NAD and NADP-dependent glutamate dehydrogenase in Hydrogenomonas H 16

Summary Hydrogenomonas H 16 synthetized two chromatographically distinct forms of glutamate dehydrogenase which differed in their thermolability. One glutamate dehydrogenase utilized NAD, the other NADP as a coenzyme.Low specific activity of NAD-dependent glutamate dehydrogenase was found in cells grown with glutamate as sole nitrogen source or in cells grown with a high concentration of ammonium ions. In the presence of a low concentration of ammonium ions or in a nitrogen free medium, the specific activity of the NAD-dependent enzyme increased. Corresponding to the formation of the NAD-dependent glutamate dehydrogenase the enzyme glutamine synthetase was synthesized. The ratio of NAD-dependent glutamate dehydrogenase to glutamine synthetase activity differed only slightly in cells grown with different nitrogen and carbon sources.The NADP-dependent glutamate dehydrogenase was found in high specific activity in cells grown with an excess of ammonium ions. Under nitrogen starvation the formation of the NADP-dependent glutamate dehydrogenase ceased and the enzyme activity decreased.     

Isolation and characterization of xanthine dehydrogenase from Chlamydomonas reinhardtii

Xanthine dehydrogenase (XDH, EC 1.2.1.37) of Chlamydomonas reinhardtii (Sager) 6145c wild strain has been isolated and characterized for the first time in a unicellular green alga. The enzyme has an M_r of 330 kDa, and FAD, molybdenum and iron are cofactors required for its activity as deduced from results obtained using specific inhibitors, ⁵⁹Fe-labelling experiments, activity protection by FAD, physiological responses in vivo to iron and molybdenum deficiencies in the culture medium and work with mutants lacking molybdenum cofactor. Xanthine dehydrogenase exhibited Mi-chaelian kinetics typical for a bisubstrate enzyme with apparent K_m values for NAD ⁺, hypoxanthine and xanthine of 35, 160 and 70 μ M , respectively. Under phototrophic conditions enzyme activity was repressed by ammonium, but xanthine was not required for the enzyme to be induced, since high levels of enzyme activity were found in cells grown on ammonium and transferred to either N-frec media or media containing either of the nitrogen sources adenine, urea, urate, xanthine, hypoxanthine and guanine.     

Selenium requirement for active xanthine dehydrogenase from Clostridium acidiurici and Clostridium cylindrosporum

The xanthine dehydrogenase of Clostridium acidiurici and C. cylindrosporum was assayed with methyl viologen as acceptor. In C. acidiurici the basal activity level was about 0.3 mol/min x mg of protein. Cells grown on uric acid in the presence of 10^-7 M selenite showed a 14-fold increase in xanthine dehydrogenase activity, which decreased with higher selenite concentrations (10^-5 M). The supplementation with 10^-7 M molybdate or tungstate was without effect. High concentrations of tungstate decreased the xanthine dehydrogenase if selenite was also present. In comparison, high concentrations of molybdate affected only a small decrease in activity level at the optimal concentration for selenite and relieved to some degree the inhibitory effect of 10^-5 M selenite. With hypoxanthine and xanthine as substrates for growth again only the addition of selenite was necessary to show a similar increase in xanthine dehydrogenase activity. C. acidiurici could be grown in a mineral medium. Both xanthine dehydrogenase and formate dehydrogenase exhibited the highest level of activity if selenite and tungstate were present in that medium.In C. cylindrosporum the basal activity level of xanthine dehydrogenase was about 0.95 mol/min x mg of protein. The addition of 10^-7 M selenite to the growth medium increased the activity level about 3-fold, but the highest level (3.7 U/mg) was reached if 10^-7 M molybdate was also added. The presence of tungstate resulted in a decreased enzyme activity.     

