alanine

Thicknesses of films formed on the copper in alkaline solution in the presence of alanine are studied by ellipsometric method. It was found that small additives of β-alanine (10-4-10-3 mol/l) cause additional stabilization of copper passivity due to an increase in the film thickness. The adsorption kinetics of α- and β-alanine is studied. It is shown experimentally that β-alanine adsorbs on copper according to the Frumkin isotherm. By contrast, no definite type isotherm can be obtained for α-alanine due to its higher activity and ability to form soluble complexes with copper ions.

Differential nucleoside recognition during Bacillus cereus 569 (ATCC 10876) spore germination

We have tested a series of inosine analogs for their effect on germinating B. cereus 569 spores. Our results showed that although inosine (hypoxanthine nucleoside) causes spore germination by itself, the kinetic pathway exhibited complex and strongly cooperative character. Contrary to inosine’s germinating effect, the purine pathway degradation products xanthine, xanthosine, uric acid, hypoxanthine, ribose, or ribose plus hypoxanthine failed to activate spore germination. Furthermore, even small modifications of inosine’s nucleobase or sugar moieties have deleterious effects on germination efficiency. In contrast to previous work with the B. cereus 3711 strain, incubation of B. cereus 569 spores with adenosine (6-aminopurine riboside) did not trigger germination, but prevented inosine-mediated germination. The inhibitory effect was lost if adenosine was substituted with adenine alone, or ribose plus adenine. Although adenosine is able to block inosine-mediated germination, it acts as a co-germinant in the presence of alanine. Nucleosides that have substitutions in the purine base are not able to trigger germination by themselves, but can act as co-germinants in the presence of sub-germinant concentrations of alanine. In contrast, modifications of the sugar moiety precluded germination activity under all conditions tested. The data suggests that only inosine can activate germination by itself. However, when alanine is present as a co-germinant, different germination receptors are activated that recognize a larger subset of nucleoside structures.

Influence of Proteins on the Hydrothermal Gasification and Liquefaction of Biomass. 2. Model Compounds

The hydrothermal gasification is a promising process to produce hydrogen from biomass with high water content. In a lot of cases, this biomass may contain proteins, for example, in residues from the food industry or sewage sludge. In Part I of this serial, experiments on hydrothermal gasification of protein containing biomass (zoo mass) have been reported. This biomass produces lower gas yields than biomass originating exclusively from plants (phyto mass). To understand these findings, experiments with model compounds are necessary. Here, such experiments with model compounds in a tubular and a batch reactor are described. The model system for the phyto mass is glucose with a potassium salt, and the model system for the zoo mass is glucose, potassium salt, and the amino acid alanine. The model systems show a lower gas yield in the presence of alanine. So the presence of alanine in the model system has a similar effect to the presence of proteins in biomass. Additionally, the gas composition and the concentration of selected key compounds are slightly changed by alanine addition. Likely, consecutive products of carbohydrate and protein degradation react with each other. In such Maillard reactions, free radical scavengers might be formed, reducing the reaction rate of free radical chain reactions that are highly relevant for gas formation. Therefore, the gas yield is lower in the presence of proteins or amino acids compared with systems without these compounds. In addition, experiments with real biomass in a batch reactor were reported to verify the assumption of Maillard products reducing free radical reactions. As an example, the addition of urea to phyto mass leads to a decrease of the gas yield to a value similar to that found for zoo mass.

Peculiarities of electrochemical behavior of copper in alkaline solutions in the presence of alanine

Anodic oxidation of copper in alkaline solution in the presence of alanine is studied by electrochemical and ellipsometric methods. Thickness of films formed on the metal in the working solutions, are determined. Small additives of β-alanine (10−4–10−3 mol/l) cause additional stabilization of copper passivity due to an increase in the film thickness. The adsorption kinetics of α-and β-alanine is studied. It is shown experimentally that β-alanine adsorbs on copper according to the Frumkin isotherm. By contrast, no definite type isotherm can be obtained for α-alanine due to its higher activity and ability to form soluble complexes with copper ions.

