Dissimilatory hexaheme c nitrite reductase of “Spirillum” strain 5175: purification and properties

When grown with nitrate as terminal electron acceptor both the soluble (periplasm, cytoplasm) and the membrane fraction of Spirillum strain 5175 exhibited high nitrite reductase activity. The nitrite reductase obtained from the soluble fraction was purified 76-fold to electrophoretical homogeneity. The enzyme reduced nitrite to ammonia with a specific activity of 723 mol NO _inf2 ^sup- × (mg protein × min)^-1. The molecular mass was 58±1 kDa by SDS-PAGE compared to 59±2 kDa determined by size exclusion chromatography under nondenaturing conditions. The enzyme (as isolated) contained 5.97±0.15 heme c molecules/M_r 58 kDa. The absorption spectrum was typical for c-type cytochrome with maxima at 280, 408, 532 and 610 nm (oxidized) and at 420, 523 and 553 nm (dithionite-reduced). The enzyme (as isolated) exhibited a complex set of high-spin and lowspin ferric heme resonances with g-values at 9.82, 3,85, 3.31, 2.95, 2.30 and 1.49 in agreement with data reported for electron paramagnetic resonance spectra of nitrite reductases from Desulfovibrio desulfuricans, Wolinella succinogenes and Escherichia coli.Abbreviations DNRA dissimilatory nitrate reduction to ammonia - EPR electron paramagnetic resonance - PAGE polyacrylamide gel electrophoresis - NaP_i sodium phosphate - SDS sodium dodecylsulfate     

Isolation of the sulphur reductase and reconstitution of the sulphur respiration of Wolinella succinogenes

Wolinella succinogenes can grow at the expense of sulphur reduction by formate. The enzymes involved in the catalysis of this catabolic reaction have been investigated. From the results the following conclusions are drawn: 1. The enzyme isolated as a sulphide dehydrogenase from the cytoplasmic membrane of W. succinogenes is the functional sulphur reductase that operates in the electron transport from formate to sulphur. 2. The enzyme (M_r 200,000) consists essentially of one type of subunit with the M_r 85,000 and contains equal amounts of free iron and sulphide (120 mol/g protein), but no heme. It represents the first functional sulphur reductase ever isolated. 3. The electron transport chain catalyzing sulphur reduction by formate consists merely of formate dehydrogenase and sulphur reductase. A lipophilic quinone which mediates the transfer of electrons between enzymes in other chains, is apparently not involved. This is the first known example of a phosphorylative electron transport chain that operates without a quinone. 4. The same formate dehydrogenase appears to operate in the electron transport both with sulphur and with fumarate as the terminal electron acceptor in W. succinogenes.Abbreviations DMN 2,3-Dimethyl-1,4-naphthoquinone - DTT dithiothreitol - MK menaquinone (vitamin K₂) - PMSF phenylmethane sulfonylfluoride - Tricine N-[2-hydroxy-1,1-bis(hydroxymethyl)ethyl]-glycine - Tea triethanolamine - Hepes 4-(2-hydroxyethyl)-1-piperazineethane sulfonate Dedicated to Professor F. Schneider (Philipps-Universität Marburg) on the occasion of his 60th birthday     

The malonate decarboxylase enzyme system of Malonomonas rubra: evidence for the cytoplasmic location of the biotin-containing component

Malonate decarboxylase of Malonomonas rubra is a complex enzyme system involving cytoplasmic and membrane-bound components. One of these is a biotin-containing protein of M_r 120'000, the location of which in the cytoplasm was deduced from the following criteria: (i) If the cytoplasm was incubated with avidin and the malonate decarboxylase subsequently completed with the membrane fraction the decarboxylase activity was abolished. The corresponding incubation of the membrane with avidin, however, was without effect. (ii) Western blot analysis identified the single biotin-containing polypeptide of M_r 120'000 within the cytoplasm. (iii) Transmission electron micrographs of immuno-gold labeled M. rubra cells clearly showed the location of the biotinyl protein within the cytoplasm, whereas the same procedure with Propionigenium modestum cells indicated the location of the biotin enzyme methylmalonyl-CoA decarboxylase in the cell membrane. The biotin-containing protein of the M. rubra malonate decarboxylase enzyme system was not retained by monomeric avidin-Sepharose columns but could be isolated with this column in a catalytically inactive form in the presence of detergents. If the high binding affinity of tetrameric avidin towards biotin was reduced by destructing part of the tryptophan residues by irradiation or oxidation with periodate, the inhibition of malonate decarboxylase by the modified avidin was partially reversed with an excess of biotin. Attempts to purify the biotin protein in its catalytically active state using modified avidin columns were without success.     

