Inhibitor effects on the accumulation and efflux of nickel ions in plasmid pMOL28-harboring strains of Alcaligenes eutrophus

The accumulation and efflux of ⁶³Ni²⁺ ions were studied using two strains of the strictly respiratory bacterium Alcaligenes eutrophus, the wild type strain N9A and its transconjugant N9A-M243 which harbors plasmid pMOL28.1 encoding constitutive resistance to nickel. When 1 M ⁶³Ni²⁺ is added to respiring cells, strain N9A accumulates high, but M243 only negligibly small amounts of nickel. When the cells were preincubated for about 20 h under anoxic conditions and were then exposed to 1 M ⁶³Ni²⁺ anaerobically, both strains accumulated approximately the same amounts of nickel. Aeration of these preloaded cells resulted in rapid efflux by strain M243 but renewed uptake of nickel by N9A. ⁶³Ni²⁺ uptake and efflux are highly sensitive to protonophores such as CCCP, FCCP and TCS but insensitive to DCCD (each at 20 M concentrations). Measurements on the effects of the inhibitors on biosynthetic processes requiring ATP either from substrate phosphorylation or from electron transport phosphorylation made sure that at the concentrations used the inhibitor effects were specific. Thus the results suggest that in the nickelresistant strain M243 under normal aerobic conditions two constitutive energy-dependent nickel transport systems can function concomitantly, a chromosomally-determined specific uptake system and a plasmid-mediated specific nickel efflux system.     

Ganglioside-cholera toxin interactions: a binding and lipid monolayer study

Summary On t.l.c. plates ¹²⁵I-cholera toxin binds to a disialoganglioside tentatively identified as GD_lb with about 10 times less capacity than to ganglioside GM₁. Binding of labeled toxin to both gangliosides was abolished in presence of excess amounts of unlabeled B subunit. Ganglioside extracts from human or pig intestinal mucosa showed toxin binding to gangliosides GM₁ and GD_1b. In ganglioside-containing lipid monolayers the penetration of the toxin was independent of the ganglioside binding capacity.Abbreviations GM₂ Gal-NAc14Gal(3-2NeuAc)14G1c1Cer - GM₁ Gal3Ga1-NAc14Gal(32NeuAc)14G1c11Cer - GD_1a NeuAc23Ga113Gal-NAc14Gal(32NeuAc)14G1c11Cer - GD1b Gall3Gal-NAcl4Gal(32NeuAc82NeuAc)14Glc11Cer - GT_1b NeuAc23Ga113Ga1-NAcal4Gal(3-2NeuAc82NeuAc)14G1c11Cer - dpPC 1,2-hexadecanoyl-sn-glycero-3-phosphocholine - dpPE 1,2-hexadecanoyl-sn-glycero-3-phosphoethanolamine     

Effect of microrelief and vegetation on methane emission from wet polygonal tundra, Lena Delta, Northern Siberia

The effect of microrelief and vegetation on methane (CH₄) emission was investigated in a wet polygonal tundra of the Lena Delta, Northern Siberia (72.37N, 126.47E). Total and plant-mediated CH₄ fluxes were measured by closed-chamber techniques at two typical sites within a low-centred polygon. During the study period, total CH₄ flux averaged 28.0±5.4mgm^–2d^–1 in the depressed polygon centre and only 4.3±0.8mgm^–2d^–1 at the elevated polygon rim. This substantial small-scale spatial variability of CH₄ emission was caused by strong differences of hydrologic conditions within the microrelief of the polygon, which affected aeration status and organic matter content of the soils as well as the vegetation cover. Beside water table position, the vegetation cover was a major factor controlling CH₄ emission from polygonal tundra. It was shown that the dominant vascular plant of the study area, Carex aquatilis, possesses large aerenchyma, which serve as pathways for substantial plant-mediated CH₄ transport. The importance of plant-mediated CH₄ flux was strongly influenced by the position of the water table relative to the main root horizon. Plant-mediated CH₄ transport accounted for about two-thirds of the total flux in the wet polygon centre and for less than one-third of the total flux at the moist polygon rim. A clipping experiment and microscopic-anatomical studies suggested that plant-mediated CH₄ transport via C. aquatilis plants is driven only by diffusion and is limited by the high diffusion resistance of the dense root exodermes.     