L-Cysteine Influx and Efflux in Human Erythrocytes: the Role of Red Blood Cells in Redox and Metabolite Homeostasis in the Plasma

treated with L-cysteine 1 mM displayed efflux and increased the free-SH concentrations up to 0.184 ± 0.010 mM in the incubation media in 1 hr. While this concentration reached 0.843 ± 0.012 mM in 10 mM-L-cysteine pretreated erythrocytes. Our results also showed that the L-cysteine efflux is partly mediated by the Alanine-Serine-Cysteine (ASC) system. The presence of alanine or serine in the incubation media decreased the rate of efflux by about 16%. Our results also showed that the L-cysteine efflux process is not a simple diffusion but a carrier-mediated process. When compared with N-acetyl-L-cysteine (NAC), which is known to diffuse through the membranes, L-cysteine displayed a higher efflux rate under the same conditions. Pretreatment of erythrocytes with L-cysteine 4 mM increased the free-SH concentration to 0.48 ± 0.005 mM whereas the same concentration of NAC brought the free-SH concentration to 0.36 ± 0.01 mM in the incubation media. Our results suggest that erythrocytes may contribute to redox and metabolite homeostasis of the plasma.

N‐terminal acetylation and protonation of individual hemoglobin subunits: Position‐dependent effects on tetramer strength and cooperativity

The presence of alanine (Ala) or acetyl serine (AcSer) instead of the normal Val residues at the N-terminals of either the alpha- or the beta-subunits of human adult hemoglobin confers some novel and unexpected features on the protein. Mass spectrometric analysis confirmed that these substitutions were correct and that they were the only ones. Circular dichroism studies indicated no global protein conformational changes, and isoelectric focusing showed the absence of impurities. The presence of Ala at the N-terminals of the alpha-subunits of liganded hemoglobin results in a significantly increased basicity (increased pK(a) values) and a reduction in the strength of subunit interactions at the allosteric tetramer-dimer interface. Cooperativity in O(2) binding is also decreased. Substitution of Ala at the N-terminals of the beta-subunits gives neither of these effects. The substitution of Ser at the N terminus of either subunit leads to its complete acetylation (during expression) and a large decrease in the strength of the tetramer-dimer allosteric interface. When either Ala or AcSer is present at the N terminus of the alpha-subunit, the slope of the plot of the tetramer-dimer association/dissociation constant as a function of pH is decreased by 60%. It is suggested that since the network of interactions involving the N and C termini of the alpha-subunits is less extensive than that of the beta-subunits in liganded human hemoglobin disruptions there are likely to have a profound effect on hemoglobin function such as the increased basicity, the effects on tetramer strength, and on cooperativity.

AMINO-ACID-DEPENDENT, DIFFERENTIAL EFFECTS OF ETHANOL ON GLUCOSE PRODUCTION IN RABBIT KIDNEY-CORTEX TUBULES

The effect of ethanol on glucose synthesis in kidney-cortex tubules of control and diabetic rabbits has been investigated. Both freshly isolated and grown in primary cultures, kidney-cortex tubules were incubated with alanine or aspartate plus lactate or glycerol plus octanoate in the absence and presence of 100 mmol/l ethanol. In freshly isolated renal tubules incubated in the presence of alanine plus lactate or glycerol plus octanoate, and in tubules grown in primary culture in the medium containing alanine plus lactate plus octanoate alcohol, resulted in about 30% decrease in glucose formation. A diminished glucose production in freshly isolated tubules was accompanied by: (i) a decrease in alanine utilization, (ii) an increase in lactate or glycerol consumptions and (iii) a decline in GSH:GSSG ratio. The ethanol action was not abolished by 4-methylpyrazole, an inhibitor of alcohol dehydrogenase (ADH). In view of ethanol-induced changes in gluconeogenic intermediates it is likely that in the presence of alanine plus glycerol plus octanoate ethanol causes a decline in flux through phosphoenolpyruvate carboxykinase, probably due to either an increase in intracellular content of 2-oxoglutarate, inhibitor of this key gluconeogenic enzyme and/or an enhanced flux through pyruvate kinase, as concluded from an increased lactate formation in the presence of glycerol in the incubation medium. In renal tubules grown in primary cultures in the presence of alanine plus lactate plus octanoate a decrease in GSH:GSSG ratio was accompanied by elevated generation of reactive oxygen species (ROS). Upon replacement of alanine by aspartate ethanol affected neither glucose production, substrate uptake, ROS accumulation nor GSH:GSSG ratio. In the presence of alanine ethanol-induced decrease in glucose production and elevation of ROS might cause a limited NADPH generation resulting in a decrease in the intracellular GSH:GSSG ratio. On the contrary, aspartate might protect against ROS generation, so intensive gluconeogenesis supports NADPH generation and in consequence high values of the intracellular GSH:GSSG ratio are maintained.