Modulation of glyceraldehyde-3-phosphate dehydrogenase inAnacystis nidulans by glutathione

Glyceraldehyde-3-phosphate dehydrogenase (EC 1.2.1.13; GAPDH) from the cyanobacteriumAnacystis nidulans was activated up to five-fold by reduced glutathione (GSH) in the physiological concentration range (0.1–2 mM GSH). Non-physiological reductants, like dithiothreitol (DTT) and -mercaptoethanol, also activated the enzyme. Oxidized glutathione (GSSG) had no effect on the cyanobacterial GAPDH but treatment with H₂O₂ led to a rapid, reversible deactivation of both untreated and GSH-treated enzyme preparations. GSH reversed the inhibition induced by H₂O₂. An oligomeric form of the enzyme (apparentM _r440,000) was dissociated by GSH into a lower-M _r, more active enzyme form (M _r200,000). The enzyme was shown to obey regular Michaelis-Menten kinetics. The activation of GAPDH by GSH was associated with a decrease inK _m and an increase inV _max values of the enzyme for 3-phosphoglycerate. GSH had virtually no effect on a GAPDH preparation isolated from corn chloroplasts and studied for comparison.Abbreviations GAPDH glyceraldehyde-3-phosphate dehydrogenase - GSH reduced glutathione - GSSG oxidized glutathione - DTT dithiothreitol     

Purification and characterization of a (R)-2,3-butanediol dehydrogenase from Saccharomyces cerevisiae

A NAD-dependent (R)-2,3-butanediol dehydrogenase (EC 1.1.1.4), selectively catalyzing the oxidation at the (R)-center of 2,3-butanediol irrespective of the absolute configuration of the other carbinol center, was isolated from cell extracts of the yeast Saccharomyces cerevisiae. Purification was achieved by means of streptomycin sulfate treatment, Sephadex G-25 filtration, DEAE-Sepharose CL-6B chromatography, affinity chromatography on Matrex Gel Blue A and Superose 6 prep grade chromatography leading to a 70-fold enrichment of the specific activity with 44% yield. Analysis of chiral products was carried out by gas chromatographic methods via pre-chromatographic derivatization and resolution of corresponding diasteromeric derivatives. The enzyme was capable to reduce irreversibly diacetyl (2,3-butanediol) to (R)-acetoin (3-hydroxy-2-butanone) and in a subsequent reaction reversibly to (R,R)-2,3-butanediol using NADH as coenzyme. 1-Hydroxy-2-ketones and C₅-acyloins were also accepted as substrates, whereas the enzyme was inactive towards the reduction of acetone and dihydroxyacetone. The relative molecular mass (M _r) of the enzyme was estimated as 140 000 by means of gel filtration. On SDS-polyacrylamide gel the protein decomposed into 4 (identical) subunits of M _r 35 000. Optimum pH was 6.7 for the reduction of acetoin to 2,3-butanediol and 7.2 for the reverse reaction.Abbreviations GC-MS gas chromatography-mass spectrometry - i.d. internal diameter - M _r relative molecular mass - MTPA-Cl -methoxy--trifluoromethylphenyl acetic acid chloride - PEIC 1-phenylethylisocyanate     

Methanobacterium thermoautotrophicum (strain ΔH) contains a membrane-bound cyclic 2,3-diphosphoglycerate hydrolase

