Some properties of glutamine synthetase and glutamate synthase from Chlorobium vibrioforme f. thiosulfatophilum

The phototrophic green sulphur bacterium Chlorobium vibrioforme f. thiosulfatophilum assimilated ammonia via glutamine synthetase and glutamate synthase when grown with ammonia up to 30 mM, but above this level glutamate dehydrogenase was the key enzyme. Glutamine synthetase purified 42-fold was found to be adenylylated. The -glutamyltransferase activity of the enzyme was markedly inhibited by alanine, glycine, serine and lysine, and these amino acids in various combinations showed cumulative inhibition. Adenine nucleotides also inhibited enzyme activity, especially ATP. Glutamate synthase purified 222-fold had a maximum absorption at 440 nm which was reduced by sodium dithionite, and the enzyme was inhibited by atebrin indicating the presence of a flavin component. The enzyme had specific requirements for NADH, -ketoglutarate and l-glutamine, the K _m values for these were 13.5, 270 and 769 M respectively. Glutamate synthase was sensitive to feedback inhibition by amino acids, adenine nucleotides and other metabolites and the combined effects of these inhibitors was cumulative.Abbreviations GS glutamine synthetase - GOGAT glutamate synthase - GDH glutamic dehydrogenase     

Cytochrome aa 3 from Methylococcus capsulatus (Bath)

A cytochrome aa ₃-type oxidase was isolated with and without a c-type cytochrome (cytochrome c-557) from Methylococcus capsulatus Bath by ion-exchange and hydrophobic chromatography in the presence of Triton X-100. Although cytochrome c-557 was not a constitutive component of the terminal oxidase, the cytochrome c ascorbate-TMPD oxidase activity of the enzyme decreased dramatically when the ratio of cytochrome c-557 to heme a dropped below 1:3. On denaturing gels, the purified enzyme dissociated into three subunits with molecular weights of 46,000, 28,000 and 20,000. The enzyme contains two heme groups (a and a ₃), absorption maximum at 422 nm in the resting state, at 445 and 601 nm in the dithionite reduced form and at 434 and 598 nm in the dithionite reduced plus CO form. Denaturing gels of the cytochrome aa ₃-cytochrome c-557 complex showed the polypeptides associated with cytochrome aa ₃ plus a heme c-staining subunit with a molecular weight of 37,000. The complex contains approximately two heme a, one heme c, absorption maximum at 420 nm in the resting state and at 421, 445, 522, 557 and 601 nm in the dithionite reduced form. The specific activity of the purified enzyme was 130 mol O₂/min · mol heme a compared to 753 mol O₂/min · mol heme a when isolated with cytochrome c-557.Abbreviations MMO methan monooxygenase - sMMO soluble methane monooxygenase - pMMO particulate methane monooxygenase - TMPD N,N,N,N-tetramethyl-p-phenylenediamine dihydrochloride - Na₂EDTA disodium ethylenediamine-tetraacetic acid     

Stachyose synthesis in mature leaves of Cucumis melo. Purification and characterization of stachyose synthase (EC 2.4.1.67)

Galactinol: raffinose-6-galactosyltransferase (EC 2.4.1.67), a stachyose synthase, was extracted from mature leaves of Cucumis melo cv. Ranjadew and was purified to homogeneity by (NH₄)₂SO₄ precipitation, ion-exchange chromatography, gel-filtration and non-denaturing polyacrylamide gel electrophoresis. A specific activity of 516 kat · mg^-1 and a 160-fold purification was achieved. The pH optimum of the enzyme reaction was found to be 6.8 in sodium-phosphate buffer, and the temperature optimum 32° C. The purified enzyme was very sensitive towards SH-poisons but its reaction was hardly affected by changes in the ion composition of the assay medium. The two-substrate enzyme was specific for galactinol and raffmose; uridine-diphosphate galactose and p-nitrophenyl--d-galactoside as well as melibiose were not accepted by the purified enzyme. Stachyose synthesis was competitively inhibited by concentrations >4 mM raffinose as well as 2.5 mM galactinol. The K _m values determined under non-saturating conditions were 3.3 mM for raffinose and 7.7 mM for galactinol. Myoinositol was a strong competitive inhibitor with a K _i of 1.8mM. Galactinol was hydrolyzed in the absence of raffinose with a K _m of 0.8 mM. The pure enzyme is a protein with a molecular weight of at least 95 kDa and an isoelectric point of 5.1. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed the presence of two subunits of 45 and 50 kDa. Polyclonal antibodies from rabbit were obtained which were specific for the native enzyme but cross-reacted with other proteins separated under denaturing conditions.Abbreviations DEAE diethylaminoethyl - DTT dithiothreitol - FPLC fast protein liquid chromatography - HPLC high-performance liquid chromatography - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate This work was supported by Deutsche Forschungsgemeinschaft. The gift of galactinol by Dr. T. Schweizer (Nestlé, Switzerland) is gratefully acknowledged.     