Identification and characterization of a nitrate transporter gene in Neurospora crassa.

The Neurospora crassa genome database was searched for sequence similarity to crnA, a nitrate transporter in Aspergillus nidulans. A 3.9-kb fragment (contig 3.416, subsequence 183190-187090) was cloned by PCR. The gene coding for this nitrate transporter was termed nit-10. The nit-10 gene specifies a predicted polypeptide containing 541 amino acids with a molecular mass of 57 kDa. In contrast to crnA, which is clustered together with niaD, encoding nitrate reductase, and niiA, encoding nitrite reductase, nit-10 is not linked to nit-3 (nitrate reductase), nit-6 (nitrite reductase), or to nit-2, nit-4 (both are positive regulators of nit-3), or nmr (negative regulator of nit-3) in Neurospora crassa. A nit-10 rip mutant failed to grow in the medium when nitrate (< 10 mM) was used as the sole nitrogen source, but grew similarly to wild type when nitrate concentration was 10 mM or higher. In addition, it showed strong sensitivity to cesium in the presence of nitrate and resistance to chlorate in the presence of alanine, proline, or hypoxanthine. The expression of nit-10 required nitrate induction and was subject to repression by nitrogen metabolites such as glutamine. Expression of nit-10 also required functional products of nit-2 and nit-4. The half-life of nit-10 mRNA was determined to be approximately 2.5 min.

Utility of proton MR spectroscopy in the diagnosis of radiologically atypical intracranial meningiomas.

Our aim was to evaluate the usefulness of proton MR spectroscopy ((1)H MRS) in the diagnosis of radiologically atypical brain meningiomas. We studied 37 patients with intracranial meningiomas with MRI and (1)H MRS (TE 136 ms). Their spectra were quantitatively assessed and compared with those of 93 other intracranial brain neoplasms: 15 low-grade and 14 anaplastic astrocytomas, 30 glioblastomas and 34 metastases. The most characteristic features of meningiomas were the presence of alanine, high relative concentrations of choline and glutamine/glutamate and low concentrations of creatine-containing compounds, N-acetyl-containing compounds and lipids. These resonances were assembled in algorithms for two-way differentiation between meningioma and the other tumours. The performance of the algorithms was tested in the 130 patients using the leave-one-out method, with 94% success in differentiating between meningioma and other tumour. Of the 37 meningiomas, five (14%) were thought atypical on MRI, and in only one of these, found to be malignant on histology, was a diagnosis other than meningioma suggested by the algorithm. The other four were correctly classified. We suggest that (1)H MRS provides information on intracranial meningiomas which may be useful in diagnosis of radiologically atypical cases.

