Alanine Dehydrogenase Research Articles

Enzymes of ammonia assimilation and their regulation inAzospirillum lipoferum strain D-2

Azospirillum lipoferum strain D-2 possesses the following enzymes for the assimilation of N2 and NH 4 + : nitrogenase, glutamine synthetase, NADPH-dependent glutamate synthase, NADH-/NADPH-dependent glutamate dehydrogenase, and NADH-dependent alanine dehydrogenase. Nitrogenase and glutamine synthetase are repressed, whereas glutamate dehydrogenase and alanine dehydrogenase are induced by NH 4 + . Glutamine synthetase activity is modulated by both repression and depression and also by adenylylation.

Implications of salinity fluctuation for growth and nitrogen metabolism of the marine diatom Ditylum brightwellii in comparison with Skeletonema costatum

In 1987 effects of salinity fluctuations on growth of Ditylum brightwellii (West) Grunow, isolated from the Eastern Scheldt estuary (SW Netherlands) in 1981, were studied. D. brightwellii was grown in a 12 h light: dark cycle at constant salinity in brackish media. Ammonium-limited cultures were subjected to a salinity fluctuation. By decreasing the salinity to 4.8‰ photosynthesis and cell division were inhibited; cells were deformed. Protein and carbohydrate contents increased slightly, dark respiration was stimulated and cellular levels of glucose decreased at low salinity; this indicated a possible role of sugars in osmoregulation. Ammonium was accumulated in cultures, amino acids may have been stored; the role of the vacuole as a storage compartment was discussed. Both the ammonium uptake capacity and the affinity for ammonium decreased. Nitrogen limitation was relieved in the transient state. [With the activity of the nitrogen assimilation enzymes glutamine synthetase (GS) and glutamate synthase (GOGAT) being uninhibited by lower salinity.] Recovery from hypo-osmotic stress during a salinity increase was initiated by stimulated photosynthesis; chlorophyll a increased, but persistant contractions of cytoplasm (with chloroplasts) may have delayed cell growth. The glutamate dehydrogenase (GDH) activity decreased further whereas the cellular level of alanine increased in the presence of large ammonium pools; this may indicate a temporary activity of ADH (alanine dehydrogenase). Skeletonema costatum (Greville) Cleve, recovered faster from hypoosmotic stress than did D. brightwellii. Due to an osmotic shock from 13.6 to 7.1‰ S both species excreted amino acids and glucose; S. costatum accumulated more glucose, D. brightwellii accumulated more amino acids. S. costatum may with the competition for nitrogen in waters with an unstable salinity; it will replace D. brightwellii.

Thermostable alanine dehydrogenase of Bacillus sp. DSM730: gene cloning, purification, and characterization

We have cloned the thermostable alanine dehydrogenase (EC 1.4.1.1) gene from a thermophile, Bacillus sp. DSM730, into Escherichia coli C600 with a vector plasmid, pBR322. The enzyme was overproduced by the transformed cells, and purified to homogeneity with a yield of 69% by heat treatment and another step. The enzyme has a molecular weight of about 250,000 and consists of 6 subunits identical in molecular weight (43,000). It is not inactivated by heat treatment at 75° C for 60 min, or incubation in the pH range of 5.5–10.5 at 55°C for 10 min. The enzyme catalyzes the oxidative deamination of L-serine in addition to L-alanine. The oxo analogue of serine is as reactive as pyruvate. Thus, the enzyme differs markedly from alanine dehydrogenases so far studied.

Alanine dehydrogenase from Streptomyces fradiae. Purification and properties.

Alanine dehydrogenase was purified to homogeneity from a cell-free extract of Streptomyces fradiae, which produces tylosin. The enzyme was purified 1180-fold to give a 21% yield, using a combination of hydrophobic chromatography and ion-exchange fast protein liquid chromatography. The relative molecular mass of the native enzyme was determined to be 210,000 or 205,000 by equilibrium ultracentrifugation or gel filtration, respectively. The enzyme is composed of four subunits, each of Mr 51,000. Using analytical isoelectric focusing the isoelectric point of alanine dehydrogenase was found to be 6.1. The Km were 10.0 mM for L-alanine and 0.18 mM for NAD+. Km values for reductive amination were 0.23 mM for pyruvate, 11.6 mM for NH4+ and 0.05 mM for NADH. Oxidative deamination of L-alanine proceeds through a sequential-ordered binary-ternary mechanism in which NAD+ binds first to the enzyme, followed by alanine, and products are released in the order ammonia, pyruvate and NADH.