Cytochromes of the non-thiosulfate-utilizing green sulfur bacterium Chlorobium limicola

Flavocytochrome c-553 of the non-thiosulfateutilizing green sulfur bacterium Chlorobium limicola strain 6330 was partially purified by ion exchange column chromatography and ammonium sulfate fractionation (highest purity index obtained: A ₂₈₀/A _{417 red}=0.96). It is autoxidizable and located in the soluble fraction. This hemoprotein contains a flavin component and one heme per molecule. The dithionite reduced spectrum reveals the typical maxima of a c-type cytochrome: =553,5 nm; =523 nm; =417 nm, while the oxidized form shows a -band at 410 nm and two shoulders at 440 nm and 480 nm indicating the flavin component. The flavocytochrome is a basic protein with an isoelectric point at pH 9.0 (± 0.5), a redox potential of 65 mV, a molecular weight of 56,000. It participates in sulfide oxidation and shows neither adenylylsulfate reductase nor sulfite reductase activity. C. limicola further contains a soluble cytochrome c-555 (highest purity index obtained: A ₂₈₀/A _{412 ox}=0.13; isoelectric point between pH 9.5 and 10) and the non-heme iron-containing proteins rubredoxin and ferredoxin, but lacks cytochrome c-551. Besides these soluble electron transfer proteins a membrane-bound c-type cytochrome (=554,5 nm) can be detected spectrophotometrically.Non-common abbreviations HIPIP high-potential iron sulfur protein - APS adenylylsulfate     

Microbial oxidation of methane and methanol: Purification and properties of a heme-containing aldehyde dehydrogenase from Methylomonas methylovora

Procedures for the purification of an aldehyde dehydrogenase from extracts of the obligate methylotroph, Methylomonas methylovora are described. The purified enzyme is homogeneous as judged from polyacrylamide gel electrophoresis. In the presence of an artificial electron acceptor (phenazine methosulfate), the purified enzyme catalyzes the oxidation of straight chain aldehydes (C₁-C₁₀ tested), aromatic aldehydes (benzaldehyde, salicylaldehyde), glyoxylate, and glyceraldehyde. Biological electron acceptors such as NAD⁺, NADP⁺, FAD, FMN, pyridoxal phosphate, and cytochrome c cannot act as electron carriers. The activity of the enzyme is inhibited by sulfhydryl agents [p-chloromercuribenzoate, N-ethylmaleimide and 5,5-dithiobis (2-nitrobenzoic acid)], cuprous chloride, and ferrour nitrate. The molecular weight of the enzyme as estimated by gel filtration is approximately 45000 and the subunit size determined by sodium dodecyl sulfate-gel electrophoresis is approximately 23000. The purified enzyme is light brown and has an absorption peak at 410 nm. Reduction of enzyme with sodium dithionite or aldehyde substrate resulted in the appearance of peaks at 523 nm and 552 nm. These results suggest that the enzyme is a hemoprotein. There was no evidence that flavins were present as prosthetic group. The amino acid composition of the enzyme is also presented.Non-Standard Abbreviations PMS phenazine methosulfate - DCPIP 2,6-dichlorophenol indophenol - DEAE diethylaminoethyl     

Action of light,nitrate and ammonium on the levels of NADH- and ferredoxin-dependent glutamate synthases in the cotyledons of mustard seedlings