Regulation of nitrogen-metabolizing enzymes in the commercial Mushroom Agaricus bisporus

Mycelium of Agaricus bisporus strain Horst U1 was grown in batch cultures on different concentrations of ammonium, glutamate, and glucose to test the effect of these substrates on the activities of NADP-dependent glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4), NAD-dependent glutamate dehydrogenase (NAD-GDH, EC 1.4.1.2.), and glutamine synthetase (GS, EC 6.3.1.2.). When grown on ammonium, the activities of NADP-GDH and GS were repressed. NAD-GDH activity was about 10 times higher than the activities of NADP-GDH and GS. At concentrations below 8 mM ammonium, NADP-GDH and GS were slightly derepressed. When glutamate was used as the nitrogen source, activities of NADP-GDH and GS were derepressed; compared with growth on ammonium, the activities of these two enzymes were about 10 times higher. Activities of GDHs showed no variation at different glutamate concentrations. Activity of GS was slightly derepressed at low glutamate concentrations. Growth of A. bisporus on both ammonium and glutamate as nitrogen sources resulted in enzyme activities comparable to growth on ammonium alone. Activities of NADP-GDH, NAD-GDH, and GS were not influenced by the concentration of glucose in the medium. In mycelium starved for nitrogen, the activities of NADP-GDH, NAD-GDH, and GS were derepressed, while in carbon-starved mycelium the activity of GS and both GDHs was repressed.     

Derepressed synthesis of cellulase by Cellulomonas.

A Cellulomonas sp. was isolated from the soil which hydrolyzed cellulose, as shown by clear-zone formation on cellulose agar medium. Catabolite repression of cellulase synthesis occurred when moderate levels of glucose were added to the medium. A stable mutant that no longer exhibits catabolite repression was produced through treatment of the wild-type organism with N-methyl-N'-nitro-N-nitrosoguanidine. Both enzyme concentration and specific activity, as determined by the rate of hydrolysis of carboxymethylcellulose, were greater with the mutant than with the wild-type organism under various test conditions. The wild type had no measurable cellulase activity when grown in the presence of either 1.0% glucose or cellobiose. Cellobiose, but not glucose, inhibited enzyme activity towards both cellulose and carboxymethylcellulose. Cellobiose, cellulose, and sophorose at low concentrations induced cellulase synthesis in both the wild-type and the mutant organism. Cellulase regulation appears to depend upon a complex relationship involving catabolite repression, inhibition, and induction.     

Effect of Glucose on the Uricase Formation by Streptomyces sp

The effect of glucose on the formation of uricase by a strain of Streptomyces sp. incubated under conditions of nitrogen limitation was investigated. Glucose stimulated uricase formation in the presence of potassium ion and inhibited it in the absence of the ion. Glucose metabolism by the organism was altered in the absence of the ion, and this appeared to cause the inhibition of the enzyme formation. The stimulatory effect of glucose in the presence of potassium ion was to shorten the lag period. Comparisons of the enzyme formation with and without urate in the presence and absence of glucose revealed that glucose promoted the utilization of exogenous urate as the inducer. The effect of glucose appeared to require protein synthesis, since it was prevented by chloramphenicol. Cyclic adenosine 3′,5′-mono-phosphate showed apparently no effect on uricase formation of this organism.     

Influence of ammonium and extracellular pH on the amino and organic acid contents of suspension culture cells of Acer pseudoplatanus

The effects of supplied ammonium and nitrate on the amino and organic acid contents and enzyme activities of cell suspension cultures of Acer pseudoplatanus L. were examined. Regardless of nitrogen source the pH of the culture medium strongly affected the malate and citrate contents of the cells; these organic acid pools declined at pH 5, but increased at pH 7 and 8. Over a period of two days, ammonium had little effect on the responses of the organic acid pool sizes to the pH of the medium. In contrast, ammonium had a strong influence on amino acid pool sizes, and this effect was dependent on the pH of the medium. At pH 5 there was no increase in cell ammonium or amino acid contents, but at higher pH values cellular ammonium content rose, accompanied by accumulation of glutamine, glutamate and asparagine. Over several days, supplied ammonium led to an increase in activity of glutamate dehydrogenase irrespective of any changes in internal ammonium and amino acid contents. If the pH of the medium was allowed to fall below pH 4 in the presence of ammonium, phosphoenolpyruvate (PEP) carboxylase activity declined to a very low value over several days; at higher pH, the activity of this enzyme, and that of NAD malic enzyme and NAD malate dehydrogenase, remained substantial irrespective of whether the nitrogen source was NH⁺₄ or NO-₃.     

