bluuebunny/crossref_metadata_2025_split huggingface dataset:
Short‐Term Effect of Ammonium Chloride on Nitrogen Fixation by Azotobacter vinelandii and by Bacteroids of Rhizobium leguminosarum
Authors: LAANE Colja , KRONE Willy , KONINGS Wil , HAAKER Huub , VEEGER Cees 
Published: March 2005

Evidence is presented that the direct depressing effect of ammonium chloride on nitrogen fixation by Azotobacter vinelandii is due to inhibition of the electron transport system to nitrogenase. Furthermore, we were able to confirm the observation [Houwaard, F. (1979) Appl. Environ. Microbiol. in the press] that ammonium chloride has no short‐term effect on nitrogen fixation by isolated bacteroids of Rhizobium leguminosarum.
By means of the flow dialysis technique it could be demonstrated that in A. vinelandii ammonium is taken up as a cation in response to the ΔΨ and that uptake of ammonium specifically inhibits the flow of reducing equivalents to nitrogenase by lowering the ΔΨ across the cytoplasmic membrane. In A. vinelandii, like in bacteroids, the generation of reducing equivalents at a potential low enough to reduce nitrogenase was found to be extremely sensitive towards changes in ΔΨ. At ΔΨ values less than 80 mV, interior negative, no such reducing equivalents are generated, while at a ΔΨ value of 110 mV nitrogenase is supplied optimally with reducing equivalents. The nature of the ammonium transport system in A. vinelandii and its significance as a regulator for the rapid ‘switch off/switch on’ of nitrogenase activity is discussed.
Bacteroids of R. leguminosarum did not accumulate ammonium and no effect of ammonium on ΔΨ was observed. On the contrary, it could be demonstrated that bacteroids excrete ammonium in response to the ΔpH.
Studies on the Intracellular Distribution of Sindbis Messenger RNA in Infected Chick‐Embryo Fibroblasts
Authors: BONATTI Stefano , CERASUOLO Alfonso , CANCEDDA Ranieri , BORGESE Nica , MELDOLESI Jacopo 
Published: March 2005

Four hours after infection with Sindbis virus, chick embryo fibroblasts were studied by electron microscopy and cell fractionation.Electron microscopy of infected and non‐infected cells revealed that infection produced a disaggregation of polyribosomes into monomers. Apart from this observation most cells appeared well preserved, and no degranulation of the rough endoplasmic reticulum was visible.Analysis of postnuclear supernatants by sucrose density gradients showed that no change in the relative proportions of free and membrane‐bound ribosomes was produced by infection. Approximately 30% of the ribosomes and 50% of the viral RNA were found to be associated with membranes. Of the membrane‐associated viral RNA, 70% was recovered as 26‐S RNA.Similar results were obtained with fibroblasts infected by the temperature‐sensitive Sindbis mutant ts2, which is defective in the co‐translational processing of the viral gene products at the non‐permissive temperature.Sucrose gradient analysis of membrane‐bound polyribosomes solubilized by detergent indicated that as much as 50% of the membrane‐associated viral 26‐S RNA is not integrated into polyribosomes and that most of the ribosomes are present as monomers or ribosomal subunits.Treatment with puromycin of living cells or of isolated membrane fractions under a variety of ionic conditions revealed that the viral RNA‐membrane linkage is insensitive to puromycin but sensitive to high concentrations of monovalent ions. The bulk of the membrane‐bound ribosomes were detached by high salt and recovered as ribosomal subunits on sucrose gradients.These results are consistent with the idea that in chick embryo fibroblasts infected with Sindbis virus only a small percentage of the ribosomes are engaged in protein synthesis, and that the Sindbis messenger RNA may attach to endoplasmic reticulum membranes through a ribosome‐independent, salt‐sensitive link.
A Simple Procedure for Covalent Immobilization of NADH in a Soluble and Enzymically Active Form
Authors: FULLER Carl W. , RUBIN Joyce Rachel , BRIGHT Harold J. 
Published: March 2005