In mustard (Sinapis alba L.) cotyledons, NADH-dependent glutamate synthase (NADH-GOGAT, EC 1.4.1.14) is only detectable during early seedling development with a peak of enzyme activity occurring between 2 and 2.5 d after sowing. With the beginning of plastidogenesis at approximately 2 d after sowing, ferredoxindependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) appears while NADH-GOGAT drops to a very low level. The enzymes were separated by anion exchange chromatography. Both enzymes are stimulated by light operating through phytochrome. However, the extent of induction is much higher in the case of Fd-GOGAT than in the case of NADH-GOGAT. Moreover, NADH-GOGAT is inducible predominantly by red light pulses, while the light induction of Fd-GOGAT operates predominantly via the high irradiance response of phytochrome. The NADH-GOGAT level is strongly increased if mustard seedlings are grown in the presence of nitrate (15 mM KNO₃,15 mM NH₄NO₃) while the Fd-GOGAT level is only slightly affected by these treatments. No effect on NADH-GOGAT level was observed by growing the seedlings in the presence of ammonium (15 mM NH₄Cl) instead of water, whereas the level of Fd-GOGAT was considerably reduced when seedlings were grown in the presence of NH₄Cl. Inducibility of NADH-GOGAT by treatment with red light pulses or by transferring water-grown seedlings to NO ₃ ^- -containing medium follows a temporal pattern of competence. The very low Fd-GOGAT level in mustard seedlings grown under red light in the presence of the herbicide Norflurazon, which leads to photooxidative destruction of the plastids, indicates that the enzyme is located in the plastids. The NADH-GOGAT level is, in contrast, completely independent of plastid integrity which indicates that its location is cytosolic. It is concluded that NADH-GOGAT in the early seedling development is mainly concerned with metabolizing stored glutamine whereas Fd-GOGAT is involved in ammonium assimilation.Abbreviations and symbols c continuous - D darkness - Fd-GOGAT ferredoxin-dependent glutamate synthase (EC 1.4.7.1) - FR far-red light (3.5 W·m^-2) - NADH-GOGAT NADH-dependent glutamate synthase (EC 1.4.1.14) - Pfr far-red absorbing form of phytochrome - Ptot total phytochrome - R red light (6.8 W· m^-2) - RG9-light long wavelength FR (10 W·m^-2, _RG9<0.01) - () Pfr/Ptot=wavelength-dependent photoequilibrium of the phytochrome system     

Some properties of glutamate dehydrogenase,glutamine synthetase and glutamate synthase from Corynebacterium callunae

Characteristics of the three major ammonia assimilatory enzymes, glutamate dehydrogenase (GDH), glutamine synthetase (GS) and glutamate synthase (GOGAT) in Corynebacterium callunae (NCIB 10338) were examined. The GDH of C. callunae specifically required NADPH and NADP⁺ as coenzymes in the amination and deamination reactions, respectively. This enzyme showed a marked specificity for -ketoglutarate and glutamate as substrates. The optimum pH was 7.2 for NADPH-GDH activity (amination) and 9.0 for NADP⁺-GDH activity (deamination). The results showed that NADPH-GDH and NADP⁺-GDH activities were controlled primarily by product inhibition and that the feedback effectors alanine and valine played a minor role in the control of NADPH-GDH activity. The transferase activity of GS was dependent on Mn⁺² while the biosynthetic activity of the enzyme was dependent on Mg²⁺ as essential activators. The pH optima for transferase and biosynthetic activities were 8.0 and 7.0, respectively. In the transfer reaction, the K _m values were 15.2 mM for glutamine, 1.46 mM for hydroxylamine, 3.5×10^-3 mM for ADP and 1.03 mM for arsenate. Feedback inhibition by alanine, glycine and serine was also found to play an important role in controlling GS activity. In addition, the enzyme activity was sensitive to ATP. The transferase activity of the enzyme was responsive to ionic strength as well as the specific monovalent cation present. GOGAT of C. callunae utilized either NADPH or NADH as coenzymes, although the latter was less effective. The enzyme specifically required -ketoglutarate and glutamine as substrates. In cells grown in a medium with glutamate as the nitrogen source, the optimum pH was 7.6 for NADPH-GOGAT activity and 6.8 for NADH-GOGAT activity. Findings showed that NADPH-GOGAT and NADH-GOGAT activities were controlled by product inhibition caused by NADP⁺ and NAD⁺, respectively, and that ATP also had an important role in the control of NADPH-GOGAT activity. Both activities of GOGAT were found to be inhibited by azaserine.Abbreviations GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase     