Cyclic 2,3-diphosphoglycerate (cDPG) hydrolase activity was demonstrated in cofactor-free extract of Methanobacterium thermoautotrophicum (strain H), but not in crude extract. Only after ultrafiltration or dialysis of crude extract cDPG hydrolase activity could be shown. cCPG hydrolysis was optimal at pH 6.0 and 60°C. Hydrolysis of cDPG occurred under nitrogen or hydrogen atmosphere and was completely inhibited by oxygen. Phosphate and potassium chloride were also strong inhibitors: 50% inhibition occurred at 0.6–0.7 mM phosphate or 0.2 M KCl. The enzyme was localized in the membrane fraction and could be solubilized for approximately 60% by treatment with 25 mM of the detergent CHAPS. The K _m and the V _max for cDPG were determined at 60°C and were 59 mM and 216 mU/mg, respectively. Furthermore, cDPG hydrolase was dependent on the presence of Co²⁺. The role of cDPG and cDPG hydrolase is discussed.Abbreviations cDPG cyclic 2,3-diphosphoglycerate - 2,3-DPG 2,3-diphosphoglycerate - 2-PG 2-phosphoglycerate - 3-PG 3-phosphoglycerate - PG phosphoglycerate - PEP phosphoenolpyruvate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - TRIS tris(hydroxymethyl)-aminomethane - DTT dithiothreitol - CHAPS 3-([3-cholamidopropyl]-dimethylammonio)-1-propanesulfonate - MOPS 3-(N-morpholino) propanesulfonic acid     

The menaquinol oxidase of Bacillus subtilis W23

The quinol oxidase appears to be mainly responsible for the oxidation of bacterial MKH₂ in Bacillus subtilis W23 growing with either glucose or succinate. The activity of the enzyme was maximum with dimethylnaphthoquinol, a water-soluble analogue of the bacterial menaquinol. Menadiol or duroquinol were less actively respired, and naphthoquinol was not oxidized at all. After fourtyfold purification the isolated enzyme contained 5.3 mol cytochrome aa ₃ per gram of protein and negligible amounts of cytochrome b and c. The turnover number based on cytochrome aa ₃ was about 10³ electrons · s^-1 at pH 7 and 37°C. The preparation consisted mainly of a M _r 57000 and a M _r 36000 polypeptide. The N-terminal amino acid sequence of the latter polypeptide differed from that predicted by the qoxA gene of B. subtilis strain 168 (Santana et al. 1992), in that asp-14 predicted by qoxA was missing in the M _r 36000 polypeptide.Abbreviations DMN 2,3-dimethyl-1,4-naphthoquinone - DMNH₂ 2,3-dimethyl-1,4-naphthoquinol - Duroquinol 2,3,5,6-tetramethyl-1,4-benzoquinol - MK menaquinone - MKH₂ menaquinol - NBH₂ 2,3-dimethoxy-5-methyl-6-(n-nonyl)-1,4-benzoquinol - TMPD N,N,N, N,-tetramethyl-1,4-phenylenediamine     

Characteristics of intracellular proteolytic activities ofBacillus megaterium

Intracellular proteolytic activities ofB. megaterium KM occur soluble in the cytoplasm and periplasm and insoluble in the membrane. Two proteolytic enzymes were found in the cytoplasmic fraction by gel filtration on Sephadex G 150 and by polyacrylamide gel electrophoresis. The first enzyme called C_I was stable, had a relative molecular mass ofM _r=105000 (M=105 kg/mol) and was inhibited by EDTA and PMSF, whereas the second, designated C_II, was labile and had a relative molecular mass ofM _r=46000 (M=46 kg/mol). Because of its lability it could not be characterized in detail. In the “periplasm” only a single proteolytic enzyme P (M _r=28000;M=28 kg/mol) inhibited by EDTA could be demonstrated. The extracellular enzyme exhibited similar properties. The membrane proteolytic activity was sensitive to PMSF and EDTA. The membrane enzymes have not yet been solubilized. In cells of the mutant KM 12 that does not produce the extracellular proteinase, only one type of proteinase, in all its properties identical with the cytoplasmic proteinase C_I, could be demonstrated.     