Using a new and simplified technique for covalent immobilization of adenine nucleotides [Fuller, C. W. and Bright, H. J., J. Biol. Chem. 252, 6631 (1977)], we have prepared immobilized NADH using two water‐soluble polymers and tested these preparations for activity with several dehydrogenases. The first polymer used, having a weight‐average molecular weight of about 350000, is a copolymer of methacrylyl choline and the epoxide‐containing monomer 3‐[4‐(2,3‐epoxypropoxy)butoxy]‐2‐hydroxypropyl acrylate. Immobilization of NADH onto this copolymer was accomplished in three steps, namely, alkylation of NAD at N‐1, reduction of the nicotinamide moiety with dithionite and Dimroth rearrangement of the alkyl linkage from the N‐1 to the C‐6 amino position. The second copolymer tested was a copolymer of the same epoxide‐containing monomer and N‐methacrylyl‐2‐glucosamine. Using this copolymer, immobilization of NADH through the adenine C‐6 amino position was accomplished in a single step.Measurements of the steady‐state kinetics of five dehydrogenases at pH 7 and pH 9 showed that, on the average, V and Km values obtained with the immobilized NADH were, respectively, about one third and twice those found for the free coenzyme. In general, when compared with the free coenzyme, the immobilized NADH had greater relative enzymic reactivity at pH 9 than at pH 7.The simplicity of this method, the general enzymic reactivity, and the ability to be recycled enzymically suggest that this immobilized NADH may be useful in creating enzyme reactors for synthetic, analytical and other purposes.
Studies on Interactions between Immobilized Lysine Residues and Oligomers of Thymidylic and Deoxyadenylic Acids
Authors: SCHOTT Herbert , ECKSTEIN Heiner 
Published: March 2005

Two groups of crosslinked polyacrylic gels with immobilized lysine and lysine peptides (Lys)5 and (Lys)5‐Pro have been used as models for the chromatographic investigation of lysine‐peptide–oligonucleotide interactions. One group carries carboxylic groups in addition to the peptide residues in the gel matrix; the other gel type contains no such carboxylic groups in the gel matrix.Nucleotides of the series (dT)2–5, p(dT)1–4, p(dT)1–4p, (dA)2–5, p(dA)1–5 and p(dA)1–4p were chromatographed on these gels under various conditions in an aqueous buffer. On the gels of the first group the nucleotides were retarded only slighly, the positive charges of the ɛ‐amino groups being compensated partially or totally by the negatively charged carboxyl groups of the polymer matrix. On the gels of the second group, however, the oligonucleotides underwent specific interactions. These interactions were based primarily upon electrostatic forces between the positively charged ɛ‐amino groups of the immobilized peptides and the negatively charged phosphate groups of the oligonucleotides.Our results indicate that, in addition to the electrostatic interaction, the conformation plays a crucial role. We explain the selectivity of the interaction with a conformation‐fit mechanism. The origin of this mechanism, which creates specific interactions from unspecific forces, is discussed.
Phosphoenolpyruvate Carboxykinase in Cultured Foetal Hepatocytes from the Rat
Authors: STEELE John G. , MCGRATH Malcolm C. , YEOH George C. T. , OLIVER Ivan T. 
Published: March 2005

The involvement of glucocorticoids and insulin in the induction of cytosolic Ppyruvate carboxykinase was studied in cultured hepatocytes derived from 19‐day foetal rat liver. Dexamethasone did not increase Ppyruvate carboxykinase activity, but had a synergistic effect on the induction of the enzyme by N6,O2′‐dibutyryladenosine 3′,5′‐monophosphate (Bt2cAMP). Insulin partially inhibited the induction of Ppyruvate carboxykinase by glucagon, epinephrine or Bt2cAMP. The inhibitory effect of insulin was dose‐dependent: significant inhibition was obtained with 0.1 nM insulin. However, the effect of insulin was independent of the dose of glucagon or epinephrine added to the culture medium.The ontogeny of inducibility of Ppyruvate carboxykinase by Bt2cAMP or hormones was tested in foetal hepatocytes after one day of culture. Ppyruvate carboxykinase activity could be induced by epinephrine, glucagon or Bt2cAMP in hepatocytes isolated from day‐19, day‐16 or day‐14 foetuses. Similarly, hepatocytes isolated from day‐12 foetal liver were competent to respond to the hormones and Bt2AMP by the accumulation of Ppyruvate carboxykinase. Binding proteins for adenosine 3′,5′‐monophosphate (cyclic AMP) were demonstrated in the cytosol from hepatocytes isolated from day‐14 and day‐19 foetal liver.The results are discussed in terms of the acquisition during hepatocyte differentiation of the competence to respond to hormonal signals, and the role of hormones in the induction of hepatic Ppyruvate carboxykinase at birth.
Glutathione Metabolism in Relation to the Amino‐Acid Permeation Systems of the Yeast Saccharomyces cerevisiae
Authors: PENNINCKX Michel , JASPERS Charles , WIAME Jean‐Marie 
Published: March 2005