Effect of Endogenous and Exogenous Urate on the Uricase Formation by Streptomyces sp.

Cells of a strain of Streptomyces sp. were incubated with an equivalent quantity of urate, xanthine, 6,8-dihydroxypurine or hypoxanthine in a medium deprived of other nitrogen source. The amount of uricase produced by these cells was shown to differ significantly, increasing in the following order of purine bases added to the medium: urate, xanthine, 6,8-dihydroxypurine and hypoxanthine. Of these was only urate indicated to be the inducer of uricase formation, and the difference in the quantity of uricase produced was found to be based on the duration of enzyme formation. The rate of uricase formation was essentially identical regardless of the purine bases supplied to cells.

Allantoin was accumulated in medium in remarkably different manners depending on the purine bases, which suggested the diversity in the mode of generation of urate in cells. Urate was generated at the slowest rate in the cells incubated with hypoxanthine, although the largest amount of uricase was produced, However, urate supplied to cells at the same rate but from medium failed to support the enzyme formation when the activity increased to a certain level. In order that the same amount of uricase was produced by the cells incubated with the different purine bases, the initial concentration of the purine bases should be raised so that they could remain in medium for the same incubation time.

Intracellular compartmentalization that might segregate endogenous and exogenous urate and might cause the difference in “effeciency” of these urate molecules as the inducer of uricase formation has been discussed.     

Regulation of aldehyde oxidase and nitrate reductase in roots of barley (Hordeum vulgare L.) by nitrogen source and salinity

The molybdenum cofactor (MoCo) is a component of aldehyde oxidase (AO EC 1.2.3.1), xanthine dehydrogenase (XDH EC 1.2.1.37) and nitrate reductase (NR, EC 1.6.6.1). The activity of AO, which catalyses the last step of the synthesis of abscisic acid (ABA), was studied in leaves and roots of barley (Hordeum vulgare L.) plants grown on nitrate or ammonia with or without salinity. The activity of AO in roots was enhanced in plants grown with ammonium while nitrate-grown plants exhibited only traces. Root AO in barley was enhanced by salinity in the presence of nitrate or ammonia in the nutrient medium while leaf AO was not significantly affected by the nitrogen source or salinity of the medium.Salinity and ammonium decreased NR activity in roots while increasing the overall MoCo content of the tissue. The highest level of AO in barley roots was observed in plants grown with ammonium and NaCl, treatments that had only a marginal effect on leaf AO. ABA concentration in leaves of plants increased with salinity and ammonium.Keywords: ABA, aldehyde oxidase, ammonium, nitrate, salinity.      

Regulation of avilamycin biosynthesis in Streptomyces viridochromogenes: effects of glucose, ammonium ion, and inorganic phosphate

Effects of glucose, ammonium ions and phosphate on avilamycin biosynthesis in Streptomyces viridochromogenes AS4.126 were investigated. Twenty grams per liter of glucose, 10 mmol/L ammonium ions, and 10 mmol/L phosphate in the basal medium stimulated avilamycin biosynthesis. When the concentrations of glucose, ammonium ions, and phosphate in the basal medium exceeded 20 g/L, 10 mmol/L, and 10 mmol/L, respectively, avilamycin biosynthesis greatly decreased. When 20 g/L glucose was added at 32 h, avilamycin yield decreased by 70.2%. Avilamycin biosynthesis hardly continued when 2-deoxy-glucose was added into the basal medium at 32 h. There was little influence on avilamycin biosynthesis with the addition of the 3-methyl-glucose (20 g/L) at 32 h. In the presence of excess (NH₄)₂SO₄ (20 mmol/L), the activities of valine dehydrogenase and glucose-6-phosphate dehydrogenase were depressed 47.7 and 58.3%, respectively, of that of the control at 48 h. The activity of succinate dehydrogenase increased 49.5% compared to the control at 48 h. The intracellular adenosine triphosphate level and 6-phosphate glucose content of S. viridochromogenes were 128 and 129%, respectively, of that of the control at 48 h, with the addition of the 40 mmol/L of KH₂PO₄. As a result, high concentrations of glucose, ammonium ions, and inorganic phosphate all led to the absence of the precursors for avilamycin biosynthesis and affected antibiotic synthesis.     