Bacterial mesosomes: Method dependent artifacts

The occurrence of mesosomes was investigated during septum formation of vegetative and sporulating cells of Bacillus cereus. It has been demonstrated that bacterial mesosomes which are considered by numerous microbiologists as an integrated constituent of Gram positive bacteria, are in reality artifacts arising during the preparation for electron microscopy. The conventional fixation methods allowed enough time for the cytoplasmic membrane to react to the changed conditions and to form the typical pocket-like membrane invaginations. With cryofixation followed by freeze-substitution it was shown in ultrathin sections that mesosomes do not occur. The extremely rapid freezing and the substitution of the ice by an organic solvent containing the fixative prevented the formation of membraneous artifacts.Non-standard abbreviations OsO₄ osmium tetroxide - UO₂Ac uranylacetate - PHB poly--hydroxy-butyric acid - M mesosome - CW cell wall - CM cytoplasmic membrane - PF plasmatic fracture of the cytoplasmic membrane     

Phosphorylation of the plasma-membrane H+-ATPase of oat roots by a calcium-stimulated protein kinase

When plasma-membrane vesicles isolated from oat (Avena sativa L.) root cells were incubated with [-³²P]ATP, the H⁺-ATPase was found to be phosphorylated at serine and threonine residues. Phosphotyrosine was not detected. Endogenous ATPase kinase activity was also observed in plasma-membrane vesicles isolated from potato (Solanum tuberosum L.) root cells as well as from yeast (Saccharomyces cerevisiae). Identity of the phosphorylated oat root M_r=100 000 polypeptide as the ATPase was confirmed using conventional glycerol density-gradient centrifugation to purify the native enzyme and by a new procedure for purifying the denatured polypeptide using reversephase high-performance liquid chromatography. Kinase-mediated phosphorylation of the oat root plasma-membrane H⁺-ATPase was stimulated by the addition of low concentrations of Ca²⁺ and by a decrease in pH, from 7.2 to 6.2. These results demonstrate that kinase-mediated phosphorylation of the H⁺-ATPase is a plausible mechanism for regulating activity. They further indicate that changes in the cytoplasmic [Ca²⁺] and pH are potentially important elements in modulating the kinase-mediated phosphorylation.Abbreviations EDTA ethylenediaminetetraacetic acid - EGTA ethylene glycol-bis-(-aminoethyl ether)-N,N,N,N-tetraacetic acid - M_r relative molecular mass - RP-HPLC reverse-phase high-performance liquid chromatography - SDS-PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis     

Redox-state of intactNitella cells: dependency on intracellular pH and photosynthesis

Summary The present paper describes the construction and properties of a Pt/Ir-semi-microelectrode and its application as a redoxsensitive electrode in intact cells of the giant algaNitella. For compartmental analysis of the stationary redox-state voltage (E_RED), a value reflecting the interaction of the dominant redox couples with a Pt/Ir-electrode, the redox-sensitive electrode was inserted into the vacuole of leaf cells or cytoplasm enriched fragments (CEF) fromNitella internodal cells. After correction for the membrane voltage, measured with a second, conventional voltage electrode, E_RED values of+237±93mVand+419±51 mV with respect to a normal H⁺-electrode were obtained for cytoplasm and vacuole, respectively. The redox-state of the cell culture medium was+604 mV. The steady state E_RED in the cytoplasm can be perturbed by experimental treatments: indirect acidification of the cytoplasm by an external pH jump from 7.5 to 5.8 and direct acidification, by acid loading with 5 mM butyrate, both resulted in a positive shift of E_RED, i.e., to an increase in cytoplasmic oxidation. At the same time the membrane depolarized electrically following the external pH jump, but hyperpolarized in response to acid loading. The data demonstrate the direct dependence of cytoplasmic redox state on intracellular pH, probably due to enhanced oxidation of protonated redox couples favoured by mass action. The electrical membrane voltage changes were not correlated with the shift in cytoplasmic E_RED. This demonstrated that redox energy does not determine the electrical membrane voltage. Cytoplasmic E_RED was also affected by photosynthesis. When CEFs were transferred from light to dark, or exposed to 10M 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU), E_RED shifted negatively (more reduced) by 6.4±4.5mV or 4.2±2mV, respectively. These data compare favourably with biochemical estimates of cytoplasmic pyridin nucleotides which also show an increase in cytoplasmic reduction in the dark. Therefore, it is unlikely that diffusable reducing equivalents are supplied to the cytoplasm from photosynthetically-active chloroplasts to act as secondary messengers.Abbreviations E_M transmembrane voltage - E_RED redox-state voltage - E₀ midpoint-redox-voltage - APW artificial pond water - CEF cytoplasm enriched fragment     