The role of β-hydroxyamino acids in the Maillard reaction—transamination route to Amadori products

Analyses of degradation products of β-hydroxyamino acids—l-serine and l-threonine have indicated that the formation of 2-ketoacids and 2-amino alcohols such as pyruvic acid and ethanolamine constitutes a major degradation pathway. Simple mechanisms of decarboxylations and dehydrations can explain their formation in these systems as confirmed by labeling studies. Interestingly, the amino carbonyl interaction between the resulting pyruvic acid and the amino ethanol can lead to the formation of an Amadori product identical in structure to that formed from alanine and glycolaldehyde, indicating the existence of another route to this important Maillard intermediate. In this study, using variously [13C]-labeled pyruvic acids, amino ethanol and alanine, detailed investigation of this interaction was carried out. The initial imine formed during this reaction undergoes an isomerization through a 1,3-prototropic shift followed by enolization and formation of an Amadori product. Aternatively, the isomerized imine can undergo hydrolysis to produce an amino acid and an α-hydroxyaldehyde. The results of these labeling studies have also confirmed the formation of pyruvic acid in the reaction mixture of alanine/glycolaldehyde and the presence of alanine and glycolaldehyde in pyruvic acid/ethanolamine mixture, consistent with the proposed mechanism. It can be concluded therefore that in addition to sugar/amino acid mixtures, 2-ketoacid and α-amino alcohol interaction can also lead to the formation of Amadori product and initiation of Maillard reaction through transamination process.

A nuclear magnetic resonance-based demonstration of substantial oxidative L-alanine metabolism and L-alanine-enhanced glucose metabolism in a clonal pancreatic beta-cell line: metabolism of L-alanine is important to the regulation of insulin secretion.

Early experiments indicated that islet beta-cells substantially metabolized L-alanine but that insulin secretion was largely unaffected by the amino acid. It was subsequently demonstrated using more intricate studies that L-alanine is a strong stimulus to insulin secretion in the presence of glucose in normal rodent islets and beta-cell lines. Using (13)C nuclear magnetic resonance (NMR), we have demonstrated substantial oxidative metabolism of L-alanine by the clonal beta-cell line BRIN-BD11, with time-dependent increases in production of cellular glutamate and aspartate. Stimulatory effects of L-alanine on insulin secretion were attenuated by the inhibition of beta-cell oxidative phosphorylation using oligomycin. Additionally, we detected substantial production of lactate, alanine, and glutamate from glucose (16.7 mmol/l) after 60 min. On addition of 10 mmol/l L-alanine to a stimulus of 16.7 mmol/l glucose, the utilization rate of glucose increased approximately 2.4-fold. L-Alanine dramatically enhanced NMR-measurable aspects of glucose metabolism (both oxidative and nonoxidative). The enhanced rate of entry of glucose-derived pyruvate into the tricarboxylic acid (TCA) cycle in the presence of alanine may have stimulated rates of generation of key metabolites, including ATP, which affect the insulin secretory process. Thus L-alanine metabolism, in addition to the enhancing effect on glucose metabolism, contributes to the stimulatory effects of this amino acid on insulin secretion in vitro.

Caramelization of maltose solution in presence of alanine.

Two solutions of maltose in water were used to prepare caramels. Alanine as a catalyst was added to one of these solutions. The caramelization was conducted at 130 degrees C for total time period 90 minutes. Convenient samples were taken of each caramel solution every 30 min and subjected to sensory analysis and isolation of volatile components. The odour and colour sensory tests were evaluated according to the international standard methods (ISO). The results showed that, the presence of alanine gave rise to a high significant (P < 0.01) decrease in acid attributes and remarkable increase in the sweet and caramel attributes, which are the most important caramel notes. On the other hand the increase in heating time in presence of alanine as a catalyst resulted in a high significant (P < 0.01) increase in the browning rate of caramel solution. The new technique Solid Phase Micro Extraction (SPME) was used for trapping the volatile components in the headspace of each caramel samples followed by thermal desorption and GC and GC - MS analysis. The 5-hydroxymethyl-2-furfural (HMF), the main characteristic caramel product, showed its highest value in sample containing alanine after heating for 60 minutes. The best sensory results of the sample contains alanine were confirmed by the presence of high concentrations of the most potent odorants of caramel besides to the formation of some volatile compounds have caramel like flavours such as 2-acetyl pyrrole, 2-furanones and 1-(2-furanyl)1,2-propandione.