The effect of various culture conditions on the levels of ammonia assimilatory enzymes of Corynebacterium callunae

Corynebacterium callunae (NCIB 10338) grows faster on glutamate than ammonia when used as sole nitrogen sources. The levels of glutamine synthetase (GS; EC 6.3.1.2) and glutamate synthase (GOGAT; EC 1.4.1.13) of C. callunae were found to be influenced by the nitrogen source. Accordingly, the levels of GS and GOGAT activities were decreased markedly under conditions of ammonia excess and increased under low nitrogen conditions. In contrast, glutamate dehydrogenase (GDH; EC 1.4.1.4) activities were not significantly affected by the type or the concentration of the nitrogen source supplied. The carbon source in the growth medium could also affect GDH, GS and GOGAT levels. Of the carbon sources tested in the presence of 2 mM or 10 mM ammonium chloride as the nitrogen source pyruvate, acetate, fumarate and malate caused a decrease in the levels of all three enzymes as compared with glucose. GDH, GS and GOGAT levels were slightly influenced by aeration. Also, the enzyme levels varied with the growth phase. Methionine sulfoximine, an analogue of glutamine, markedly inhibited both the growth of C. callunae cells and the transferase activity of GS. The apparent K _m values of GDH for ammonia and glutamate were 17.2 mM and 69.1 mM, respectively. In the NADPH-dependent reaction of GOGAT, the apparent K _m values were 0.1 mM for -ketoglutarate and 0.22 mM for glutamine.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase     

Ammonium assimilation in an Arthrobacter sp. “fluorescens”

Ammonium assimilation was studied in a nitrogenfixing Arthrobacter strain grown in both batch and ammonium-limited continuous cultures. Arthrobacter sp. fluorescens grown in nitrogen-free medium or at low ammonium levels assimilated NH ₄ ⁺ via the glutamine synthetase/glutamate synthase pathway. When ammonium was in excess it was assimilated via the alanine dehydrogenase pathway. Very low levels of glutamate dehydrogenase were found, irrespective of growth conditions.Abbreviations GS glutamine synthetase - GOGAT glutamine oxoglutarate aminotransferase - GDH glutamate dehydrogenase - ADH alanine dehydrogenase - GOT glutamate oxaloacetate transaminase - GPT glutamate pyruvate transaminase     

Assimilation of exogenous and dinitrogen-derived13NH 4 + byAnabaena azollae separated fromAzolla caroliniana Willd

Anabaena azollae was isolated fromAzolla caroliniana by the gentle roller method and differential centrifugation. Incubation of suchAnabaena preparations for 10 min with [¹³N]N₂ resulted in the formation of four radioactive compounds; ammonium, glutamine, glutamate and alanine. Ammonium accounted for 66% of the total radioactivity recovered and 58% of the ammonium was in an extracellular fraction. Since essentially no extracellular¹³N-labeled organic compounds were found, it appears that ammonium is the compound most probably made available toAzolla during dinitrogen-dependent growth of the association.The kinetics of incorporation of exogenous¹³NH ₄ ⁺ into glutamine and glutamate were characteristic of a precursor (glutamine)-product (glutamate) relationship and consistent with assimilation by the glutamine synthetase-glutamate synthase pathway. The results of experiments using the glutamine synthetase inhibitor, methionine sulfoximine, the glutamate synthase inhibitor, diazo-oxonorleucine, and increasing the ammonium concentration to greater than 1 mM, provided evidence for assimilation primarily by the glutamine synthetase-glutamate synthase pathway with little or no contribution from biosynthetic glutamate dehydrogenase.While showing that N₂ fixation and NH ₄ ⁺ assimilation were not tightly coupled metabolic processes in symbioticAnabaena, these results reflect a composite picture and do not indicate the extent to which ammonium assimilatory enzymes might be regulated in filaments associated with specific stages in theAzolla-Anabaena developmental profile.Non-standard abbreviations DON 6-Diazo-5-oxo-l-norleucine - GDH glutamate dehydrogenase - GOGAT glutamate synthase - GS glutamine synthetase - MSX l-methionine-Dl-sulfoximine     

Purification and properties of tobacco ferredoxin-dependent glutamate synthase,and isolation of corresponding cDNA clones