A further characterization of alanine dehydrogenase from Streptomyces aureofaciens

Homogeneous alanine dehydrogenase isolated from Streptomyces aureofaciens, a producer of tetracycline, was characterized from the point of its molecular and catalytic properties. Using analytical ultracentrifugation the molecular weight of alanine dehydrogenase was found to be 198,000. The enzyme could use as cofactors apart from NAD+ also 1,N6-etheno-NAD+, 3-acetylpyridine-NAD+, deamino-NAD+ and nicotinamide guanine dinucleotide. The enzyme activity in the direction of oxidative deamination was not affected by the addition of nonsubstrate amino acids, however, it was sensitive to inhibitors of SH-groups. Reductive amination of pyruvate was inhibited by L-alanine, L-serine and D-alanine. The inhibition by L-alanine and L-serine was uncompetitive with respect to NADH and noncompetitive with regard to pyruvate and ammonium ions.

Alanine metabolism of the lugworm arenicola marina l. (annelida, polychaeta) during adaptation to reduced salinity

1. The alanine metabolism of the euryhaline osmoconformer Arenicola marina after hyposmotic shock (half-diluted seawater) was investigate. 2. 14C- or 15N-labelled amino acids were injected and the appearance of labelled intermediates and end-products was detected. 3. The importance of pyridoxal phosphate-dependent enzymes (aminotransferases, racemases) was estimated by application of aminooxyacetate. 4. An involvement of d-amino acid oxydase, l-amino acid oxydase, alanine dehydrogenase, alanine carboxylase and the purine nucleotide cycle in alanine catabolism could be excluded. 5. The first step of the alanine degradation observed during adaptation to reduced salinity is the transfer of the amino-group by alanine/2-oxoglutarate aminotransferase [EC 2.6.1.2] yielding glutamate. 6. The second step most likely is the deamination of glutamate by catalysis of glutamate dehydrogenase.

Ammonium-assimilating enzymes and erythromycin formation in Saccharolypospora erythrea.

Ammonium assimilation in Saccharolypospora erythrea was mediated by the glutamine synthetase/glutamate synthase pathway depending on the NH4 concentration in the culture medium. Glutamine synthetase formation was repressed by high ammonium concentration, and a rapid loss of its activity occurred after ammonium addition. There was difference in erythromycin biosynthesis and glutamine synthetase levels when L-glutamate of L-glutamine was added as the nitrogen source. When glutamic acid was used as the nitrogen source, the glutamine synthetase activity was high and there was no erythromycin production. But when glutamine was added, the glutamine synthetase level was low and the yield of the antibiotic was high.Glutamate synthase was formed independently of the nitrogen sources used, but glutamate dehydrogenase was present when ammonium was added as a nitrogen source, and it was NADH-dependent.There were high levels of alanine dehydrogenase in cultures grown with glycine, NH4Cl, alanine and glutamine. Alanine most effectively induced the enzyme formation.The results suggest that there was no correlation between the levels of nitrogen-assimilating enzymes and erythromycin biosynthesis in this microorganism.

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. KM values were 5.0 mM for L-alanine and 0.11 mM for NAD+. KM values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH4Cl.

Use of primary deuterium and 15N isotope effects to deduce the relative rates of steps in the mechanisms of alanine and glutamate dehydrogenases.