Active transport of alanine by the neutral amino-acid exchanger ASCT1

ASCT1 protein is a member of the glutamate transporter superfamily, which shows system ASC selectivity and properties and has been characterized as a Na+-dependent neutral amino-acid exchanger. Here, by using ASCT1-expressing oocytes, the uptake of alanine and glutamate was measured to investigate ASCT1's ability to mediate a concentrative transport of alanine, ASCT1's sodium dependence, and the influence of pH on the mutual inhibition between alanine and glutamate. Alanine uptake was measured after 30 min incubation. Kinetic analysis of the Na+ dependence of alanine uptake showed an apparent K0.5 (affinity constant) value for Na+ of 23.1 +/- 4.3 mM (mean +/- SE). Concentration dependence of alanine uptake was tested at 100 and 1 mM Na+, with apparent K0.5 values of 0.16 +/- 0.04 and 1.8 +/- 0.4 mM, respectively, at pH 7.5, and 0.21 +/- 0.06 and 1.9 +/- 0.3 mM at pH 6. Vmax was not modified between 100 and 1 mM Na+ at either pH. ASCT1 actively transports alanine and accumulates it in the cytosol even when the Na+ concentration in the medium was as low as 1-3 mM. 22Na uptake studies revealed that Na+ transport was stimulated by the presence of alanine in the medium. Our results demonstrate that ASCT1 is able to mediate a concentrative transport of alanine, which is Na+-dependent but not coupled to the Na+ gradient.

Active site mapping, biochemical properties and subcellular localization of rhodesain, the major cysteine protease of Trypanosoma brucei rhodesiense

Cysteine protease activity of African trypanosome parasites is a target for new chemotherapy using synthetic protease inhibitors. To support this effort and further characterize the enzyme, we expressed and purified rhodesain, the target protease of Trypanosoma brucei rhodesiense (MVAT4 strain), in reagent quantities from Pichia pastoris. Rhodesain was secreted as an active, mature protease. Site-directed mutagenesis of a cryptic glycosylation motif not previously identified allowed production of rhodesain suitable for crystallization. An invariable ER(A/V)FNAA motif in the pro-peptide sequence of rhodesain was identified as being unique to the genus Trypanosoma. Antibodies to rhodesain localized the protease in the lysosome and identified a 40-kDa protein in long slender forms of T. b. rhodesiense and all life-cycle stages of T. b. brucei. With the latter parasite, protease expression was five times greater in short stumpy trypanosomes than in the other stages. Radiolabeled active site-directed inhibitors identified brucipain as the major cysteine protease in T. b. brucei. Peptidomimetic vinyl sulfone and epoxide inhibitors designed to interact with the S 2, S 1 and S′ subsites of the active site cleft revealed differences between rhodesain and the related trypanosome protease cruzain. Using fluorogenic dipeptidyl substrates, rhodesain and cruzain had acid pH optima, but unlike some mammalian cathepsins retained significant activity and stability up to pH 8.0, consistent with a possible extracellular function. S 2 subsite mapping of rhodesain and cruzain with fluorogenic peptidyl substrates demonstrates that the presence of alanine rather than glutamate at S 2 prevents rhodesain from cleaving substrates in which P 2 is arginine.

13C isotopomer analysis of glucose and alanine metabolism reveals cytosolic pyruvate compartmentation as part of energy metabolism in astrocytes.