假丝酵母尿酸酶形成条件

选出了一株产尿酸酶的产朊候丝酵母(Candida utilis)AS2．117。此菌株尿酸酶形成条件的研究表明：尿酸、黄嘌呤和鸟嘌呤对酶形成起诱导作用;玉米浆对菌株生长和酶形成起十分重要的作用;蔗糖、葡萄塘、D-甘露糖和果糖是酶形成的适合碳源;生物素对酶产生有促进作用;在含有玉米浆培养基中加入无机氮源对产酶无作用,添加有机氮略增加产酶量。尿酸酶形成最适培养基组成为(％)：蔗糖;,玉米浆3,尿酸0．1,蛋白胨0．1,生物素0．05,KCI0．1,NaCl 0.1。最适pH为6．2。在250ml三角瓶中装30ml培养基为最适。在200r／min的旋转摇床上25℃振荡培养21h,在此条件下最终酶活力可达0．6u／ml。     

Glutamate Dehydrogenase Activity in Lemna perpusilla 6746: The Effects of Sucrose,Glucose and Fructose Addition to Growth Media

Lemna perpusilla Torr, strain 6746 clones were maintained under conditions of continuous illumination with various concentrations of sucrose, glucose or fructose added to the growth, medium. After two weeks of growth, plants were harvested and either assayed for total glutamate dehydrogenase activity or fractionated into one chloroplast-rich and one mitochondria-rich preparation and then assayed for glutamate dehydrogenase activity. In all assays for glutamate dehydrogenase it was necessary to add bovine serum albumin to the extraction medium in order to obtain sufficient enzyme activity for accurate and reproducible results. The presence of sucrose in the growth medium reduced glutamate dehydrogenase activity in all studies. When samples containing intact organelles were assayed, sucrose inhibition of activity appeared to occur primarily in the chloroplast fraction. Glucose, on the other hand, increased glutamate dehydrogenase activity in the chloroplast-rich fractions. Upon freeze-thawing differences between the various treatments were less obvious. The results from these studies indicate possible differences in sugar uptake and/or utilization in Lemna perpusilla.     

Distribution of the 7S Proteins in Soybean Globulins by Gel Filtration with Sephadex G-200

The level of isocitrate lyase, an enzyme of glyoxylate cycle, in Candida tropicalis was enhanced at the later period of growth when the yeast was cultivated in a semisynthetic glucose medium. On the other hand, such increase in the enzyme activity was not observed in C. lipolytica grown under the same conditions. In the case of C. tropicalis, high concentrations of glucose remaining in the medium permitted the increase in the enzyme activity and the addition of ethanol, one of the major products from glucose, to the glucose medium did not stimulate the enzyme formation, indicating that the enhanced enzyme level in the yeast was not merely attributable to the release from the repression by glucose or to the induction by ethanol. Biotin, one of the growth-stimulating factors for C. tropicalis, affected markedly the level of isocitrate lyase. That is, the supplementation of biotin to the synthetic glucose medium inhibited completely the increase in the enzyme activity, and reversely the absence of biotin stimulated the enzyme formation in the glucose-assimilating cells. Thiamine, another growth-stimulating factor for C. tropicalis, did not show any effect on the level of isocitrate lyase in the yeast. The level of isocitrate lyase in C. lipolytica growing on glucose was not affected by biotin added exogenously.     

Inhibition of amino acid transport by ammonium ion in Saccharomyces cerevisiae.