Ferredoxin-dependent glutamate synthase (Fd-GOGAT, EC 1.4.7.1) was purified to electrophoretic homogeneity from leaves of tobacco (Nicotiana tabacum L.). The holoenzyme is a monomeric flavoprotein with a molecular weight of 164 kDa. Polyclonal rabbit antibodies against the purified enzyme were used to isolate a 450-bp Fd-GOGAT cDNA clone (C₁₆) from a tobacco gt11 expression library. A longer Fd-GOGAT cDNA clone (C₃₅) encoding about 70% of the amino acids of tobacco Fd-GOGAT was isolated from a tobacco gt10 cDNA library using C₁₆ as the probe. The amino-acid sequence of the protein encoded by the Fd-GOGAT cDNA clone C₃₅ was delineated. It is very likely that Fd-GOGAT is encoded by two genes in the amphidiploid genome of tobacco while only a single Fd-GOGAT gene appears to be present in the diploid genome of Nicotiana sylvestris. Two Fd-GOGAT isoenzymes could be distinguished in extracts of tobacco leaf protein. In contrast, a single Fd-GOGAT protein species was detected in leaves of Nicotiana sylvestris speg. et Comes. In tobacco leaves, the 6-kb Fd-GOGAT mRNA is about 50-fold less abundant than chloroplastic glutamine synthetase (EC 6.3.1.2) mRNA. Both Fd-GOGAT mRNA and Fd-GOGAT protein accumulated during greening of etiolated tobacco leaves, and a concomitant increase in Fd-GOGAT activity was observed. These results indicate that tobacco Fd-GOGAT gene expression is light-inducible. Levels of Fd-GOGAT mRNA in tobacco organs other than leaves were below the detection limit of our Northern-blot analysis. Polypeptides of Fd-GOGAT were present in tobacco leaves and, to a lesser extent, in pistils and anthers, but not in corollas, stems and roots. These results support organ specificity in tobacco Fd-GOGAT gene expression.Abbreviations bp base pairs - Fd-GOGAT ferredoxin-dependent glutamate synthase - GS glutamine synthetase - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate The authors wish to thank Juan Luis Gómez Pinchetti (Marine Plant Biotechnology Laboratory) for his assistance during the experiments. This study was supported by grants received from SAREC (Swedish Agency for Research Cooperation with Developing Countries), Carl Tryggers Fund for Scientific Research (K. Haglund), SJFR (Swedish Council for Forestry and Agricultural Research) (M. Björk, M. Pedersén), CITYT Spain (SAB 89-0091 and MAR 91-1237, M. Pedersén) and CICYT Spain (Z. Ramazanov, invited professor of Ministerio de Educatión y Ciencia, Spain). The planning of this cooperation was facilitated by COST-48.     

Location of two isoenzymes of NADH-dependent glutamate synthase in root nodules of Phaseolus vulgaris L.

The two isoenzymes of NADH-dependent glutamate synthase (NADH-GOGAT; EC 1.4.1.14), previously identified in root nodules of Phaseolus vulgaris L., have both been shown to be located in root-nodule plastids. The nodule specific NADH-GOGAT II accounts for the majority of the activity in root nodules, and is present almost exclusively in the central tissue of the nodule. However about 20% of NADH-GOGAT I activity is present in the nodule cortex, at about the same specific activity as this isoenzyme is found in the central tissue. Glutamine synthetase (GS; EC 6.3.1.2) occurs predominantly as the polypeptide in the central tissue, whereas in the cortex, the enzyme is represented mainly by the polypeptide. Over 90% of both GS and NADH-GOGAT activities are located in the central tissue of the nodule and GS activity exceeds NADH-GOGAT activity by about twofold in this region. Using the above information, a model for the subcellular location and stoichiometry of nitrogen metabolism in the central tissue of P. vulgaris root nodules is presented.Abbreviations Fd-GOGAT ferredoxin-dependent glutamate synthase - GOGAT glutamate synthase - GS glutamine synthetase - NADH-GOGAT NADH-dependent glutamate synthase - IEX-HPLC ion-exchange high-performance liquid chromatography     