After incubation of glial cells with both (13)C-labeled and unlabeled glucose and alanine, (13)C isotopomer analysis indicates two cytosolic pyruvate compartments in astrocytes. One pyruvate pool is in an exchange equilibrium with exogenous alanine and preferentially synthesizes releasable lactate. The second pyruvate pool, which is of glycolytic origin, is more closely related to mitochondrial pyruvate, which is oxidized via tri carbonic acid (TCA) cycle activity. In order to provide 2-oxoglutarate as a substrate for cytosolic alanine aminotransferase, glycolytic activity is increased in the presence of exogenous alanine. Furthermore, in the presence of alanine, glutamate is accumulated in astrocytes without subsequent glutamine synthesis. We suggest that the conversion of alanine to releasable lactate proceeds at the expense of flux of glycolytic pyruvate through lactate dehydrogenase, which is used for ammonia fixation by alanine synthesis in the cytosol and for mitochondrial TCA cycle activity. In addition, an intracellular trafficking occurs between cytosol and mitochondria, by which these two cytosolic pyruvate pools are partly connected. Thus, exogenous alanine modifies astrocytic glucose metabolism for the synthesis of releasable lactate disconnected from glycolysis. The data are discussed in terms of astrocytic energy metabolism and the metabolic trafficking via a putative alanine-lactate shuttle between astrocytes and neurons.

Stimulation of Na+-alanine cotransport activates a voltage-dependent conductance in single proximal tubule cells isolated from frog kidney.

1. The swelling induced by Na+-alanine cotransport in proximal tubule cells of the frog kidney is followed by regulatory volume decrease (RVD). This RVD is inhibited by gadolinium (Gd3+), an inhibitor of stretch-activated channels, but is independent of extracellular Ca2+. 2. In this study, the whole cell patch clamp technique was utilized to examine the effect of Na+-alanine cotransport on two previously identified volume- and Gd3+-sensitive conductances. One conductance is voltage dependent and anion selective (GVD) whilst the other is voltage independent and cation selective (GVI). 3. Addition of 5 mM L-alanine to the bathing solution increased the whole cell conductance and gave a positive (depolarizing) shift in the reversal potential (Vrev, equivalent to the membrane potential in current-clamped cells) consistent with activation of Na+-alanine cotransport. Vrev shifted from -36 +/- 4.9 to +12.9 +/- 4.2 mV (n = 15). 4. In the presence of alanine, the total whole cell conductance had several components including the cotransporter conductance and GVD and GVI. These conductances were separated using Gd3+, which inhibits both GVD and GVI, and the time dependency of GVD. Of these two volume-sensitive conductances, L-alanine elicited a specific increase in GVD, whereas GVI was unaffected. 5. The L-alanine-induced activation of GVD was significantly reduced when cells were incubated in a hypertonic bathing solution. 6. In summary, in single proximal tubule cells isolated from frog kidney, on stimulation of Na+-alanine cotransport GVD is activated, while GVI is unaffected. Taken with other evidence, this suggests that GVD is activated by cell swelling, consequent upon alanine entry, and may play a role as an anion efflux pathway during alanine-induced volume regulation.

Fatty acids and glycerol or lactate are required to induce gluconeogenesis from alanine in isolated rabbit renal cortical tubules.

In isolated rabbit renal cortical tubules, glucose synthesis from 1 mM alanine is negligible, while the amino acid is metabolized to glutamine and glutamate. The addition of 0.5 mM octanoate plus 2 mM glycerol induces incorporation of [U-14C]alanine into glucose and decreases glutamine synthesis, whereas oleate and palmitate in the presence of glycerol are less potent than octanoate. Gluconeogenesis is also significantly accelerated when glycerol is substituted by lactate. In view of an increase in 14CO2 fixation and elevation of both cytosolic and mitochondrial NADH/NAD+ ratios, the activation of glucose formation from alanine upon the addition of glycerol and octanoate is likely due to (i) stimulation of pyruvate carboxylation, (ii) increased availability of NADH for glyceraldehyde-3-phosphate dehydrogenase and (iii) elevation of mitochondrial redox state causing a diminished provision of ammonium for glutamine synthesis. The induction of gluconeogenesis in the presence of alanine, glycerol and octanoate is not related to cell volume changes. The results presented in this paper show the importance of free fatty acids and glycerol for regulation of renal gluconeogenesis from alanine. The possible physiological significance of the data is discussed.

Amino acid transformation of oscillatory Ca2+ signals in mouse pancreatic beta-cells.