The rate of transport of L-amino acids by Saccharomyces cerevisiae epsilon 1278b increased with time in response to nitrogen starvation. This increase could be prevented by the addition of ammonium sulfate or cycloheximide. A slow time-dependent loss of transport activity was observed when ammonium sulfate (or ammonium sulfate plus cycloheximide) was added to cells after 3 h of nitrogen starvation. This loss of activity was not observed in the presence of cycloheximide alone. In a mutant yeast strain which lacks the nicotinamide adenine dinucleotide phosphate-dependent (anabolic) glutamate dehydrogenase, no significant decrease in amino acid transport was observed when ammonium sulfate was added to nitrogen-starved cells. A double mutant, which lacks the nicotinamide adenine dinucleotide phosphate-dependent enzyme and in addition has a depressed level of the nicotinamide adenine dinucleotide-dependent (catabolic) glutamate dehydrogenase, shows the same sensitivity to ammonium ion as the wild-type strain. These data suggest that the inhibition of amino acid transport by ammonium ion results from the uptake of this metabolite into the cell and its subsequent incorporation into the alpha-amino groups of glutamate and other amino acids.     

Ammonium-assimilating enzymes and their regulation in wild and NADP-glutamate dehydrogenase-deficient strains of the ectomycorrhizal fungus Hebeloma cylindrosporum

Hebeloma cylindrosporum strain h 17 was grown on media containing either glutamate or ammonium as nitrogen source. Growth tests and in vitro activity measurements revealed that both glutamine synthetase (GS. EC 6.3.1.2) and NADP-specific glutamate dehydrogenase (NADP-GDH, EC 1.4.1.4) are fully functional in wild type mycelia grown on glutamate or ammonium as sole nitrogen source. However, NADP-GDH appeared to be more active than GS in stationary growing mycelia. NADP-GDH is also able to sustain adequate ammonium assimilation in methionine sulfoximine (MSX)-treated mycelia since they grew as well as mycelia fed with ammonium alone. The NADP-GDH also appeared to be L-glutamate inducible whereas GS was repressed by ammonium. The NADP-GDH deficient strain, when transferred from a glutamate containing medium to an ammonium containing medium, exhibited a derepressed GS, although this enzyme did not fully substitute for the deficiency of NADP-GDH in ammonium assimilation. The low NADP-GDH activity of the mutant strain exhibited a reduced mobility on a 6% constant polyacrylamide gel. By contrast, the two enzymes had identical molecular weights, estimated to be ca 295 kDa on gradient polyacrylamide gel. The involvement of NADP-GDH and GS enzymes in nitrogen assimilation is discussed.     

Quantitative Studies on Glycolytic Enzymes in Lactobacillus plantarum

The intracellular activity of glyceraldehyde-3-phosphate dehydrogenase was estimated. The dehydrogenase was purified about 70-fold by ammonium sulfate fractionation and DEAE cellulose chromatography. Basal properties of the purified enzyme were examined. From the activity of the enzyme in the sonicate, the intracellular activity was calculated by correcting the value for the intracellular conditions which were known previously. The intracellular activity of the dehydrogenase was found to be nearly equal to that of the lactate-forming activity from glucose in original washed cells, showing a significant participation of the dehydrogenase in the metabolic pathway of lactic acid fermentation in this organism.     

Studies on the Formation of Uricase by Streptomyces

The induced formation of uricase by the cultured cells of Streptomyces sp. and the effect of purine bases on the enzyme formation were studied. The microorganism was grown in media containing urate and/or purine bases (adenine, guanine, hypoxanthine or xanthine) and the development of the uricase activity of the cells were measured at intervals. The disappearance of urate and purine bases from the media was also determined. Without the purine bases, the production of uricase was significantly low even in the presence of urate and the disappearance of urate from the medium was in a slow rate. Upon the addition of hypoxanthine or xanthine in the presence of urate, a significant increase in the uricase activity of the cells and a concomitant rapid decrease of urate in the medium were observed. The purine bases added to the media were incorporated into the cells at a relatively early period of the culture and appeared to be converted into urate within the cells. The repression of uricase formation in the cultured cells and the derepression by the addition of the purine bases were discussed.     