Valine inhibition of β-isopropylmalate dehydrogenase takes part in the regulation of leucine biosynthesis inCandida maltosa

The -isopropylmalate (IPM) dehydrogenase (EC 1.1.1.85) ofCandida maltosa, the third pathway-specific enzyme of leucine biosynthesis, was purified, some properties of the enzyme were studied and a novel regulatory pattern was found. The K_m values of the enzyme were estimated to be 0.42 mM for -IPM and 0.34 mM for NAD⁺. It is demonstrated that the enzyme can be regulated by L-valine. The inhibition was competitive with respect to -IPM (K_i=1.84 mM) and non-competitive with respect to NAD⁺ (K_i=5.67 mM). Exogenous addition of L-valine toC. maltosa cells increased the intracellular pool of some intermediates of leucine biosynthesis (-ketoisovalerate, -IPM, -IPM), but has hardly influence on the leucine pool.     

Purification and properties of the ATP: adenylylsulphate 3′-phosphotransferase from Chlamydomonas reinhardii

APS-kinase (ATP: adenylylsulphate 3-phosphotransferase, EC 2.7.1.25) has been purified from the alga Chlamydomonas reinhardii, strain CW 15 by means of chromatofocussing and affinity chromatography. The isolated protein showed an apparent molecular mass of 44,000 upon sodium dodecylsulphate polyacrylamide gel electrophoresis. The transfer of phosphate groups from ATP onto APS required a pH of 6.8, the presence of Mg²⁺ ions and a reducing thiol. Its catalytical activity was destroyed by sulphhydryl group inhibitors (phenyl-mercuri compounds, dithiopyridine) and alkylating reagents.The purified enzyme attained a V _max of 360 pkat under optimal reaction conditions declining to v _limit of 260 pkat in the presence of excess substrate APS. This sensitivity towards changes in substrate concentrations was parallelled by a high affinity and specificity: apparent K _m APS: 2 · 10^-6 mol · l^-1, and K _m ATP: 7 · 10^-6 mol · l^-1. The enzyme was found specific for ATP, d-ATP and CTP, while UTP, ITP and GTP showed marginal activity. The Hill coefficients suggested 4 binding sites for APS and 1 for ATP. Excessive APS resulted in a negative slope indicating 3 inhibiting sites of the substrate.Abbreviations APS Adenosine 5-phosphosulphate - dATP 2-deoxyadenosine 5-triphosphate - p-CMB p-chloromercuribenzoate - DTE dithioerythritol - DTT dithiothreitol - -MSH -mercaptoethanol - PAPS 3-phosphoadenosine 5-phosphosulphate - PAP 3-phosphoadenosine 5-phosphate - SDS sodium dodecyl sulphate This work is part of a dissertation submitted by H. G. J., Bochum 1982     

Urease from Staphylococcus saprophyticus: purification,characterization and comparison to Staphylococcus xylosus urease

Urease from Staphylococcus saprophyticus was purified more than 800-fold by liquid chromatography reaching homogeneity, as shown by isoelectric focussing, at a maximum specific activity of 1979 U/mg. The molecular weight of the native enzyme was 420000; it consisted of subunits with molecular weights of 72400 (), 20400 (), and 13900 () in an estimated ()₄ stoichiometry. In native gradient polyacrylamide gel electrophoresis urease exhibited a multiple activity band pattern with molecular weights ranging from 420000 to 100000. In the native enzyme, 4.09 (±0.25) atoms of nickel per molecule were detected. The N-terminal amino acids of the urease subunits were identical to those from Staphylococcus xylosus, and amino acid analysis revealed high similarities in both enzymes; no cysteine was detected after acid hydrolysis of vinylpyridinylated urease. Electron micrographs of negatively stained urease specimens from both staphylococci showed identical size and structure.Abbreviation PAGE polyacrylamide gel electrophoresis     

Changes of cytoplasmic free Ca2+ in the green alga Mougeotia scalaris as monitored with indo-1, and their effect on the velocity of chloroplast movements

The fluorescent calcium-sensitive dye 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,N-tetraacetic acid (indo-1) was loaded by a transplasmalemma pH gradient into filamentous cells and protoplasts of Mougeotia scalaris, such that most of the indo-1 fluorescence originated from the cytoplasm. Incubation of M. scalaris filaments in ethylene glycol-bis(-aminoethyl ether)-N,N,N,N-tetraacetic acid (EGTA)-buffered media (-log [Ca²⁺] (=pCa) 8 versus pCa 3) caused a consistent and significant decrease in the cytoplasmic free [Ca²⁺]. Pulses of the fluorescence excitation light (UV-A 365 nm, 0.7 s) caused an increase in cytoplasmic free [Ca²⁺] in M. scalaris that was nearly independent of the external [Ca²⁺] and of chloroplast dislocation by centrifugation. This calcium flux, highest in UV-A light, compared with blue or red light, probably resulted from a release of Ca²⁺ from intracellular stores. Increased cytoplasmic [Ca²⁺] may affect the velocity of chloroplast rotation since UV-A-light-mediated chloroplast movement was faster than in blue or red light. Consistently, the calcium ionophore A23187 and the calcium-channel agonist Bay-K8644 both increased the velocity of the red-light-mediated chloroplast rotation. Based on these and other observations, a Ca²⁺-induced decrease in cytoplasmic viscosity in Mougeotia is presumed to occur.Abbreviations EGTA ethylene glycol-bis-(-aminoethyl ether)N,N,N,N-tetraacetic acid - indo-1 1-[2-amino-5-(6-carboxyindol-2-yl)-phenoxy]-2-(2-amino-5-methylphenoxy)-ethane-N,N,N,Ntetraacetic acid - pCa log [Ca²⁺] - P_fr far-red-absorbing form of phytochrome - P_r red-absorbing form of phytochrome - x_G geometric mean Dedicated to Professor Wolfgang Haupt on the occasion of his 70th birthdayThis paper is part of the Ph.D. thesis of U. Russ at the Justus-Liebig-Universitat Giessen (FRG). Part of this work has been presented at a meeting on Calcium and intracellular signalling in plants in Plymouth, UK, Dec. 1990We are indebted to Dr. G. Seibold and Dipl. Phys. H. Weintraut for their advice on the technique of microspectrofluorometry and for allowing access to the microspectrophotometric facilities in the Strahlenzentrum der Justus-Liebig-Universität, Giessen, FRG. We thank Mrs. A. Quanz for reliable culture of the algae and evaluation of the videotapes. Bay-K8644 was a generous gift of Bayer AG, Wuppertal, FRG. U. russ was supported by a scholarship according to the Hessisches Graduierten Förderungsgesetz. This work was supported by the Deutsche Forschungsgemeinschaft.     

The electrochemical H+ gradient in the yeastRhodotorula glutinis

The electrochemical gradient of protons, , was estimated in the obligatory aerobic yeastRhodotorula glutinis in the pH₀ range from 3 to 8.5. The membrane potential, , was measured by steady-state distribution of the hydrophobic ions, tetraphenylphosphonium (TPP⁺) for negative above pH₀ 4.5, and thiocyanate (SCN^–) for positive below pH₀ 4.5. The chemical gradient of H⁺ was determined by measuring the chemical shift of intracellular P_i by³¹P-NMR at given pH₀ values. The values of pH_i increased almost linearly from 7.3 at pH₀ 3 to 7.8 at pH₀ 8.5. In the physiological pH₀ range from 3.5 to 6, was fairly constant at values between 17–18 KJ mol^–1, gradually decreasing at pH₀ above 6. In deenergized cells, the intracellular pH_i decreased to values as low as 6, regardless of whether the cell suspension was buffered at pH₀ 4.5 or 7.5. There was no membrane potential detectable in deenergized cells.     

Cell recycling studies for α-amylase production by Bacillus amyloliquefaciens

Production of -amylase by a strain of Bacillus amyloliquefaciens was investigated in a cell recycle bioreactor incorporating a membrane filtration module for cell separation. Experimental fermentation studies with the B. amyloliquefaciens strain WA-4 clearly showed that incorporating cell recycling increased -amylase yield and volumetric productivity as compared to conventional continuous fermentation. The effect of operating conditions on -amylase production was difficult to demonstrate experimentally due to the problems of keeping the permeate and bleed rates constant over an extended period of time. Computer simulations were therefore undertaken to support the experimental data, as well as to elucidate the dynamics of -amylase production in the cell recycle bioreactor as compared to conventional chemostat and batch fermentations. Taken together, the simulations and experiments clearly showed that low bleed rate (high recycling ratio) various a high level of -amylase activity. The simulated fermentations revealed that this was especially pronounced at high recycling ratios. Volumetric productivity was maximum at a dilution rate of around 0.4 h^–1 and a high recycling ratio. The latter had to exceed 0.75 before volumetric productivity was significantly greater than with conventional chemostat fermentation.List of Symbols a proportionality constant relating the specific growth rate to the logarithm of G (h) - a ₁ reaction order with respect to starch concentration - a ₂ reaction order with respect to glucose concentration - B bleed rate (h^–1) - C starch concentration (g/l) - C ₀ starch concentration in the feed (g/l) - D dilution rate (h^–1) - D _E volumetric productivity (KNU/(mlh)) - e intracellular -amylase concentration (g/g cell mass) - E extracellular -amylase concentration (KNU/ml) - F volumetric flow rate (l/h) - G average number of genome equivalents of DNA per cell - k _l intracellular equilibrium constant - k ₂ intracellular equilibrium constant - k _s Monod saturation constant (g/l) - k ₃ excretion rate constant (h^–1) - k _d first order decay constant (h^–1) - k _gl rate constant for glucose production - k _st rate constant for starch hydrolysis - k _t1 proportionality constant for -amylase production (gmRNA/g substrate) - k ₁ translation constant (g/(g mRNAh)) - KNU kilo Novo unit - m maintenance coefficient (g substrate/(g cell massh)) - n number of binding sites for the co-repressor on the cytoplasmic repressor - Q repression function K₁/K₂Q1.0 - R ratio of recycling - R _s rate of glucose production (g/lh) - r _c rate of starch hydrolysis (g/(lh)) - R _eX retention by the filter of the compounds X: starch or -amylase - r intracellular -amylase mRNA concentration (g/g cell mass) - r _C volumetric productivity of starch (g/lh) - r _E volumetric productivity of intracellular -amylase (KNU/(g cell massh)) - r _r volumetric productivity of intracellular mRNA (g/(g cell massh)) - r _e volumetric productivity of extracellular -amylase (KNU/(mlh)) - r _s volumetric productivity of glucose (g/(lh)) - r _X volumetric productivity of cell mass (g/(lh)) - S ₀ free reducing sugar concentration in the feed (g/l) - S extracellular concentration of reducing sugar (g/1) - t time (h) - V volume (l) - X cell mass concentration (g/l) - Y yield coefficient (g cell mass/g substrate) - Y _E/S yield coefficient (KNU -amylase/g substrate) - Y _E total amount of -amylase produced (KNU) - substrate uptake (g substrate/(g cell massh)) - specific growth rate of cell mass (h^–1) - _d specific death rate of cells (h^–1) - _m maximum specific growth rate of cell mass (h^–1) This study was supported by Bioprocess Engineering Programme of the Nordic Industrial Foundation and the Center for Process Biotechnology, the Technical University of Denmark.     

Interaction between photosynthetic and respiratory electron-transfer chains in the membranes of Anabaena variabilis

The rate of CO₂- and p-benzoquione-dependent photosynthetic O₂ evolution by Anabaena variabilis cells remained unaltered and the rate of O₂ uptake observed after switching off the light (endogenous respiration) was enhanced by a factor of 6–8 when the O₂ concentration was increased from 200 to 400 M. Photosystem-I-linked O₂ uptake and respiration of the cells incubated with ascorbate and N,N,NN-tetramethyl-p-phenylenediamine was not appreciable influenced by the O₂ concentration. 2-Iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether, blocking electron transfer at the plastoquinone level, suppressed O₂ evolution and had no influence on endogenous respiration. 2-n-Heptyl-4-hydroxyquinoline-N-oxide, an inhibitor of electron transfer between photosystems II and I, as well as the cytochrome-oxidase inhibitors N ₃ ^- , CN^- and NH₂OH, caused a 35–50% retardation of endogenous respiration and blocked photosynthetic O₂ evolution. The molar ratio of cytochromes b₆, f, c-553, aa₃ and photosystem-I reaction centers in the isolated membranes equalled approx. 2:1:2:0.7:2. It is inferred that endogenous respiration of A. variabilis cells is inhibited by the light-induced electron flow through both photosystems at the level of the plastoquinone-plastocyanin-oxidoreductase complex.Abbreviations DCMU 3-(3,4-dichlorophenyl)-1,1-dimethylurea - DNP-INT 2-iodo-6-isopropyl-3-methyl-2,4,4-trinitrodiphenyl ether - Hepes 4-(2-hydroxyethyl)-1-piperazine ethansulfonic acid - TMPD N,N,NN-tetramethyl-p-phenylenediamine     

Measurement of internal pH in the coccolithophoreEmiliania huxleyi using 2′,7′-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein acetoxymethylester and digital imaging microscopy

Internal pH (pH_i) was determined inEmiliania huxleyi (Lohmann) using the probe 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluoresceinacetoxymethylester (BCEF-AM) and digital imaging microscopy. The probe BCECF-AM was taken up and hydrolysed to the free acid by the cells. A linear relationship was established between pH_i and the 490/450 fluorescence ratio of BCECF-AM over the pH range 6.0 to 8.0 using the ionophore nigericin. Two distinct pH domains were identified within the cell, the cytoplasmic domain (approx. pH 7.0) and the chloroplast domain (approx. pH 8.0). The average pH_i was 7.29 (±0.11) for cells in the presence of 2 mM HCO ₃ ^– . In the absence of HCO ₃ ^– the pH_i was decreased by 0.8 pH unit. The importance of these changes in pH_i is considered in relation to inorganic-carbon uptake.Abbreviations AM acetoxymethylester - BCECF 2,7-bis-(2-carboxyethyl)-5(and-6)carboxyfluorescein - Hepes 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid - pH_i intracellular pH     

Salmonella typhimurium LT2 metF operator mutations

Summary Using an Escherichia coli lac deletion strain lysogenized with lambda phage carrying a metF-lacZ gene fusion (Flac), in which -galactosidase levels are dependent on metF gene expression, cis-acting mutations were isolated that affect regulation of the Salmonella typhimurium metF gene. The mutations were located in a region previously defined as the metF operator by its similarity to the E. coli metF operator sequence. Regulation of the metF gene was examined by measuring -galactosidase levels in E. coli strains lysogenized with the wild-type Flac phage and mutant Flac phage. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product, but not the vitamin B₁₂ control system mediated by the metH gene product. The results also demonstrate that negative control of the metF gene by the metH gene product and vitamin B₁₂ is dependent on a functional metJ gene product.Abbreviations Ap ampicillin - dNTP deoxyribonucleoside triphosphates - GM glucose minimal - Km kanamycin - L-agar Luria agar - LM lactose minimal - SAM s-adenosyl-L-methionine - TPEG phenylethyl -D-thiogalactoside - X-gal 5-bromo-4-chloro-3-indolyl -D-galactopyranoside - [] designates plasmid-carrier state - :: novel joint     

Energieabhängige 63Ni-Aufnahme bei Alcaligenes eutrophus Stamm H 1 und H 16

Zusammenfassung Die Kinetik der Aufnahme von ⁶³Ni wurde an zwei Stämmen von Alcaligenes eutrophus untersucht, die Nickelionen für das chemolithotrophe Wachstum benötigen. Mit Kohlendioxid als einziger Kohlenstoffquelle wird das Wachstum durch niedrige Konzentrationen von Nickel gefördert, wobei das Optimum der Wachstumsförderung bei 0,3 M Nickel lag. Höhere Nickelkonzentrationen wirkten hemmend. Das heterotrophe Wachstum mit Fructose wurde durch Nickelionen nicht gefördert. — Übertragen in Phosphatpuffer, der von Schwermetallionen befreit worden war, zeigten autotroph gewachsene Zellen eine rasche Aufnahme von ⁶³Ni, sofern Wasserstoff, Sauerstoff und Kohlendioxid zugegen waren. Dabei wurde Nickel innerhalb von 60 min aus dem umgebenden Medium bis zur 280 fachen Konzentration in den Zellen angehäuft. Die beobachtete Ni-Aufnahme zeigte ein Temperaturoptimum bei etwa 29° C und wurde durch Hemmstoffe wie Arsenit, Jodacetat, Methylenblau, Natriumazid und Natriumcyanid stark beeinträchtigt. Andere Schwermetallionen (Zn, Co, Mn und Cu) verminderten die Nickelaufnahme nur geringfügig. Durch ⁵⁸NiCl₂ und Toluol wurde der Efflux von ⁶³Ni aus den Zellen gefördert. Die Beobachtungen lassen darauf schließen, daß Nickelionen durch einen energieabhängigen Prozeß in chemolithotroph gewachsenen Zellen dieser Stämme angehäuft werden.

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Energy-dependent ⁶³Ni-uptake by Alcaligenes eutrophus strains H 1 and H 16

Kinetic studies of the uptake of ⁶³Ni were undertaken with two strains of Alcaligenes eutrophus, known to require nickel ions for chemolithotrophic growth. Using carbon dioxide as sole carbon source, growth is stimulated by low concentrations of nickel with optimum concentration for growth stimulation at about 0.3 M nickel. Higher nickel concentrations were inhibitory. Heterotrophic growth on fructose was not stimulated by nickel ions.-Upon transfer into phosphate buffer freed of heavy metal ions, autotrophically grown cells exhibited rapid uptake of ⁶³Ni which was dependent upon th presence of hydrogen, oxygen and carbon dioxide. Within 60 min nickel was accumulated from the medium, reaching 280-fold concentration in the cells. The observed uptake exhibited a temperature optimum at about 29° C and was markedly inhibited by metabolic inhibitors such as arsenite, iodoacetate, methylene-blue, sodium azide and sodium cyanide. Other heavy metal ions (Zn, Co, Mn and Cu) only slightly inhibited ⁶³Ni-uptake. The efflux of ⁶³Ni from the cells was stimulated by ⁵⁸NiCl₂ and by toluene. These data indicate that nickel ions are accumulated by an energy dependent mechanism in chemolithotrophically grown cells of these strains.

     

Increased erythritol production in Torula sp. by Mn2+and Cu2+

The production of erythritol and the erythritol yield from glucose by Torula sp. were improved, in increasing order, by supplementing with 10 mg MnSO₄4H₂O l^–1, 2 mg CuSO₄5H₂O l^–1, and both 10 mg MnSO₄4H₂O l^–1 and 2 mg CuSO₄5H₂O l^–1. Mn²⁺ decreased the intracellular concentration of erythritol, whereas Cu²⁺ increased the activity of erythrose reductase in cells. These results suggest that Mn²⁺ altered the permeability of cells, whereas Cu²⁺ increased the activity of erythrose reductase in cells.     

Ammonia inhibition of nitrogenase activity in purple and green bacteria

Ammonia reversibly inhibits the nitrogenase activity not only in purple nonsulfur bacteria but in purple (Thiocapsa roseopersicina) and green (Chlorobium limicola forma thiosulfatophilum) sulfur bacteria as well.The complete inhibition of nitrogenase activity (acetylene reduction) is observed about 30 s after addition of NH ₄ ⁺ (2.5×10^-6 M) to cell suspensions. The pattern of ammonia inhibition of acetylene reduction in T. roseopersicina does not differ from the action of tetrabutylammonium and tetraphenylphosphonium (3 · 10^-6-5·10^-5 M) on nitrogenase activity of this bacterium.Simultaneously with the switch-off effect of NH ₄ ⁺ a considerable increase of ATP in cells of Rhodobacter sphaeroides and C. limicola f. thiosulphatophilum was observed.     

High frequency transformation of Alcaligenes eutrophus producing poly-β-hydroxybutyric acid by electroporation

Summary A strain of Alcaligenes eutrophus producing poly--hydroxybutyric acid was successfully transformed by the electroporation. The plasmid used was a broad host range plasmid pKT230 conferring kanamycin resistance. The optimum yield of transformant was 0.8×10²/g DNA when 50 l competent cells at 10¹⁰/ml were pulsed by 11.5 kV/cm for 5 ms with 1 g DNA. Plasmid DNA in the A. eutrophus transformant was stably maintained as a monomeric structure.     

Partial purification and properties of guanosine triphosphate cyclohydrolase from Drosophila melanogaster

The first enzyme (named GTP cyclohydrolase) in the pathway for the biosynthesis of pteridines has been partially purified from extracts of late pupae and young adults of Drosophila melanogaster. This enzyme catalyzes the hydrolytic removal from GTP of carbon 8 as formate and the synthesis of 2-amino-4-hydroxy-6-(d-erythro-1,2,3-trihydroxypropyl)-7,8-dihydropteridine triphosphate (dihydroneopterin triphosphate). Some of the properties of the enzyme are as follows: it functions optimally at pH 7.8 and at 42 C; activity is unaffected by KCl and NaCl, but divalent cations (Mg²⁺, Mn²⁺, Zn²⁺, and Ca²⁺) are inhibitory; the K _m for GTP is 22 m; and the molecular weight is estimated at 345,000 from gel filtration experiments. Of a number of nucleotides tested, only GDP and dGTP were used to any extent as substrate in place of GTP, and these respective compounds were used only 1.8% and 1.5% as well as GTP.This work was supported by research grants from the National Institutes of Health (AM03442) and the National Science Foundation (GB33929).     

Substitutions of the Conserved Thr42 Increased the Roles of the ε-Subunit of Maize CF1 as CF1 Inhibitor and Proton Gate

The conserved residue Thr42 of -subunit of the chloroplast ATP synthase of maize (Zea mays L.) was substituted with Cys, Arg, and Ile, respectively, through site-directed mutagenesis. The over-expressed and refolded -proteins were purified by chromatography on DEAE-cellulose and FPLC on mono-Q column, which were as biologically active (inhibiting Ca²⁺-ATPase activity and blocking proton gate) as the native subunit isolated from chloroplasts. The T42C and T42R showed higher inhibitory activities on the soluble CF₁(–) Ca²⁺-ATPase than the WT. The T42I inhibited the Ca²⁺-ATPase activity of soluble CF₁ and restored photophosphorylation activity of membrane-bound CF₁ deficient in the most efficiently. Far-ultraviolet CD spectra showed that the portions of -helix and -sheet structures of the three mutants were somewhat different from WT. Thus the conserved residue Thr42 may be important for maintaining the structure and function of the -subunit and the basic functions of the -subunit as far as an inhibitor of Ca²⁺-ATPase and the proton gate are related.     

Isolation and characterization of a toxic intracellular protein from Vibrio parahaemolyticus

A toxic factor released from disrupted cells of Vibrio parahaemolyticus was partially purified by gel filtration after precipitation with (NH₄)₂SO₄ at 40% saturation. The factor, which was a thermostable protein of 63 kDa, lysed human erythrocytes at a concentration of 0.15 g ml^-1. Its LD₅₀ by intravenous injection into mice was 6.4 g. Fluid accumulated in suckling mice force-fed with the toxic material (1 to 25 g). Haemolytic activity, which occurred maximall at 37°C and pH 7.0 was enhanced by Ca²⁺, Cu²⁺ and Zn²⁺, each at 1 mm. Anti-toxic-factor serum agglutinated V. parahaemolyticus cells. The factor may play a role in the pathogenesis of V. parahaemolyticus infections and in the host's defence mechanisms against infection by the microorganism.     

Energetic basis of cadmium toxicity in Staphylococcus aureus

In washed cells of cadmium-sensitive Staphylococcus aureus 17810S oxidizing glutamate, initial Cd²⁺⁺⁺ influx via the Mn²⁺ porter down membrane potential () was fast due to involvement of energy generated by two proton pumps—the respiratory chain and the ATP synthetase complex working in the hydrolytic direction. Such an unusual energy drain for rapid initial Cd²⁺ influx is suggested to be due to a series of toxic events elicited by Cd²⁺ accumulation down generated via the redox proton pump: (i) strong inhibition of glutamate oxidation accompanied by a decrease of electrochemical proton gradient ( _H ⁺) formation via the respiratory chain, (ii) automatic reversal of ATP synthetase from biosynthetic to hydrolytic mode, which was monitored by a decrease of _H ⁺-dependent ATP synthesis, (iii) acceleration of the initial Cd²⁺ influx down generated the reversed ATP synthetase, the alternative proton pump hydrolyzing endogenous ATP. The primary, cadmium-sensitive targets in strain 17810S seem to be dithiols located in the cytoplasmic glutamate oxidizing system, prior to the membrane-embedded NADH oxidation system. Inhibition by Cd²⁺ of _H ⁺-dependent ATP synthesis and of pH gradient (pH)-linked [¹⁴C]glutamate transport is a secondary effect due to cadmium-mediated inhibition of _H ⁺ generation at the cytoplasmic level. In washed cells of cadmium-resistant S. aureus 17810R oxidizing glutamate, Cd²⁺ accumulation was prevented due to activity of the plasmid-coded Cd²⁺ efflux system. Consequently, _H ⁺-producing and -requiring processes were not affected by Cd²⁺.