Aromatic amino acid biosynthesis in Alcaligenes eutrophus H 16

Properties and regulation of anthranilate synthase from Alcaligenes eutrophus H 16 were investigated. Anthranilate synthase was partially purified from crude extracts by affinity chromatography on tryptophan-substituted Sepharose, and was used for kinetic measurements. During the purification procedure the enzyme was stabilized by 50 mM l-glutamine or during chromatography on DEAE-cellulose and Sephadex G-200 with 30% glycerol, respectively.The glutamine dependent activity of anthranilate synthase was examined; it showed little change between pH 8.4 and pH 9.1. The Arrhenius plot was broken and the activation energy, H, calculated therefrom amounted to 8.9 kcal/mole up to 30°C and 5.5 kcal/mole at higher temperatures. The molecular weight determined by gelfiltration on Sephadex G-200 and by sucrose density gradient centrifugation resulted in 158000 and 126000, respectively. The K _m -values for the two substrates chorismate and glutamine were found to be 5 M and 560 M, respectively.Anthranilate synthase was strongly inhibited by l-tryptophan; the only amino acid that affected enzyme activity. Homotropic interactions for chorismate (Hill coefficient n=1.4) were obtained in the presence of l-tryptophan. 50% inhibition were caused by 10 M l-tryptophan at 100 M chorismate. The inhibition with respect to l-glutamine was noncompetitive.Anthranilate synthase was not associated to phosphoribosyl transferase and easily separable from the latter by different chromatographic methods.Abbreviation TEA triethanolamine     

A theoretical study of glucosamine synthase

Glucosamine synthase transfers the -amino group of glutamine to fructose, producing 1-glucosamine which is the key constituent of bacterial and fungal cell walls. In this study, model calculations were performed on substrate binding to the enzyme active site. Two models of the active site of glucosamine synthase were proposed, which assume two different sequences of aminoacids, Cys-Gly-Ile and Cys-Ala-Cys, the first one being the N-terminal sequence of the Escherichia coli enzyme. Several initial geometries were assumed for these tripeptides, the energy was then optimized by means of molecular mechanics. It has been found that the structure which is both energy optimal and satisfies the assumed cysteine sulphur arrangement consists of combinations of C ₇ ^eq and C ₇ ^ax conformations of single residues. Molecular mechanics calculations were then performed on glutamine and d-fructose-6-phosphate, which are the substrates of the enzymatic satalysis, and on their complex with the enzyme glutamine-binding site. The spatial configuration of the compounds under study, which is optimal as far as the reaction path is concerned, also turned out to be an energy minimum.     

Characterization and localization of phosphatidylglycerophosphate and phosphatidylserine synthases in Rhodobacter sphaeroides

Catalytic properties and membrane associations of the phosphatidylglycerophosphate (PGP) and phosphatidylserine (PS) synthases of Rhodobacter sphaeroides were examined to further characterize sites of phospholipid biosynthesis. In preparations of cytoplasmic membrane (CM) enriched in these activities, apparent K _m values of PGP synthase were 90 M for sn-glycerol-3-phosphate and 60 M for CDP-diacylglycerol; the apparent K _m of PS synthase for l-serine was near 165 M. Both enzymes required Triton X-100 with optimal PS synthase activity at a detergent/CDP-diacylglycerol (mol/mol) ratio of 7.5:1.0, while for optimal PGP synthase, a range of 10–50:1.0 was observed. Unlike the enzyme in Escherichia coli and several other Gram-negative bacteria, the PS synthase activity had a specific requirement for magnesium and was tightly associated with membranes rather than ribosomes in crude cell extracts. Sedimentation studies suggested that the PGP synthase ws distributed uniformly over the CM in both chemoheterotrophically and photoheterotrophically grown cells, while the PS synthase was confined mainly to a vesicular CM fraction. Solubilized PGP synthase activity migrated as a single band with a pI value near 5.5 in a chromatofocusing column and 5.8 on isoelectric focusing; in the latter procedure, the pI was shifted to 5.3 in the presence of CDP-diacylglycerol. The PGP synthase activity gave rise to a single polypeptide band in lithium dodecyl sulfatepolyacrylamide gel electrophoresis at 4°C.Abbreviations CM cytoplasmic membrane - ICM intracytoplasmic photosynthetic membrane - OM outer membrane - PGP phosphatidylglycerophosphate - PS phosphatidylserine - TLC thin-layer chromatography Supported in part by a Fellowship Awards from the Charles and Johanna Busch Memorial Fund Award to the Rutgers Bureau of Biological Research     