Ammonium uptake and metabolism by nitrogen fixing bacteria

The primary steps of N₂, ammonia and nitrate metabolism in Klebsiella pneumoniae grown in a continuous culture are regulated by the kind and supply of the nitrogenous compound. Cultures growing on N₂ as the only nitrogen source have high activities of nitrogenase, unadenylated glutamine synthetase and glutamate synthase and low levels of glutamate dehydrogenase. If small amounts of ammonium salts are added continuously, initially only part of it is absorbed by the organisms. After 2–3 h complete absorption of ammonia against an ammonium gradient coinciding with an increased growth rate of the bacteria is observed. The change in the extracellular ammonium level is paralleled by the intracellular glutamine concentration which in turn regulates the glutamine synthetase activity. An increase in the degree of adenylation correlates with a repression of nitrogenase synthesis and an induction of glutamate dehydrogenase synthesis. Upon deadenylation these events are reversed.—After addition of nitrate ammonia appears in the medium, probably due to the action of a membrane bound dissimilatory nitrate reductase.—Addition of dinitrophenol causes transient leakage of intracellular ammonium into the medium.     

Regulation of glutamate dehydrogenases in nit-2 and am mutants of Neurospora crassa.

The regulation of the glutamate dehydrogenases was investigated in wild-type Neurospora crassa and two classes of mutants altered in the assimilation of inorganic nitrogen, as either nitrate or ammonium. In the wild-type strain, a high nutrient carbon concentration increased the activity of reduced nicotinamide adenine dinucleotide phosphate (NADPH)-glutamate dehydrogenase and decreased the activity of reduced nicotinamide adenine dinucleotide (NADH)-glutamate dehydrogenase. A high nutrient nitrogen concentration had the opposite effect, increasing NADH-glutamate dehydrogenase and decreasing NADPH-glutamate dehydrogenase. The nit-2 mutants, defective in many nitrogen-utilizing enzymes and transport systems, exhibited low enzyme activities after growth on a high sucrose concentration: NADPH-glutamate dehydrogenase activity was reduced 4-fold on NH(4)Cl medium, and NADH-glutamate dehydrogenase, 20-fold on urea medium. Unlike the other affected enzymes of nit-2, which are present only in basal levels, the NADH-glutamate dehydrogenase activity was found to be moderately enhanced when cells were grown on a low carbon concentration. This finding suggests that the control of this enzyme in nit-2 is hypersensitive to catabolite repression. The am mutants, which lack NADPH-glutamate dehydrogenase activity, possessed basal levels of NADH-glutamate dehydrogenase activity after growth on urea or l-aspartic acid media, like the wild-type strain, and possessed moderate levels (although three- to fourfold lower than the wild-type strain) on l-asparagine medium or l-aspartic acid medium containing NH(4)Cl. These regulatory patterns are identical to those of the nit-2 mutants. Thus, the two classes of mutants exhibit a common defect in NADH-glutamate dehydrogenase regulation. Double mutants of nit-2 and am had lower NADH-glutamate dehydrogenase activities than either parent. A carbon metabolite is proposed to be the repressor of NADH-glutamate dehydrogenase in N. crassa.     

Regulation by ammonium and glucose of Arthrobacter fluorescens alanine dehydrogenase

Alanine dehydrogenase in Arthrobacter fluorescens exhibited an allosteric behaviour and two K _m values for ammonium were estimated. In batch cultures at different ammonium concentrations and in continuous culture following an NH₄ ⁺ pulse, the level of ADH activity seems to be regulated by the ammonium concentration, high activities being observed when extracellular ammonium was in excess. The response to the growth rate of an ammonium-limited chemostat culture of A. fluorescens seems to indicate that alanine dehydrogenase and glutamine synthetase activities were inversely related. High activities of glutamate oxaloacetate transaminase and glutamate pyruvate transaminase have been found in crude extract of ammonium-limited cultures. From the results obtained in batch cultures grown at different glucose concentrations and in carbon-limited chemostat culture it appeared that the limitation by glucose influenced alanine dehydrogenase activity negatively. No glutamate dehydrogenase activity and no glutamate synthase activity could be detected with either NADH or NADPH as coenzymes.Abbreviations ADH alanine dehydrogenase - GS glutamine synthetase - GDH glutamate dehydrogenase - GOGAT glutamine oxoglutarate aminotransferase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase