Cooperative Modulation of [3H]MK-801 Binding to the N-Methyl-d-Aspartate Receptor-Ion Channel Complex by l-Glutamate, Glycine, and Polyamines

In extensively washed rat cortical membranes [3H](+)-5-methyl-10,11-dihydro-5 H-dibenzo [a,d]cyclohepten-5,10-imine ([3H]MK-801) labeled a homogeneous set of sites (Bmax = 1.86 pmol/mg protein) with relatively low affinity (KD = 45 nM). L-Glutamate, glycine, and spermidine produced concentration-dependent increases in specific [3H]MK-801 binding due to a reduction in the KD of the radioligand. In the presence of high concentrations of L-glutamate, glycine, or spermidine, the KD values for [3H]MK-801 were reduced to 11 nM, 18 nM, and 15 nM, respectively. Maximally effective concentrations of combinations of the three compounds further increased [3H]MK-801 binding affinity as follows: L-glutamate + glycine, KD = 6.2 nM; L-glutamate + spermidine, KD = 2.2 nM; glycine + spermidine, KD = 8.3 nM. High concentrations of spermidine did not inhibit either [3H]glycine orf [3H]3-(2-carboxypiperazin-4-yl)propyl-1-phosphonic acid binding to the N-methyl-D-aspartate (NMDA) receptor complex. The concentration of L-glutamate required to produce half-maximal enhancement (EC50) of [3H]MK-801 binding was reduced from 218 nM to 52 nM in the presence of 30 microM glycine and to 41 nM in the presence of 50 microM spermidine. The EC50 value for glycine enhancement of [3H]MK-801 binding was 184 nM. This was lowered to 47 nM in the presence of L-glutamate and to 59 nM in the presence of spermidine. Spermidine enhanced [3H]MK-801 binding with an EC50 value of 19.4 microM which was significantly reduced by high concentrations of L-glutamate (EC50 = 3.9 microM) or glycine (EC50 = 6.2 microM).(ABSTRACT TRUNCATED AT 250 WORDS)     

Covalent modification of bacterial glutamine synthetase: physiological significance

Summary Stadtman, Holzer and their colleagues (reviewed in Stadtman and Ginsburg 1974) demonstrated that the enzyme glutamine synthetase (GS) [L-glutamate: ammonia ligase (ADP-forming), EC 6.3.1.2] is covalently modified by adenylylation in a variety of bacterial genera and that the modification is reversible. These studies further indicated that adenylylated GS is the less active form in vitro. To assess the physiological significance of adenylylation of GS we have determined the growth defects of mutant strains (glnE) of S. typhimurium that are unable to modify GS and we have determined the basis for these growth defects. The glnE strains, which lack GS adenylyl transferase activity (ATP: [L-glutamate: ammonia ligase (ADP-forming)] adenylyltransferase, EC 2.7.7.42), show a large growth defect specifically upon shift from a nitrogen-limited growth medium to medium containing excess ammonium (NH₄ ⁺). The growth defect appears to be due to very high catalytic activity of GS after shift, which lowers the intracellular glutamate pool to 10% that under preshift conditions. Consistent with this view, recovery of a rapid growth rate on NH₄ ⁺ is accompanied by an increase in the glutamate pool. The glnE strains have normal ATP pools after shift. They synthesize very large amounts of glutamine and excrete glutamine into the medium, but excess glutamine does not seem to inhibit growth. We hypothesize that a major function for adenylylation of bacterial GS is to protect the cellular glutamate pool upon shift to NH₄ ⁺-excess conditions and thereby to allow rapid growth.     

The purine nucleotide cycle inHelix hepatopancreas

Summary The enzymes adenylosuccinate synthetase (EC 6.3.4.4 IMP: L-aspartate ligase [GDP-forming]), adenylosuccinate lyase (EC 4.3.2.2) and AMP deaminase (EC 3.5.4.6 AMP aminohydrolase) were demonstrated inHelix aspersa hepatopancreas tissue. The presence of these enzymes along with high levels of aspartate transaminase is presumptive evidence for the operation in this tissue of the purine nucleotide cycle. In the absence of evidence that glutamate dehydrogenase acts to release ammonia during amino acid catabolism, it is suggested that the purine nucleotide cycle serves this function. Glutamine synthetase (EC 6.3.1.2 L-glutamate: ammonia ligase [ADP-forming]) was shown to be present primarily in the cytosolic fraction ofHelix hepatopancreas. Since the operation of the purine nucleotide cycle results in the release of ammonia in the cytosol, the localization of glutamine synthetase in this compartment indicates that it is the primary ammonia-detoxifying enzyme and is consistent with the suggestion that the purine nucleotide cycle serves as the major pathway for amino acid catabolism.Supported by grants from the USPHS National Institute of Allergic and Infections Diseases (AI 05006) and the National Science Foundation (PCM-75-13161)     

O-methyltransferase(s)-suppressed plants produce lower amounts of phenolic vir inducers and are less susceptible to Agrobacterium tumefaciens infection

The first step of Agrobacterium tumefaciens/plant interaction corresponds to the activation of a transduction pathway of the bacterium by plant exudate. Phenolic compounds rapidly secreted by wounded plant cells induce the expression of bacterial virulence (vir) genes; however, little is known about their biosynthesis in plant. Here we show that inoculation of an Agrobacterium tumefaciens virulent strain on orthodiphenol-O-methyltransferases-suppressed tobacco plants leads to significantly smaller tumors compared to control plants. These transgenic plants are inhibited for caffeic acid O-methyltransferase class I or II (OMT; EC 2.1.1.6) and/or caffeoyl-coenzyme A O-methyltransferase (CCoAOMT; EC 2.1.1.104) that are involved in monolignol biosynthesis. The significant decrease of tumor size could be suppressed by the pre-activation of bacterial virulence, before inoculation, using acetosyringone a known vir inducer. Total soluble phenolic amounts and cell wall composition analyzed by FT-IR analysis did not show significant differences between transgenic and control plants. The potential of phenolic extracts from control and OMT-suppressed plants to induce virulence was evaluated using an Agrobacterium tumefaciens reporter strain carrying a vir::LacZ gene fusion plasmid. Lower vir-inducing activities were recorded for plants that show inhibition to caffeic acid O-methyltransferase activity. HPLC analysis confirmed that the levels of several phenolic compounds were differently affected by wounding and/or by bacterial inoculation. Statistical correlations were established between tumor sizes, vir-inducing activities, O-methyltransferases proteins accumulations and the levels of various soluble phenolic compounds such as acetosyringone. These results demonstrate the role of the O-methyltransferases of the phenylpropanoid pathway in the early production of soluble Agrobacterium tumefaciens vir inducers.     

Characterization of Peptostreptococcus asaccharolyticus glutamate dehydrogenase purified by dye-ligand chromatography

Glutamate dehydrogenase (L-glutamate:NAD+ oxidoreductase (deaminating); EC 1.4.1.2) has been purified from Peptostreptococcus asaccharolyticus in a single step using dye-ligand chromatography. The enzyme (GDH) was present in high yields and was stabilized in crude extracts. A subunit molecular weight of 49000 +/- 500 was determined by SDS polyacrylamide gel electrophoresis and six bands were obtained after cross-linking the subunits with dimethyl suberimidate. This bacterial GDH was predominantly NAD+-linked, but was able to utilize both NADP+ and NADPH at 4% of the rates with NAD+ and NADH, respectively. An investigation of the amino acid specificity revealed some similarities with GDH from mammalian sources and some clear differences. The values of apparent Km for the substrates ammonia, 2-oxoglutarate, NADH, NAD+ and glutamate were 18.4, 0.82, 0.066, 0.031 and 6 mM, respectively. The P. asaccharolyticus GDH was not regulated by purine nucleotides, but was subject to strong inhibition with increasing ionic strength.     

Effects of organic acids on the synthesis of citrulline by intact rat liver mitochondria

Citrulline synthesis, mostly regulated at the carbamoyl-phosphate synthase I (EC 6.3.4.16) step by the intramitochondrial concentration of ATP and/or N-acetylglutamate is tested with four organic acids: propionate, alpha-ketobutyrate, dipropyl-acetate and 4-pentenoate. In the presence of 10 mM succinate, as the oxidizable substrate, citrullinogenesis was only inhibited by propionate and 4-pentenoate. With 10 mM L-glutamate, a significant inhibition was observed with the four acids. After the addition of ATP and N-acetylglutamate to uncoupled mitochondria, no inhibition could be demonstrated with dipropylacetate and 4-pentenoate. However, a slight inhibition remained with propionate and alpha-ketobutyrate. When mitochondria were incubated with 10 mM L-glutamate, ATP decreased with propionate, dipropylacetate and 4-pentenoate. Under the same conditions, N-acetylglutamate synthesis was strongly inhibited by each organic acid. The decrease of N-acetylglutamate synthesis was related to the constant diminution of intramitochondrial acetyl-coenzyme A (CoA) and to the increase of propionyl-CoA with propionate and alpha-ketobutyrate. Acetyl-CoA and propionyl-CoA are respectively substrate and competitive inhibitor of the N-acetylglutamate synthase (EC 2.3.1.1). Each acid displayed its optimum inhibition at concentrations between 1 and 2 mM. At these acid concentrations, mitochondria had the lowest acetyl-CoA content and the highest propionyl-CoA content.     

Glutamine synthesis in germinating seeds of Cucurbita moschata

During germination, an increase in glutamine and glutamine synthetase[L-glutamate: ammonia ligase (ADP), EC 6.3.1.2 [EC] ] occurred inthe cotyledons reaching a maximum at 4 to 6 days. The enzymehad a Km of 4.5 nun for L-glutamate, and 0.67 mu for hydroxylamine.Hydroxylamine exhibited substrate inhibition kinetics. The enzymewas inhibited by calcium ion, fluoride ion and p-hydroxymercuribenzoatebut not by EDTA, sodium ion or chloride ion. The sulf hydrylinhibition was reversed by dithiothreitol. In vivo synthesisof glutamine-¹⁴C from glutamate-¹⁴C was found to parallel theincrease in glutamine content and the in vitro glutamine synthetaseactivity during germination. ¹ Present address: Department of Biology, Mercyhurst College,Erie, Pennsylvania 16501, U.S.A. (Received June 12, 1971; )     

Effects of L-glutamate/D-aspartate and monensin on lactic acid production in retina and cultured retinal Müller cells

We have investigated the dependence of the rate of lactic acid production on the rate of Na(+) entry in cultured transformed rat Müller cells and in normal and dystrophic (RCS) rat retinas that lack photoreceptors. To modulate the rate of Na(+) entry, two approaches were employed: (i) the addition of L-glutamate (D-aspartate) to stimulate coupled uptake of Na(+) and the amino acid; and (ii) the addition of monensin to enhance Na(+) exchange. Müller cells produced lactate aerobically and anaerobically at high rates. Incubation of the cells for 2-4 h with 0.1-1 mM L-glutamate or D-aspartate did not alter the rate of production of lactate. ATP content in the cells at the end of the incubation period was unchanged by addition of L-glutamate or D-aspartate to the incubation media. Na(+)-dependent L-glutamate uptake was observed in the Müller cells, but the rate of uptake was very low relative to the rate of lactic acid production. Ouabain (1 mM) decreased the rate of lactic acid production by 30-35% in Müller cells, indicating that energy demand is enhanced by the activity of the Na(+)-K(+) pump or depressed by its inhibition. Incubation of Müller cells with 0.01 mM monensin, a Na(+) ionophore, caused a twofold increase in aerobic lactic acid production, but monensin did not alter the rate of anaerobic lactic acid production. Aerobic ATP content in cells incubated with monensin was not different from that found in control cells, but anaerobic ATP content decreased by 40%. These results show that Na(+)-dependent L-glutamate/D-aspartate uptake by cultured retinal Müller cells causes negligible changes in lactic acid production, apparently because the rates of uptake are low relative to the basal rates of lactic acid production. In contrast, the marked stimulation of aerobic lactic acid production caused by monensin opening Na(+) channels shows that glycolysis is an effective source of ATP production for the Na(+)-K(+) ATPase. A previous report suggests that coupled Na(+)-L-glutamate transport stimulates glycolysis in freshly dissociated salamander Müller cells by activation of glutamine synthetase. The Müller cell line used in this study does not express glutamine synthetase; consequently these cells could only be used to examine the linkage between Na(+) entry and the Na(+) pump. As normal and RCS retinas express glutamine synthetase, the role of this enzyme was examined by coapplication of L-glutamate and NH(4) (+) in the presence and absence of methionine sulfoximine, an inhibitor of glutamine synthetase. In normal retinas, neither the addition of L-glutamate alone or together with NH(4) (+) caused a significant change in the glycolytic rate, an effect linked to the low rate of uptake of this amino acid relative to the basal rate of retinal glycolysis. However, incubation of the RCS retinas in media containing L-glutamate and NH(4)(+) did produce a small (15%) increase in the rate of glycolysis above the rate found with L-glutamate alone and controls. It is unlikely that this increase was the result of conversion of L-glutamate to L-glutamine, as it was not suppressed by inhibition of glutamine synthetase with 5 mm methionine sulfoximine. It appears that the magnitude of Müller cell glycolysis required to sustain the coupled transport of Na(+) and L-glutamate and synthesis of L-glutamine is small relative to the basal glycolytic activity in a rat retina.     

A COMPARATIVE STUDY OF L-GLUTAMATE DECARBOXYLASE FROM MOUSE BRAIN AND BOVINE HEART WITH PURIFIED PREPARATIONS1

Abstract— L-Glutamate decarboxylase (EC 4.1.1.15) (GAD), the enzyme responsible for the formation of GABA, has been purified to homogeneity from mouse brain (Wu et at., 1973) and antibodies specific for neuronal GAD have been obtained (SAITO et al., 1974a). The present report describes the purification of GAD from bovine heart more than 2000-fold over the homogenate by initial solubilization with Triton X-100. subsequent fractionation with ammonium sulfate, column chromatography on DEAE cellulose, calcium phosphate gel, and DEAE-Sephadex, and gel filtration. At least two forms of GAD have been observed in bovine heart preparations; one of them appears as a high molecular weight form (Peak I, MW 360,000) and the other one as a low molecular weight form (Peak II, MW 105,000). Cysteine sulfinic acid and cysteic acid, both precursors of taurine, had no effect on the purified heart enzyme or on neuronal GAD at 10 mM, suggesting that cysteine sulfinic acid and cysteic acid probably are not substrates for any species of GAD described above. The heart enzyme and neuronal GAD differ in several respects. First, they are different immunochemically as judged by the lack of cross reactivity between the purified heart enzyme and the antibody against purified neuronal GAD. Second, they are different biochemically. 5,5′-Dithiobis[2-nitrobenzoic acid] (DTNB). one of the most potent inhibitors of neuronal GAD [K_i= 1.0 × 10^?8M] inhibits the heart enzyme only to a small extent at 1 mM. On the other hand, pyruvic acid, which inhibits the heart enzyme to an extent of 90% at 10 mM, only inhibits the neuronal enzyme slightly. Third, they are different in their substrate specificity. The neuronal enzyme can catalyze α-decarboxylation of both L-glutamate and L-aspartate while the heart enzyme can use only L-glutamate as substrate. Moreover, an unidentified product probably derived from L-glutamate is obtained in the reaction mixture of the heart enzyme but is not observed with the brain enzyme, suggesting that the heart enzyme may catalyze a reaction converting L-glutamate to products other than GABA. It is therefore concluded that heart GAD and neuronal GAD are two different entities. Work is in progress to determine whether the heart enzyme is related to the glial enzyme. Should the antibody against the heart enzyme cross-react with the glial enzyme, the role of the glial enzyme in GABA function can then be studied by immunochemical and immunocytochemical methods.     

Biodegradation of 4-chlorobenzoic acid by <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> PA01 NC

A bacterial consortium capable of degrading chloroaromatic compounds was isolated from pulp and paper mill effluents by selective enrichment on 4-chlorobenzoic acid as sole source of carbon and energy. The four different bacterial isolates obtained from bacterial consortium were identified as Pseudomonas aeruginosa AY792969 (A), P. aeruginosa PA01 NC (B), Pseudomonas sp. ZZ5 DQ113452 (C) and Pseudomonas sp. AY762360 (D) based on their morphological and biochemical characteristics and by phylogenetic analysis based on 16S rRNA gene sequences. These bacterial isolates were found to be versatile in degrading a variety of chloroaromatic compounds including fluoro- and iodobenzoic acids. P. aeruginosa PA01 NC utilized 4-chlorobenzoic acid at 2 g/l as growth substrate. Biodegradation studies have revealed that this organism degraded 4-chlorobenzoic acid through 4-chlorocatechol which was further metabolized by ortho-cleavage pathway and the dechlorination occurred after the ring-cleavage.     

Wine phenolic compounds influence the production of volatile phenols by wine-related lactic acid bacteria

Aims: To evaluate the effect of wine phenolic compounds on the production of volatile phenols (4‐vinylphenol [4VP] and 4‐ethylphenol [4EP]) from the metabolism of p‐coumaric acid by lactic acid bacteria (LAB). Methods and Results: Lactobacillus plantarum, Lactobacillus collinoides and Pediococcus pentosaceus were grown in MRS medium supplemented with p‐coumaric acid, in the presence of different phenolic compounds: nonflavonoids (hydroxycinnamic and benzoic acids) and flavonoids (flavonols and flavanols). The inducibility of the enzymes involved in the p‐coumaric acid metabolism was studied in resting cells. The hydroxycinnamic acids tested stimulated the capacity of LAB to synthesize volatile phenols. Growth in the presence of hydroxycinnamic acids, especially caffeic acid, induced the production of 4VP by resting cells. The hydroxybenzoic acids did not significantly affect the behaviour of the studied strains. Some of the flavonoids showed an effect on the production of volatile phenols, although strongly dependent on the bacterial species. Relatively high concentrations (1 g l^?1) of tannins inhibited the synthesis of 4VP by Lact. plantarum. Conclusions: Hydroxycinnamic acids were the main compounds stimulating the production of volatile phenols by LAB. The results suggest that caffeic and ferulic acids induce the synthesis of the cinnamate decarboxylase involved in the metabolism of p‐coumaric acid. On the other hand, tannins exert an inhibitory effect. Significance and Impact of the Study: This study highlights the capacity of LAB to produce volatile phenols and that this activity is markedly influenced by the phenolic composition of the medium.     

N-glycosyl-N-hydroxysulfamides as potent inhibitors of Brucella suis carbonic anhydrases

Abstract

We investigated a series of N-hydroxysulfamides obtained by Ferrier sulfamidoglycosylation for the inhibition of two bacterial carbonic anhydrases (CAs, EC 4.2.1.1) present in the pathogen Brucella suis. bsCA I was moderately inhibited by these compounds with inhibition constants ranging between 522 and 958?nM and no notable differences of activity between the acetylated or the corresponding deacetylated derivatives. The compounds incorporating two trans-acetates and the corresponding deprotected ones were the most effective inhibitors in the series. bsCA II was better inhibited, with inhibition constants ranging between 59.8 and 799?nM. The acetylated derivatives were generally better bsCA II inhibitors compared to the corresponding deacetylated compounds. Although these compounds were not highly isoform-selective CA inhibitors (CAIs) for the bacterial over the human CA isoforms, some of them possess inhibition profiles that make them interesting leads for obtaining better and more isoform-selective CAIs targeting bacterial enzymes.     

Regulation of glutamine synthetase in the blue-green alga Anabaena L-31

In N2-grown cultures of Anabaena L-31, in which protein synthesis was prevented by chloramphenicol, presence of NH+4 caused a drastic decrease of glutamine synthetase (L-glutamate:ammonia ligase (ADP-forming), EC 6.3.1.2) activity indicating NH+4-mediated inactivation or degradation of the enzyme. The half-life of glutamine synthetase was more than 24 h, whereas that of nitrogenase (reduced ferredoxin:dinitrogen oxidoreductase (ATP-hydrolysing), EC 1.18.2.1) was less than 4 h, suggesting that glutamine synthetase may not act as positive regulator of nitrogenase synthesis in Anabaena. Glutamine synthetase purified to homogeneity was subject to cumulative inhibition by alanine, serine and glycine. The amino acids, however, exhibited partial antagonism in this behaviour. Glyoxylate, an intermediate in photorespiration, virtually prevented the amino acid inhibition. Kinetic studies revealed inhibition of the enzyme activity by high Mg2+ concentration under limiting glutamate level and by high glutamate in limiting Mg2+. Maximum enzyme activity occurred when the ratio of glutamate to free Mg2+ was 0.5 to 1.0. The results demonstrate that the enzyme is subject to multiple regulation by various metabolites involved in nitrogen assimilation.     