A careful enzyme specificity analysis has revealed the presence of a typical γ‐glutamyltranspeptidase in the yeast Saccharomyces cerevisiae. The enzyme cellular level is low in the presence of NH+4 as a sole nitrogen source and rises when individual amino acids are used as nitrogen sources. The γ‐glutamyltranspeptidase appears to be repressed by NH+4 and escapes to the regulatory circuits under the control of glutamine and the glutamate‐dehydrogenase · NH+4 complex. The transpeptidase cellular level is unaffected in mutants which have lost the general amino acid permease and specific systems for l‐arginine and l‐lysine. In contrast, a low enzyme level is observed when growing an apf mutant on urea; this mutant is most probably affected in a common element shared by all the amino acid permeation systems. Urea appears to be a nitrogen source which promotes a high transpeptidase level in the wild‐type strain.The reported data are discussed in the light of the current theories about the intervention of glutathione metabolism in the translocation of amino acids.
Subcytoplasmic Distribution of Thyroglobulin mRNA in Normal Sheep Thyroid
Authors: CHABAUD Odile , CHEBATH Judith , JACQUET Michel 
Published: March 2005

The thyroglobulin 33‐S mRNA was isolated from sheep thyroid total polysomes. The 33‐S RNA, twice purified on a 1% sodium dodecylsulfate/sucrose gradient, was 30‐fold enriched in thyroglobulin messenger activity and was estimated as 50% pure by its messenger activity and 80% pure by the electrophoretic profile. It was used as template for complementary DNA synthesis and hybridized up to 85% of the DNA copy with pseudo‐first‐order kinetics. Back‐hybridization kinetics showed that the purified mRNA corresponds to a major kinetic component with a base sequence complexity of 10000 nucleotides as determined by comparison to globin mRNA. Cross‐reactivity of [3H]cDNA with liver RNA is less than 10%. Restriction endonuclease digestion of [3H]cDNA yielded a discrete band pattern.The distribution of thyroglobulin mRNA among free polysomes, membrane‐bound polysomes and extrapolysomal pools was analyzed using hybridization to the specific [3H]cDNA probe. Free particles were recovered in the supernatant and membrane‐bound particles in the pellet after a brief centrifugation of detergent‐free homogenate (5 min at 27000 ×g: procedure A; 12 min at 130000 ×g: procedure B) with precautions taken to avoid cross‐contamination. Using procedure A, 80% of thyroglobulin mRNA sequences were found in the membrane‐bound fraction. Using procedure B, where contamination of free particles by membrane‐bound particles was avoided by high‐speed initial centrifugation and further isolation through a discontinuous sucrose gradient, 95–98% of thyroglobulin mRNA sequences were recovered in membrane‐bound polysomes. In total polysomes, 89% of thyroglobulin mRNA sequences were in the polysomal area and shifted to ribosomal subunits after EDTA treatment.
The Peroxidatic Reaction Catalyzed by Horse Liver Alcohol Dehydrogenase
Authors: FAVILLA Roberto , MAZZINI Alberto , FAVA Adriano , CAVATORTA Paolo 
Published: March 2005

The main results obtained during the investigation of the peroxidatic reaction between NAD+ and H2O2, catalyzed by horse liver alcohol dehydrogenase, are here presented.

As previously reported [Favilla, R. & Cavatorta, P. (1975) FEBS Lett. 50, 324–329], the reaction can be followed spectrophotometrically around 300 nm. It consists of a biphasic pattern: the faster step reflects the enzyme‐catalyzed formation of a NAD+ derivative, here called ‘compound I’ (ɛ300= 15000 M−1 cm−1), from the reagents NAD+ and H2O2. The slower step is enzyme independent and consists of at least two distinct processes, each of them leading independently to the disappearance of the intermediate compound I. These two processes are competitive and conditions can be selected such that either one or the other occurs preferentially: one of them leads to the formation of NADX (ɛ300= 23000 M−1 cm−1), the final stable product of the overall reaction, which can be recovered quantitatively only if catalase is added soon after the end of the faster enzymatic step. The second process of the slow step is due to further reaction of compound I with excess H2O2 and the final product Y can be recovered almost quantitatively only if catalase is not added after the fast step.
A careful re‐examination of the stoichiometry of the reaction unequivocally indicated that one molecule of NAD+ is converted to compound I per molecule of H2O2 reacted at the enzyme active site during the fast step.
One proton has been found to be released per mole of NAD+ converted to compound I during the enzymatic fast step. This very significant observation allowed us to deduce the complete stoichiometric balance of the overall reaction.
An initial activation of the peroxidatic activity of horse liver alcohol dehydrogenase, followed by a slow progressive decrease of the same activity, was observed when the enzyme was preincubated with 1 mM EDTA. This behaviour has been interpreted as being due to the essential role played by the active‐site zinc ion in catalysing the peroxidatic reaction.
The peroxidatic reaction has been found to be catalyzed also by yeast alcohol dehydrogenase, thus suggesting a possible physiological meaning to this general ability of alcohol dehydrogenases.
Interpretation of the Mössbauer Spectra of the High‐Potential Iron Protein from Chromatium
Authors: MIDDLETON Peter , DICKSON Dominic P. E. , JOHNSON Charles E. , RUSH James D. 
Published: March 2005

The Mössbauer spectra of both reduced and oxidized high‐potential iron protein (Hipip) from Chromatium have been analysed using computer fits to theoretical spectra derived from a spin Hamiltonian. Fits to spectra obtained over a range of temperatures between 4.2 and 195 K and in applied magnetic fields up to 10.0 T lead to a consistent set of hyperfine parameters.These results are interpreted in terms of a model of the four‐iron four‐sulphide active centre which is consistent with its electronic and magnetic properties in both redox states. In the model for the reduced centre all four iron atoms have essentially the same valence, intermediate between ferric and ferrous, with the spins being coupled antiferromagnetically to give the centre zero net spin. The oxidized centre has one less electron which at low temperatures appears to have come predominantly from one pair of iron atoms which thus become ferric with the other pair remaining substantially unchanged. It is clear from the Mössbauer hyperfine parameters obtained from the computer fits to the low‐temperature spectra that a larger magnetic moment is associated with the ferric/ferrous pair of iron atoms than with the ferric pair of iron atoms. This also explains the g values with an average of greater than 2 which are observed in electron paramagnetic resonance (EPR) measurements. At higher temperatures the differences between the electron charge density at the different iron atoms in the oxidized centre appear to become smeared out.
Polystyrene‐Adsorbed Gangliosides for Investigation of the Structure of the Tetanus‐Toxin Receptor
Authors: HOLMGREN Jan , ELWING Hans , FREDMAN Pam , SVENNERHOLM Lars 
Published: March 2005

This paper describes the elaboration, using cholera toxin and GM ganglioside as a model system, of versatile, sensitive methods allowing simple determinations of the specific binding of protein ligands to polystyrene‐adsorbed gangliosides, and, secondly, the application of these methods to the tentative identification of the receptor structure for tetanus toxin.The adsorption characteristics and the ligand‐binding properties of the adsorbed gangliosides were determined, and the specificity and sensitivity of the various ligand‐detection methods (radio‐isotope; enzyme‐linked antibody or water‐vapour condensation) defined. The adsorption did not vary with the type of ganglioside and was found to be linearly proportional to the square root of the incubation concentration and to the log of the incubation time. No difference in adsorption was seen between temperatures of 4 to 37 deg;C. As analyzed with cholera toxin, the polystyrene‐adsorbed ganglioside was structurally specific in binding; i.e. only GM1 was active while after hydrolysis by Vibrio cholerae sialidase also GD1a, GD1b and GT1b, for example, had the same activity as GM1. The affinity of cholera toxin for polystyrene‐adsorbed GM1 was similar to that for viable cells (Ka= 1.2 nM−1).Tetanus toxin was found to bind specifically to gangliosides of the G1b series (GD1b, GT1b and GQ1b) and though with less affinity, also to GT1a, GM1, GD1a and GD3. The binding affinity of tetanus toxin for the G1b gangliosides was less than that of cholera toxin for GM1. The studies showed that tetanus toxin has specific binding affinity for tetraose‐gangliosides with a disialosyl group linked to the inner galactose. It is conceivable that the natural binding structure for the tetanus toxin has the end sequence Galβ1 → 3Ga1NAcβ1 → 4Ga1(2← 8αNeuAc2 ← 3αNeuAc) which occurs in the ganglioside G1b series and possibly also in the oligosaccharide chains of certain cell membrane glycoproteins.
Transmembrane Orientation of Proteins Present in Acetylcholine Receptor‐Rich Membranes from Torpedo marmorata Studied by Selective Proteolysis
Authors: WENNOGLE Lawrence P. , CHANGEUX Jean‐Pierre 
Published: March 2005

The transmembrane orientation of the polypeptide chains present in acetylcholine receptor‐rich membranes from Torpedo marmorata was studied by selective proteolysis. These membranes contain two major polypeptides of Mr 40000 and 43000 and two minor ones of Mr 50000 and 66000. With intact membranes all four proteins were found remarkably resistant to the action of the proteolytic enzymes pronase and trypsin. In contrast, when membranes were opened by sonication in the presence of trypsin, all four proteins were degraded. Stable degradation products of Mr 47000, 38000, 35000 and 32000 were found. Addition of protease after sonication was not effective; sonication alone did not remove the four major proteins from the membrane. It is concluded that: (a) these four chains were cut at sites normally exposed to the inside surface of the membrane and (b) the membrane fragments were sealed in vitro with the same orientation as in vivoVarious treatments were found to render the intact receptor‐rich membranes susceptible to proteolytic attack: incubation at pH 4 with the protease pepsin led to the selective degradation of the 40000‐Mr, 50000‐Mr and 66000‐Mr chains; the same results were obtained when the membranes were heated at 62°C for 1 min and then digested with trypsin. The 43000‐Mr chain remained resistant in both cases. No degradation fragments with Mr greater than 20000 were found. Furthermore, when these membranes were sonicated in the presence of pepsin, the 43000‐Mr polypeptide was degraded. It is concluded that (a) the 43000‐Mr polypeptide is entirely enclosed by the lipid bilayer, and (b) that the 40000‐Mr, 50000‐Mr and 66000‐Mr polypeptides are transmembrane proteins.Trypsin digestion of the receptor‐rich membranes opened by phospholipase A2 (crotoxin) or alkaline treatment, or solubilization by 1% Triton X‐100 gave the same series of degradation products as the sonicated membranes. The 47000‐Mr fragment was shown to be derived from the 50000‐Mr chain; and the 35000‐Mr and 32000‐Mr fragments, which were labelled by the affinity reagent 4(N‐maleimido)‐phenyl‐[3H]trimethylammonium, were derived from the 40000‐Mr chain. After dodecylsulfate/polyacrylamide gel electrophoresis, fragments of Mr 47000, 38000, 35000 and 32000 were labelled by 25I‐concanavalin A, like the native 40000‐Mr, 50000‐Mr and 66000‐Mr chains. The NH2‐terminal sequence of the 35000‐Mr fragment was identical to that of the 40000‐Mr chain.
Presence of Cyclic‐AMP‐Independent Protein Kinase Activity in RNA‐Binding Proteins of Embryonic Chicken Muscle
Authors: BAG Jnanankur , SELLS Bruce H. 
Published: March 2005

A class of protein(s) with high affinity for RNA was isolated by affinity chromatography on poly(U)‐Sepharose from an extract of embryonic chicken muscle which had been freed of ribosomes and cytoplasmic messenger ribonucleoprotein complexes. The RNA‐binding protein comprised about 1% of the total protein of the post‐ribosomal extract. Analysis of the polypeptide composition of the RNA‐binding proteins by two‐dimensional gel electrophoresis, using separation according to charge in the first dimension and molecular weight in the second, showed the presence of at least 37 polypeptides. These polypeptides were non‐ribosomal in nature. The major class of polypeptides falls into three molecular weight groups with Mr of 34000–41000, 46000–54000 and 68000–78000. Nine of these polypeptides showed similar electrophoretic migration properties to that of polypeptides from nonpolysomal cytoplasmic messenger ribonucleoprotein. Furthermore, four polypeptides, including one acidic 38000‐Mr polypeptide, displayed electrophoretic migration properties similar to those of polypeptides present in crude initiation factor preparation.The RNA‐binding proteins can form a ribonucleoprotein complex(es) when incubated with either synthetic or natural RNAs. Furthermore, in competition binding studies, mRNAs were found to be more efficient in complexing with the RNA‐binding proteins than were non‐mRNA molecules.A cyclic‐AMP‐independent protein kinase activity was detected in the RNA‐binding proteins. This protein kinase phosphorylates several polypeptides of the RNA‐binding proteins. Among these, the 38000‐Mr acidic polypeptide was the best acceptor of phosphate group(s). In contrast, ribosomal proteins were found to be poor substrates for the protein kinase. The acidic 38000‐Mr polypeptide of RNA‐binding protein, which is most intensely phosphorylated by the endogenous protein kinase, also appeared to be the common polypeptide found in the crude initiation factor preparation. The similarity of this protein kinase with the cyclic‐AMP‐independent protein kinase activity, previously reported in the nonpolysomal cytoplasmic messenger ribonucleoprotein complexes of embryonic chicken muscle [Bag, J. and Sells, B. H. (1979) J. Biol. Chem. 254, 3137–3140] has been discussed.These RNA‐binding proteins interfered with translation of both homologous and heterologous mRNAs in the rabbit reticulocyte cell‐free system. The effects obtained, however, were concentration‐dependent in the range 7–30 μg/ml: the proteins inhibited polypeptide formation while at higher concentrations (70 μg/ml) they stimulated translation. The presence of both inhibitor and activator of mRNA translation in RNA‐binding protein has been discussed.