Aspects of nitrogen fixation in Chlorobium

Four strains of the green sulfur bacterium Chlorobium were studied in respect to nitrogen nutrition and nitrogen fixation. All strains grew on ammonia, N₂, or glutamine as sole nitrogen sources; certain strains also grew on other amino acids. Acetylene-reducing activity was detectable in all strains grown on N₂ or on amino acids (except for glutamine). In N₂ grown Chlorobium thiosulfatophilum strain 8327 1 mM ammonia served to switch-off nitrogenase activity, but the effect of ammonia was much less dramatic in glutamate or limiting ammonia grown cells. The glutamine synthetase inhibitor methionine sulfoximine inhibited ammonia switch-off in all but one strain. Cell extracts of glutamate grown strain 8327 reduced acetylene and required Mg²⁺ and dithionite, but not Mn²⁺, for activity. Partially purified preparations of Rhodospirillum rubrum nitrogenase reductase (iron protein) activating enzyme slightly stimulated acetylene reduction in extracts of strain 8327, but no evidence for an indigenous Chlorobium activating enzyme was obtained. The results suggest that certain Chlorobium strains are fairly versatile in their nitrogen nutrition and that at least in vivo, nitrogenase activity in green bacteria is controlled by ammonia in a fashion similar to that described in nonsulfur purple bacteria and in Chromatium.Non-common abbreviations MSX Methionine sulfoximine - MOPS 3-(N-morpholino) propane sulfonic acid This paper is dedicated to Professor Norbert Pfennig on the occasion of his 60th birthday     

Recovery of plastids from photooxidative damage: Significance of a plastidic factor

Glyoxysomal citrate synthase (gCS) was purified from crude extracts of watermelon (Citrullus vulgaris Schrad.) cotyledons, yielding a homogenous protein with a subunit MW of 48 kDa. The enzyme was selectively inhibited by 5,5-dithiobis-(2-nitrobenzoic acid), allowing quantification in the presence of the mitochondrial isoenzyme (mCS). Differences were also observed with respect to inhibition by ATP (k _i=2.6 mmol · l^-1 for gCS, k _i=0.33 mmol · l^-1 for mCS). The antibodies prepared against gCS did not cross-react with mCS. The immunocytochemical localization of gCS by the indirect protein A-gold procedure was restricted to the glyoxysomal membrane or the peripheral matrix of glyoxysomes. Other compartments, e.g. the endoplasmic reticulum, were not labeled. Xenopus oocytes were used for the translation of watermelon polyadenylated RNA (poly(A)⁺RNA). A translation product with a MW of 51 kDa was immunoprecipitated by the anti-gCS antibodies. It was absent in controls without poly(A)⁺RNA or with preimmune serum. A similar translation product was also immunoprecipitated after cell-free synthesis of watermelon poly(A)⁺RNA in a reticulocyte system, in contrast to the in-vivo labeled gCS (48 kDa). It was concluded that gCS is synthesized as a higher-molecular-weight precursor.Abbreviations DTNB 5,5-dithiobis-(2-nitrobenzoic acid) - gCS glyoxysomal citrate synthase - gMDH glyoxysomal malate dehydrogenase - k _i inhibitor constant - mCS mitochondrial citrate synthase - OAA oxaloacetate - poly(A)⁺RNA polyadenylated RNA - SDS-PAGE sodium dodecyl sulfate-polyacrylamide gel electrophoresis     

Nitrogen metabolism inRhodopseudomonas globiformis

Rhodopseudomonas globiformis strain 7950 grew with a variety of amino acids, urea, or N₂ as sole nitrogen sources. Cultures grown on N₂ reduced acetylene to ethylene; this activity was absent from cells grown on nonlimiting NH ₄ ⁺ . Glutamate dehydrogenase could not be detected in extracts of cells of strain 7950, although low levels of an alanine dehydrogenase were present. Growth ofR. globiformis on NH ₄ ⁺ was severely inhibited by the glutamate analogue and glutamine synthetase inhibitor, methionine sulfoximine. High levels of glutamine synthetase (as measured in the -glutamyl transferase assay) were observed in cell extracts of strain 7950 regardless of the nitrogen source, although N₂ and amino acid grown cells contained somewhat higher glutamine synthetase contents than cells grown on excess NH ₄ ⁺ . Levels of glutamate synthase inR. globiformis were consistent with that reported from other phototrophic bacteria. Both glutamate synthase and alanine dehydrogenase were linked to NADH as coenzyme. We conclude thatR. globiformis is capable of fixing N₂, and assimilates NH ₄ ⁺ primarily via the glutamine synthetase/glutamate synthase pathway.Abbreviations GS glutamine synthetase - GOGAT Glutamineoxoglutarate aminotransferase - GDH Glutamate dehydrogenase - ADH Alanine dehydrogenase - MSO Methionine sulfoximine     

Nitrogen assimilation in Rhodopseudomonas acidophila

Rhodopseudomonas acidophila strain 7050 assimilated ammonia via a constitutive glutamine synthetase/glutamate synthase enzyme system.Glutamine synthetase had a K _m for NH ₄ ⁺ of 0.38 mM whilst the nicotinamide adenine dinucleotide linked glutamate synthase had a K _m for glutamine of 0.55 mM. R. acidophila utilized only a limited range of amino acids as sole nitrogen sources: l-alanine, glutamine and asparagine. The bacterium did not grow on glutamate as sole nitrogen source and lacked glutamate dehydrogenase. When R. acidophila was grown on l-alanine as the sole nitrogen source in the absence of N₂ low levels of a nicotinamide adenine dinucleotide linked l-alanine dehydrogenase were produced. It is concluded, therefore, that this reaction was not a significant route of ammonia assimilation in this bacterium except when glutamine synthetase was inhibited by methionine sulphoximine. In l-alanine grown cells the presence of an active alanine-glyoxylate aminotransferase and, on occasions, low levels of an alanine-oxaloacetate aminotransferase were detected. Alanine-2-oxo-glutarate aminotransferase could not be demonstrated in this bacterium.Abreviations ADH alanine dehydrogenase - GDH glutamate dehydrogenase - GS glutamine synthetase - GOGAT glutamate synthase - MSO methionine sulphoximine     

Cytochromes,rubredoxin, and sulfur metabolism of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum

Two soluble c-type cytochromes (c-553 and c-555) and the nonheme iron-containing protein rubredoxin of the non-thiosulfate-utilizing green sulfur bacterium Pelodictyon luteolum were highly purified by ion exchange column chromatography, gel filtration and ammonium sulfate fractionation. Both cytochrome are small and basic hemoproteins, while rubredoxin is an acidic small nonheme iron protein. Cytochrome c-553 has a molecular weight of 13,000 determined by Sephacryl S-200 chromatography and of 10,700 by electrophoresis on SDS acrylamide gel, an isoelectric point at pH 10.2, a redox-potential of +220 mV. It shows maxima at 413 nm in the oxidized form, and the characteristic three maxima in the reduced state (-band at 553 nm, -band at 523 nm, -band at 417 nm). The best purity index (A ₂₈₀/A ₄₁₇) obtained was 0.18. Cytochrome c-555 (best purity index obtained: A ₂₈₀/A ₄₁₈=0.17) has an isoelectric point at pH 10.5, a molecular weight of 9,500 (by electrophoresis on SDS acrylamide gel) and a redox-potential of +160mV. The reduced form of this cytochrome shows the typical bands of c-type cytochromes at 555 (551) nm (-band), 523 nm (-band) and 418 nm (-band), while the oxidized form has the -band at 413 nm.Rubredoxin (best purity index obtained: A ₂₈₀/A ₄₉₀=3.5) is an acidic small protein. Its molecular weight estimated by gel filtration and SDS acrylamide gel electrophoresis is 27,000 and 6,300 respectively. The monomer of this protein contains one iron atom per molecule. Rubredoxin has an isoelectric point at pH 2.8 and shows maxima at 570 nm, 490 nm and 370 nm in the oxidized form.During anaerobic sulfide oxidation of a growing culture of Pelodictyon luteolum elemental sulfur is the first main product, which appears in the medium. Elemental sulfur is further oxidized to sulfate, after the available sulfide is completely consumed by the cells.Non-common abbreviations C Chlorobium - P Pelodictyon - SDS sodium dodecylsulfate - HIPIP high-potential-iron-sulfur-protein Offprint requests to: U. Fischer