Characterization of the cell wall of the unicellular cyanobacterium Synechocystis PCC 6714

Cell walls free of cytoplasmic- and thylakoid membranes were isolated from Synechocystis PCC 6714 by sucrose density gradient centrifugation and extraction with Triton X-100. The Triton-insoluble cell wall fraction retained the multilayered fine structure. Peptidoglycan, proteins, polysaccharides, lipopolysaccharides, lipids and carotenoids were found as constituents of the cell wall. Polypeptide and lipid patterns of cell walls were completely different from that of the cytoplasmic/thylakoid membrane fraction. The purified cell walls contained about twelve outer membrane proteins. The two major polypeptides (M_r 67,000 and 61,000) were found to be associated with the peptidoglycan by ionic interactions.Myxoxanthophyll (major carotenoid), related carotenoid-glycosides and zeaxanthin were the predominating carotenoids of the cell wall of Synechocystis PCC 6714 over echinenone and -carotene. A polar unknown carotenoid was observed, the absorption spectrum of which resembled that of myxoxanthophyll. It was exclusively found in cell walls, but not in the cytoplasmic/thylakoid membrane fraction.Abbreviations Hep heptose - DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SL sulfolipid - PC phosphatidylcholin - PG phosphatidylglyceride Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (M_r 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.Abbreviations A₂pm diaminopimelic acid - ATCC American Type Culture Collection - CE cell envelope - CM cytoplasmic membrane - CW cell wall - dOcla 3-deoxy-d-manno-2-octulosonic acid - GalN galactosamine - GlcN glucosamine - GlcUA glucuronic acid - HF hydrofluoric acid - LPS lipopolysaccharide - ManN mannosamine - M relative molecular mass - MurN muramic acid - MurN-6-P muramic acid-6-phosphate - OMe O-methyl - PAGE polyacrylamide gel electrophoresis - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - SH sheath     

The purification and properties of a cd-cytochrome nitrite reductase from Paracoccus halodenitrificans

Paracoccus halodenitrificans, grown anaerobically in the presence of nitrite, contained membrane and cytoplasmic nitrite reductases. When assayed in the presence of phenazine methosulfate and ascorbate, the membranebound enzyme produced nitrous oxide whereas the cytoplasmic enzyme produced nitric oxide. When both enzymes were assayed in the presence of methyl viologen and dithionite, the cytoplasmic enzyme produced ammonia. Following solubilization, the membrane-bound enzyme behaved like the cytoplasmic enzyme, producing nitric oxide in the presence of phenazine methosulfate and ascorbate, and ammonia when assayed in the presence of methyl viologen and dithionite. The cytoplasmic and membranebound enzymes were purified to essentially the same specific activity. Only a single nitrite-reductase activity was detected on electrophoretic gels and the electrophoretic behavior of both enzymes suggested they were identical. The spectral properties of both enzymes suggested they were cd-type cytochromes. These data suggest that the products of nitrite reduction by the cd-cytochrome nitrite reductase are determined by the location of the enzyme and the redox potential of the electron donor.Abbreviations PMS phenazine methosulfate - MV methyl viologen - HEPES N-2-hydroxyethylpiperazine-N-2-ethane-sulfonic acid - CHAPSO [3-(3-cholamidopropyldimethylammonia)-1-(2-hydroxy-1-propanesulfonate)] National Research Council Research Fellow     

Vectorial electron transport in Degulfovibrio vulgaris (Marburg) growing on hydrogen plus sulfate as sole energy source