Glucose-induced increase of cytoplasmic Ca2+ in pancreatic beta-cells is usually manifested as slow oscillations from the basal level. The significance of this rhythmicity for maintaining normal beta-cell function with periodic variations of circulating insulin made it of interest to investigate how the oscillatory Ca2+ signal was affected by various amino acids. Individual mouse beta-cells were very sensitive to alanine, glycine and arginine, sometimes responding with a transformation of the oscillations into sustained elevation of cytoplasmic Ca2+ at amino acid concentrations as low as 0.1 mM. Stimulation of the entry of Ca2+, obtained either by raising the extracellular concentration or by prolonging the open state of the voltage-dependent Ca2+ channels with BAY K 8644, resulted in reappearance of the rhythmic activity in the presence of the amino acids. Oscillatory Ca2+ signals in intact islets were more resistant to transformation by amino acids than those of individual beta-cells. It is therefore suggested that signals from the adjacent cells make it possible for beta-cells situated in islets to overcome a suppression of the oscillatory activity otherwise seen in the presence of alanine, glycine or arginine.

Importance of glutamate dehydrogenase stimulation for glucose and glutamine synthesis in rabbit renal tubules incubated with various amino acids.

The effect of 2-aminobicyclo[2.2.1]heptan-2-carboxylic acid (BCH), an L-leucine nonmetabolizable analogue and an allosteric activator of glutamate dehydrogenase, on glucose and glutamine synthesis was studied in rabbit renal tubules incubated with alanine, aspartate or proline in the presence of glycerol and octanoate, i.e. under conditions of efficient glucose formation. With alanine+glycerol+octanoate the addition of BCH resulted in a stimulation of alanine and glycerol consumption, accompanied by an increased glucose, lactate and glutamine synthesis. In contrast, when alanine was substituted by either aspartate or proline, BCH altered neither glucose formation nor glutamine and glutamate synthesis, while an accelerated glycerol utilization was accompanied by a small increase in lactate production. In view of the BCH-induced changes in intracellular metabolite levels the acceleration of gluconeogenesis by BCH in the presence of alanine+glycerol+octanoate is probably due to (i) increased uptake of alanine via alanine aminotransferase, (ii) stimulation of phosphoenolpyruvate carboxykinase, a key-enzyme of gluconeogenesis, (iii) rise of glucose-6-phosphatase activity, as well as (iv) activation of the malate-aspartate shuttle resulting in an augmented glycerol utilization for lactate and glucose synthesis.

Alanine catabolism in Klebsiella aerogenes: molecular characterization of the dadAB operon and its regulation by the nitrogen assimilation control protein.

Klebsiella aerogenes strains with reduced levels of D-amino acid dehydrogenase not only fail to use alanine as a growth substrate but also become sensitive to alanine in minimal media supplemented with glucose and ammonium. The inability of these mutant strains to catabolize the alanine provided in the medium interferes with both pathways of glutamate production. Alanine derepresses the nitrogen regulatory system (Ntr), which in turn represses glutamate dehydrogenase, one pathway of glutamate production. Alanine also inhibits the enzyme glutamine synthetase, the first enzyme in the other pathway of glutamate production. Therefore, in the presence of alanine, strains with mutations in dadA (the gene that codes for a subunit of the dehydrogenase) exhibit a glutamate auxotrophy when ammonium is the sole source of nitrogen. The alanine catabolic operon of Klebsiella aerogenes, dadAB, was cloned, and its DNA sequence was determined. The clone complemented the alanine defects of dadA strains. The operon has a high similarity to the dadAB operon of Salmonella typhimurium and the dadAX operon of Escherichia coli, each of which codes for the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase. Unlike the cases for E. coli and S. typhimurium, the dad operon of K. aerogenes is activated by the Ntr system, mediated in this case by the nitrogen assimilation control protein (NAC). A sequence matching the DNA consensus for NAC-binding sites is located centered at position -44 with respect to the start of transcription. The promoter of this operon also contains consensus binding sites for the catabolite activator protein and the leucine-responsive regulatory protein.