Inhibitor studies of methionyl-tRNA transformylase of Euglena gracilis

Inhibitor studies of the only known eukaryotic methionyl-tRNA transformylase (10-formyltetrahydrofolate:L-methionyl-tRNA N-transformylase, EC 2.1.2.9) were carried out. All the natural pteroylglutamic acid derivatives examined, with the exception of pteroylglutamic acid, are inhibitors. The most effective is 5-methyltetrahydrofolate (5-CH3-H4PteGlu) (KI = 3 . 10(-6) M), which is the only noncompetitive inhibitor of the enzyme. All the other derivatives tested are competitive, and H4PteGlu shows a cooperative inhibition. These and other data obtained with pteroylglutamic analogues show that, in contrast to the bacterial enzyme, Euglena transformylase is also inhibited by compounds without a fully reduced pyrazine ring and is very sensitive to compounds with a methyl group in position 5 or 10 of the pteridine ring.     

Fungal elicitor-mediated responses in pine cell cultures

A tissue culture system has been developed to examine phenylpropanoid metabolism induced in pine tissues by an ectomycorrhizal symbiont. An elicitor preparation from the ectomycorrhizal fungus Thelephora terrestris Fr. induced enhanced phenolic metabolism in suspension cultured cells of Pinus banksiana Lamb., as indicated by tissue lignification and accumulation of specific methanol-extractable compounds in the cells. Induction of lignification was observed as early as 12 h after elicitation. The activity of phenylalanine ammonia-lyase (PAL, EC 4.3.1.5), the entry-point enzyme into phenylpropanoid metabolism, also increased within the same time-frame in elicited cells. Significant increases in PAL activity were evident by 6 h after elicitation, and, by 12 h after elicitation, PAL activity in elicited cells was ten times greater than that in the corresponding controls. Lignification of the elicited tissue was also accompanied by an increase in the activity of other enzymes associated with lignin synthesis, including caffeic acid O-methyl transferase (EC 2.1.1.46), hydroxycinnamate:CoA ligase (EC 6.2.1.12), cinnamyl alcohol dehydrogenase (EC 1.1.1.-), coniferin glucosidase (EC 3.2.1.21) and peroxidase (EC 1.11.1.7). The increase in total peroxidase activity was associated with a change in the pattern of soluble peroxidase isoforms. The pine cell culture-ectomycorrhizal elicitor system provides a good model for molecular analysis of the process of lignification in an economically important softwood species.Abbreviations 2,4-D 2,4-dichlorophenoxyacetic acid - 4CL hydroxycinnamate:Coenzyme A ligase (EC 6.2.1.12) - CAD cinnamyl alcohol dehydrogenase (EC 1.1.1.-) - COMT S-adenosyl-l-methionine:caffeate O-methyl transferase (EC 2.1.1.46) - HPLC high-pressure liquid chromatography - PAL phenylalanine ammonia-lyase (EC 4.3.1.5) - TGA thioglycolic acid To whom correspondence should be addressedFinancial assistance for this work was provided by the Natural Sciences and Engineering Research Council of Canada.     

Role of glutamate dehydrogenase and phosphoenolpyruvate carboxylase activity in ammonium nutrition tolerance in roots

Spinach (Spinacea oleracea L. “Correnta F1”) and pea (Pisum sativum L. “Macrocarpon”) plants were grown in a hydroponic culture with nitrate (5 mM), or ammonium (5 mM) as the nitrogen source. Dry matter accumulation declined dramatically in spinach plants fed with ammonium, whereas there was no change in pea plants when compared with nitrate-fed plants. Data obtained from δ¹⁵N, the organic nitrogen content, N-assimilation enzyme activity, glutamine synthetase (L-glutamate:ammonia-ligase; EC 6.3.1.2), glutamate dehydrogenase (L-glutamate:NAD⁺-oxidoreductase; EC 1.4.1.2) and enzymes from the tricarboxylic acid cycle suggest that ammonium incorporation into organic nitrogen is localized in the roots in pea plants and in the shoots in spinach plants. Distribution of incorporated ammonium (in shoots and roots) may determine ammonium tolerance. Our results show that unlike in spinach plants, in pea plants, an ammonium-tolerant species, GDH enzyme plays an important role in ammonium detoxification by its incorporation into amino acids. Furthermore, phosphoenolpyruvate carboxylase (phosphate:oxaloacetate-carboxy-lyase; EC 4.1.1.31) and pyruvate kinase (ATP:pyruvate-2-O-phosphotransferase; EC 2.7.1.40) activities reflect a major flow of carbon for ammonium assimilation through oxalacetate in pea plants and through pyruvate in spinach plants. The differences in the sensitivity to ammonium between the species are discussed in terms of differences in the site of ammonium assimilation as well as in the nitrogen assimilation ways.     