Desulfovibrio vulgaris (Marburg) was grown on hydrogen plus sulfate as sole energy source in a medium containing excess iron. The topography of electron transport components was investigated. The bacterium contained per mg cells (dry weight) 30U hydrogenase (1U=1 mol/min), 35 g desulfoviridin (= bisulfite reductase), 0.6 U adenosine phosphosulfate reductase, 30 mU thiosulfate reductase, 0.3 nmol cytochrome c ₃ (M _r=13,000), 0.04 nmol cytochrome b, 0.85 nmol menaquinone, and 0.4 nmol ferredoxin. Hydrogenase (>95%) and cytochrome c ₃ (82%) were localized on the periplasmic side and desulfoviridin (95%), adenosine phosphosulfate reductase (87%), thiosulfate reductase (74%), and ferredoxin (71%) on the cytoplasmic side of the cytoplasmic membrane; menaquinone and cytochrome b were exlusively found in the membrane fraction. The location of the oxidoreductases indicate that in D. vulgaris (Marburg) H₂ oxidation and sulfate reduction take place on opposite sides of the cytoplasmic membrane rather than on the same side, as has recently been proposed.     

Induction of nitrate reductase and membrane cytochromes in wild type and chlorate-resistant Paracoccus denitrificans

An experimental system has been devised for induction of nitrate reductase in suspensions of wild type Paracoccus denitrificans incubated with limited aeration in the presence of azide, nitrate or nitrite. Azide promoted maximum synthesis of enzyme, accompanied by formation of excess b-type cytochrome; the level of enzyme attained with nitrate was less and c-type cytochrome predominated in the membrane. The nitrate reductase was solubilized with deoxycholate from membranes of azide-induced cells and was identified as a major polypeptide M _r =150,000 by sodium dodecyl sulphate-polyacrylamide gel electrophoresis. Mutants strains lacking nitrate reductase activity were isolated on the basis of resistance to chlorate and mutant M-1 was examined in detail. When incubated in the cell suspension system M-1 formed a membrane protein M _r =150,000 similar to that attributed to nitrate reductase in the wild type. Maximum formation of the protein by M-1 occurred without inducer and it was accompanied by synthesis of excess b-type cytochrome. The observations with wild type and M-1 indicate that nitrate reductase protein and b-type cytochrome are coregulated and that the active enzyme has a role in regulating its own synthesis.Non-standard Abbreviations SDS sodium dodecyl sulphate - PAGE polyacrylamide gel electrophoresis - DOC sodlum deoxycholate     

Occurrence and biosynthesis of catalase at different stages of seed maturation

It was to be shown whether during the biogenesis of microbodies some of their components were already present in the cell prior to the organelle's assembly. To this end, the occurrence and properties of catalase in soluble and particular fractions of ripening cucumber seeds were examined. Homogenates of seeds from ripening fruits were fractionated by isopycnic density gradient centrifugation, and thus catalase was found in three different fractions: as a soluble enzyme in the gradient supernatant, as a membrane fraction at density d=1.18 kg l^-1, and in association with microbodies. In the early steps of seed formation, catalase was detected at density d=1.18 kg l^-1 and in the gradient supernatant. At a later stage of seed maturation, however, catalase was primarily associated with microbodies which exhibited an equilibrium density of d=1.23 kg l^-1. M _r as well as subunit M _r of catalase were determined, and their close immunological relationship to leaf peroxisomal catalase and glyoxysomal catalase was demonstrated. Biosynthesis of catalase at different stages of seed maturation was investigated by in vivo labeling with l-[³⁵S]methionine, l-[¹⁴C]leucine and -[³H]aminolaevulinic acid. Electrophoretic analysis of de novo synthesized catalase subunits revealed the occurrence of a heavy form (M _r 57,500) in the soluble fraction; this form was preferentially labeled. A light form, M _r 53,500, was detected in microbodies and also in the soluble fraction. The findings lend support to the hypothesis that the rate of catalase synthesis is highest in an early stage of seed formation, when globulins have already been formed, but before de novo synthesis of malate synthase has commenced. Prior to microbody assembling, a cytoplasmic pool of catalase was labeled.Abbreviations EDTA Na₂-ethylenediaminotetraacetate - Hepes 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid - M _r molecular weight     