We have used deuterium and 15N isotope effects to study the relative rates of the steps in the mechanisms of alanine and glutamate dehydrogenases. The proposed chemical mechanisms for these enzymes involve carbinolamine formation, imine formation, and reduction of the imine to the amino acid [Grimshaw, C.E., Cook, P.F., & Cleland, W.W. (1981) Biochemistry 20, 5655; Rife, J.E., & Cleland, W.W. (1980) Biochemistry 19, 2328]. These steps are almost equally rate limiting for V/Kammonia with alanine dehydrogenase, while with glutamate dehydrogenase carbinolamine formation, imine formation, and release of glutamate after hydride transfer provide most of the rate limitation of V/Kammonia. Release of oxidized nucleotide is largely rate limiting for Vmax for both enzymes. When beta-hydroxypyruvate replaces pyruvate, or 3-acetylpyridine NADH (Acpyr-NADH) or thio-NADH replaces NADH with alanine dehydrogenase, nucleotide release no longer limits Vmax, and hydride transfer becomes more rate limiting. With glutamate dehydrogenase, replacement of alpha-ketoglutarate by alpha-ketovalerate makes hydride transfer more rate limiting. Use of Acpyr-NADPH has a minimal effect with alpha-ketoglutarate but causes an 8-fold decrease in Vmax with alpha-ketovalerate, with hydride transfer the major rate-limiting step. In contrast, thio-NADPH with either alpha-keto acid causes carbinolamide formation to become almost completely rate limiting. These studies show the power of multiple isotope effects in deducing details of the chemistry and changes in rate-limiting step(s) in complicated reaction mechanisms such as those of alanine and glutamate dehydrogenases.

Oscillations in open two-michaelian-enzyme systems

Chemical oscillations are predicted theoretically in an open two-michaelian-enzyme cycling system. By numerical simulations, we analyse the conditions under which the oscillator can be stabilized. When the reaction proceeds faster than transport, as long as the H +-producing reaction is predominant at low pH values and the H +-consuming one is at high pH values, it is possible to obtain a large oscillatory domain. The period and the amplitude of the oscillations can vary within a wide range of constraints and the shape of the oscillations can itself be chosen among sinusoïdal, triangular or rectangular types. Time oscillations of pH and NADH concentration were observed experimentally in a system involving alcohol dehydrogenase and alanine dehydrogenase. The period of the oscillations was about 2 h under the experimental conditions, and, following the predictions, the oscillations were damped rapidly due to imperfect membrane selectivity and buffer production by the reactions.

Oscillations in open two-michaelian-enzyme systems

Chemical oscillations are predicted theoretically in an open two-michaelian-enzyme cycling system. By numerical simulations, we analyse the conditions under which the oscillator can be stabilized. When the reaction proceeds faster than transport, as long as the H +-producing reaction is predominant at low pH values and the H +-consuming one is at high pH values, it is possible to obtain a large oscillatory domain. The period and the amplitude of the oscillations can vary within a wide range of constraints and the shape of the oscillations can itself be chosen among sinusoïdal, triangular or rectangular types. Time oscillations of pH and NADH concentration were observed experimentally in a system involving alcohol dehydrogenase and alanine dehydrogenase. The period of the oscillations was about 2 h under the experimental conditions, and, following the predictions, the oscillations were damped rapidly due to imperfect membrane selectivity and buffer production by the reactions.

Glutamate biosynthesis in Bacillus azotofixans. 15N NMR and enzymatic studies.

Pathways of ammonia assimilation into glutamic acid in Bacillus azotofixans, a recently characterized nitrogen-fixing species of Bacillus, were investigated through observation by NMR spectroscopy of in vivo incorporation of 15N into glutamine and glutamic acid in the absence and presence of inhibitors of ammonia-assimilating enzymes, in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthase, and alanine dehydrogenase. In ammonia-grown cells, both the glutamine synthetase/glutamate synthase and the glutamate dehydrogenase pathways contribute to the assimilation of ammonia into glutamic acid. In nitrate-grown and nitrogen-fixing cells, the glutamine synthetase/glutamate synthase pathway was found to be predominant. NADPH-dependent glutamate dehydrogenase activity was detectable at low levels only in ammonia-grown and glutamate-grown cells. Thus, B. azotofixans differs from Bacillus polymyxa and Bacillus macerans, but resembles other N2-fixing prokaryotes studied previously, as to the pathway of ammonia assimilation during ammonia limitation. Implications of the results for an emerging pattern of ammonia assimilation by alternative pathways among nitrogen-fixing prokaryotes are discussed, as well as the utility of 15N NMR for measuring in vivo glutamate synthase activity in the cell.

Studies on the Biosynthesis of L-Alanine Dehydrogenase by Cuinnghamella elegans

Summary Cell-free extracts of Cunninghamella elegans grown on L-alanine as sole source of nitrogen contained L-alanine dehydrogenase that catalyzes the oxidative deamination of L-alanine to equimolar amounts of pyruvate and ammonia. The enzyme was produced during the logarithmic phase of growth, and maximum yield was obtained after 5 d of incubation. The optimum pH range for growth and enzyme synthesis was 5.0–7.0. L- Alanine dehydrogenase of C. elegans was induced by L-alanine, DL-alanine, L-aspartic acid, L-asparagine and ammonium phosphate, but L-alanine was the best inducer. Maximum growth and enzyme formation were obtained when L-alanine concentration of the medium was 2.1 g/l. Increasing ammonium phosphate concentration in the culture medium stimulated L-alanine dehydrogenase synthesis. The effect of different carbon sources and metal salts on growth and enzyme production was studied.

A spectrophotometric method for the determination of gamma-glutamyl cyclotransferase with alanine dehydrogenase in the presence of anthglutin.

gamma-Glutamyl cyclotransferase activity is assayed in tissues by a colorimetric method using gamma-glutamyl alanine as a substrate coupled with alanine dehydrogenase from B. sphericus, to measure the formation of NADH. In order to avoid interference by the reaction catalyzed by gamma-glutamyl transpeptidase, anthglutin, a specific inhibitor of the transpeptidase was included in the reaction mixture. The Km value of rat kidney gamma-glutamyl cyclotransferase with respect to gamma-glutamyl alanine appeared to be the same when determined by either the colorimetric or the radiometric method. This assay presents a reliable alternative to the use of radiolabeled substrate and is used for the assay of gamma-glutamyl cyclotransferase in a variety of physiological and experimental samples.

Preparation of 15N-labeled l-alanine by immobilized l-alanine dehydrogenase: Differential incorporation of 15N in bacterial proteins

This paper describes a system for continuous synthesis of 15 N-labeled l-alanine from lactic acid, 15NH 4Cl and NADH, which uses immobilized alanine dehydrogenase and soluble lactate dehydrogenase as enzyme sources. Lactic acid acts both as hydrogen donor for the regeneration of NADH and as pyruvate source, thus providing the carbon skeleton of l-alanine. Citrobacter freundi grown on synthetic media containing 17 unlabeled amino acids and l-( 15 N)alanine as nitrogen source, incorporated 66% of 15 N into alanine found in bacterial proteins. When 15 N-labeled glutamic acid, aspartic acid or glycocol were added to the synthetic growth media, their 15 N was “diluted” among different amino acids of bacterial proteins. Isotope enrichment of l-( 15 N)lysine found in newly synthesized proteins of C. freundi was practically unchanged as compared to the isotope content of free amino acid in the growth medium.

Conversion of phenylpyruvate to l-phenylalanine by immobilized Clostridium butyricum-alanine dehydrogenase-Micrococcus luteus under hydrogen high pressure

Alanine was the best amino donor among various amino acids and NH4Cl for the phenylalanine production of Micrococus luteus. l-Alanine was regenerated at the rate of 9.2μ moles/min/g dry cells from NH4Cl and pyruvate by immobilized Clostridium butyricum-alanine dehydrogenase. l-Phenylalanine was continuously produced from hydrogen, NH4Cl and phenylpyruvate by coupling immobilized C. butyricum, alanine dehydrogenase and M. luteus. The rate of phenylalanine production was 1.74 μ moles/min/g dry cells.

A spectrophotometric rate assay of aminoacylase

Acetamidoacrylate, a synthetic N-acetyl unsaturated amino acid, was hydrolyzed to acetate, ammonia, and pyruvate by hog kidney, fungal, and bacterial aminoacylases. A spectrophotometric procedure for rate assay of aminoacylase has been established with this substrate on the basis of the simultaneous reduction of pyruvate with NADH and alanine dehydrogenase. This assay is linear with time and enzyme concentration and is useful for kinetic studies of aminoacylase. This procedure is not influenced significantly by amino and thiol compounds and metal ions, which interfere with the ninhydrin methods traditionally used. Alanine dehydrogenase can be replaced by lactate dehydrogenase in the reaction system.

Properties of Alanine Dehydrogenase and Aspartase from Propionibacterium freudenreichii subsp. shermanii

During lactate fermentation by Propionibacterium freudenreichii subsp. shermanii ATCC 9614, the only amino acid metabolized was aspartate. After lactate exhaustion, alanine was one of the two amino acids to be metabolized. For every 3 mol of alanine metabolized, 2 mol of propionate, 1 mol each of acetate and CO(2), and 3 mol of ammonia were formed. The specific activity of alanine dehydrogenase was 0.08 U/mg of protein during lactate fermentation, and it increased to 0.9 U/mg of protein after lactate exhaustion. Alanine dehydrogenase and aspartase, key enzymes in the metabolism of alanine and aspartate, respectively, were partially purified, and some of their properties were studied. Alanine dehydrogenase had a pH optimum of 9.2 to 9.6 and high K(m) values for both NAD (1 to 4 mM) and alanine (7 to 20 mM). Activity was inhibited by low concentrations of pyruvate and NADH. The pH optimum of aspartase decreased from approximately 7.5 to approximately 6.4 when the MgCl(2) and aspartate concentrations were decreased. Plots of aspartate concentration versus activity showed either hyperbolic or sigmoidal kinetics (interaction coefficient, up to a value of 3.1), depending on pH and MgCl(2) concentration. MgCl(2) was either an activator or an inhibitor, depending on pH and its concentration. Aspartase activity was inhibited by low concentrations of fumarate. The properties of alanine dehydrogenase and aspartase are consistent with the finding that aspartate is metabolized during lactate fermentation, while alanine is only fermented after lactate exhaustion and then at a slow rate.

Ammonia assimilation in Bacillus polymyxa. 15N NMR and enzymatic studies.

Pathways of ammonia assimilation into glutamic acid and alanine in Bacillus polymyxa were investigated by 15N NMR spectroscopy in combination with measurements of the specific activities of glutamate dehydrogenase, glutamine synthetase, glutamate synthetase, alanine dehydrogenase, and glutamic-alanine transaminase. Ammonia was found to be assimilated into glutamic acid predominantly by NADPH-dependent glutamate dehydrogenase with a Km of 2.9 mM for NH4+ not only in ammonia-grown cells but also in nitrate-grown and nitrogen-fixing cells in which the intracellular NH4+ concentrations were 11.2, 1.04, and 1.5 mM, respectively. In ammonia-grown cells, the specific activity of alanine dehydrogenase was higher than that of glutamic-alanine transaminase, but the glutamate dehydrogenase/glutamic-alanine transaminase pathway was found to be the major pathway of 15NH4+ assimilation into [15N]alanine. The in vitro specific activities of glutamate dehydrogenase and glutamine synthetase, which represent the rates of synthesis of glutamic acid and glutamine, respectively, in the presence of enzyme-saturating concentrations of substrates and coenzymes are compared with the in vivo rates of biosynthesis of [15N]glutamic acid and [alpha,gamma-15N]glutamine observed by NMR, and implications of the results for factors limiting the rates of their biosynthesis in ammonia- and nitrate-grown cells are discussed.

Bioluminescent Flow Sensors: L-Alanine Determination in Serum and Urine

Abstract A continuous flow bioluminescent method for L-alanine analysis in serum and urine has been developed. Serum can be analyzed directly after simple filtration. Response is linear from 50 to 1500 pmoles in biological matrix. Alanine dehydrogenase is immobilized onto a nylon coil separated from the reactor coil containing bioluminescent enzymes. The stability of nylon immobilized enzymes is high (over three months) and more than 900 samples can be analyzed with few mg of enzymes. The results obtained with the bioluminescent sensor agree well with those obtained by ion exchange chromatography (amino acid analyzer).