The role of 4,5-dioxovaleric acid in porphyrin and vitamin B12 formation by clostridia

4,5-Dioxovalerate, which has been proposed as an intermediate in the newly discovered so-called C₅ pathway that leads from L-glutamate to δ-aminolevulinate, strongly inhibits uroporphyrin formation from δ-aminolevulinate in cells of $\text{Clostridium}\text{tetanomorphum}$ and in cell-free extracts of this organism, in spite of the presence of L-alanine: 4,5-dioxovalerate aminotransferase (aminolevulinate aminotransferase, EC 2.6.1.43). The interference by 4,5-dioxovalerate with porphyrin formation is due to strong inhibition of δ-aminolevulinate dehydratase (EC 4.2.1.24). Since 4,5-dioxovalerate hence effectively prevents the operation of the reaction sequence from L-glutamate to porphyrin, it is concluded that 4,5-dioxovalerate does not function as a physiological δ-aminolevulinate precursor.     

Identification of low molecular weight aromatic compounds by gas chromatography–mass spectrometry (GC–MS) from kraft lignin degradation by three Bacillus sp.

Three bacterial strains identified as Paenibacillus sp., Aneurinibacillus aneurinilyticus and Bacillus sp. have been shown to decolourise kraft lignin in 6 days of incubation. The release of low molecular weight aromatic compounds by these bacterial strains during degradation of kraft lignin was analysed by GC–MS analysis. The total ion chromatograph (TIC) of ethyl acetate extract from kraft lignin sample inoculated by Paenibacillus sp. was similar to control except some minor changes in the chromatographic profile indicating incapability of this bacterium to modify kraft lignin. On the other hand the TIC of ethyl acetate extract from kraft lignin inoculated by A. aneurinilyticus and Bacillus sp. caused formation of several aromatic lignin-related compound that were not present in the extract of control. The compounds identified in extract of the sample degraded by A. aneurinilyticus were guaiacol, acetoguiacone, gallic acid and ferulic acid while t-cinnamic acid, 3,4,5 trimethoxy benzaldehyde, and ferulic acid by Bacillus sp. indicating oxidization of coniferylic (G units) and sinapylic (S units) alcohol of lignin polymer. Differences between the identified compounds from different bacterial treatment were strain-specific. Among the identified aromatic compounds, ferulic acid and 3,4,5-trimethoxy benzaldehyde could be useful to the industry of preservatives, aromas and perfumes.     

Enzymes of ammonia assimilation in Rhizobium leguminosarum bacteroids.

The activities of the following enzymes were studied in connection with dinitrogen fixation in pea bacteroids: glutamine synthetase(L-glutamate: ammonia ligase (ADP-forming)(EC 6.3.1.2)(GS); glutamate dehydrogenase (NADP+)(L-glutamate: NADP+ oxidoreductase (deaminating)(EC 1.4.1.4)(GDH); glutamate synthase (L-glutamine: 2-exeglutarate aminotransferase (NADPH-oxidizing))(EC 2.6.1.53)(GOGAT). GS activity was high throughout the growth of the plant and GOGAT activity was always low. It is unlikely that GDH or the GS-GOGAT pathway can account for the incorporation of ammonia from dinitrogen fixation in the pea bacteroid,     

Penicillinamidohydrolase inEscherichia coli

Substrate specificity of the bacterial penicillinamidohydrolase (penicillinacylase, EC 3.5.1.11) fromEscherichia coli was determined by measuring initial rates of enzyme hydrolysis of different substrates within zero order kinetics. SomeN-phenylacetyl derivatives of amino acids and amides of phenylacetic acid and phenoxyacetic acid of different substituted amides of these acids or amides, structurally and chemically similar to these compounds, served as substrates. Significant differences in ratios of initial Tates of the enzyme hydrolysis of different substrates were found when using a toluenized suspension of bacterial cells or a crude enzyme preparation, in spite of the fact that the enzyme is localized between the cell wall and cytoplasmic membrane, in the so-called periplasmic space.N-phenylacetyl derivatives are the most rapidly hydrolyzed substrates. Beta-phenylpropionamide and 4-phenylbutyramide were not utilized as substrates. The substrate specificity of the enzyme is discussed with respect to a possible use of certain colourless compounds as substrates, hydrolysis of which yields chromophor products suitable for a simple and rapid assay of the enzyme activity.