Effect of iron and other nutrient limitations on the pattern of outer membrane proteins in the cyanobacterium Synechococcus PCC7942

When cells of Synechococcus PCC7942 were subjected to either iron or magnesium limitation, there was an appearance of specific proteins in the outer membrane (isolated as the cell wall fraction). Under iron limitation outer membrane polypeptides of M _r 92000, 48000–50000 and 35000 appeared. Specific iron-limited outer membrane proteins (IRMPs) of M _r 52000 and 36000 were also induced in iron-limited cultures of Synechocystis PCC6308. Under magnesium limitation polypeptides of M _r 80000, 67000, 62000, 50000, 28000 and 25000 appeared in the outer membrane. phosphate limitation caused minor changes in the outer membrane protein pattern, with polypeptides of M _r 32000 and one of over 100000 being induced, whereas calcium limitation had no apparent affect.Abbreviations EDDA ethylenediaminedihydroxyphenyl acetic acid - IRMP iron-regulated outer membrane protein - HEPES N-2-hydroxyethyl-piperazine-N-2-ethane sulphonic acid - SDS-PAGE sodium dodecyl sulphate polyacrylamide gel electrophoresis - PMSF phenylmethylsulphonyl fluoride     

The Nir1 locus in barley is tightly linked to the nitrite reductase apoprotein gene Nii

pBNiR1, a cDNA clone encoding part of the barley nitrite reductase apoprotein, was isolated from a barley (cv. Maris Mink) leaf cDNA library using the 1.85 kb insert of the maize nitrite reductase cDNA clone pCIB808 as a heterologous probe. The cDNA insert of pBNiR1 is 503 by in length. The nucleotide coding sequence could be aligned with the 3 end of other higher plant nitrite reductase apoprotein cDNA sequences but diverges in the 3 untranslated region. The whole-plant barley mutant STA3999, previously isolated from the cultivar Tweed, accumulates nitrite after nitrate treatment in the light, has very much lowered levels of nitrite reductase activity and lacks detectable nitrite reductase cross-reacting material due to a recessive mutation in a single nuclear gene which we have designated Nir1. STA3999 has the characteristics expected of a nitrite reductase apoprotein gene mutant. Here we have used pB-NiR1 in RFLP analysis to determine whether the mutation carried by STA3999 is linked to the nitrite reductase apoprotein gene locus Nii. An RFLP was identified between the wild-type barley cultivars Tweed (major hybridising band of 11.5 kb) and Golden Promise (major hybridising band of 7.5 kb) when DraI-digested DNA was probed with the insert from the partial barley nitrite reductase cDNA clone, pBNiR1. DraI-digested DNA from the mutant STA3999 also exhibited a major hybridising band of 11.5 kb after hybridisation with the insert from pBNiR1. F₁ progeny derived from the cross between the cultivar Golden Promise and the homozygous nir1 mutant STA3999 were heterozygous for these bands as anticipated. Co-segregation of the Tweed RFLP band of 11.5 kb and the mutant phenotype (leaf nitrite accumulation after nitrate treatment/loss of detectable nitrite reductase cross-reacting material at M_r 63000) was scored in an F₂ population of 312 plants derived from the cross between the cultivar Golden Promise and the homozygous mutant STA3999. The Tweed RFLP band of 11.5 kb and the mutant phenotype showed strict co-segregation (in approximately one quarter (84) of the 312 F₂ plants examined). Only those F₂ individuals heterozygous for the RFLP pattern gave rise to F₃ progeny which segregated for the mutant phenotype. We conclude that the nir1locus and the nitrite reductase apoprotein gene Nii are very tightly linked.     

Short communication: Purification and properties of a glucose-forming amylase of Lactobacillus brevis

An extracellular glucose-forming amylase was produced by Lactobacillus brevis isolated from Kagasok tea. The enzyme was purified 70-fold and had optimal activity at 55°C and pH 6.5. Its K _m value for starch was 0.27 mg ml^-1 and its M _r was approx. 75,900 Da. The activity of the enzyme was enhanced by Ca²⁺, Mg²⁺, Na⁺ or K⁺ and inhibited by EDTA, KCN, citric acid and l-cysteine.