bluuebunny/crossref_metadata_2025_split huggingface dataset:
Generation of High δɛ in Respiring Submitochondrial Particles by Steady‐State Accumulation of Oxidized N,N,N′,N′‐Tetramethyl‐p‐phenylenediamine
Authors: SAGI‐EISENBERG Ronit , GUTMAN Menachem 
Published: March 2005

Oxidation of N,N,N′,N′‐tetramethyl‐p‐phenylenediame [Ph(NMe2)2] plus ascorbate by inside out submitochondrial particles is associated with accumulation of the oxidized form [Ph(NMe2)2+] in the inner space of the vesicles. The concentration of [Ph(NMe2)2+]in, can exceed by 5–6‐fold the concentration of Ph(NMe2)2 added to the reaction mixture.The steady‐state concentration of [Ph(NMe2)2+]in, is a function of the rate of Ph(NMe2)2 influx driven by concentration gradient, the rate of Ph(NMe2)2+ oxidation, determined by its concentration, and Ph(NMe2)2+ efflux. The last rate is determined by Ph(NMe2)2+ concentration gradient and the membrane potential component (δɛ) of the proton‐motive force. Collapse of δɛ by permeant anions, ionophores or carbonylcyanide p‐trifluoromethoxyphenylhydrazone, builds up the internal concentration of [Ph(NMe2)2+]in till the concentration gradient can support an efflux identical to the rate of Ph(NMe2)2+ formation (equivalent to the rate of respiration). This relationship is analyzed according to Nernst‐Planck equation and δɛ calculated by this method amounts to 100–120 mV (positive inside). This value is corroborated by equilibrium dialysis. The measured membrane potential is built by the positive charge of Ph(NMe2)2+itself plus that of accumulated H+. Still, the contribution of Ph(NMe2)2+ is about half of the total electric force.At high Ph(NMe2)2 concentrations (0.5–5 mM), substantial electron flux through cytochrome oxidase bypasses cytochrome c and is carried by direct interaction of Ph(NMe2)2 with the terminal oxidase.
Binding of Semliki Forest Virus and Its Spike Glycoproteins to Cells
Authors: FRIES Erik , HELENIUS Ari 
Published: March 2005

We have studied the binding of the Semliki Forest virus and its isolated spike glycoproteins, in the form of water‐soluble octameric complexes, to various cells at 5°C. The number of viruses bound per cell increased strongly with increasing free concentrations of virus up to about 0.2 nM. At higher concentrations smaller increases in binding were observed but saturation was not achieved. The number of viruses bound at a given free concentration was widely different for different cells. For some cells the binding of the virus was maximal at pH 6.8 with little decrease at lower pH, for other cells it was maximal around pH 6.0.The spike protein complexes were used at 100 times higher molar concentrations than the virus. The binding increased strongly with increasing free concentrations up to about 50 nM and saturation was obtained at higher concentrations. Up to 1.3 × 106 spike protein complexes could be bound per cell but great variation could be seen between different cell types. For all cells maximal binding was found below pH 6.0.Together with earlier observations, our results suggest that the virus can bind to a cell by two different modes. Around neutral pH the virus binds to specific gycoproteins and at low pH un‐specifically to the lipids of the plasma membrane. The possible physiological roles of these two types of binding are discussed.
Nucleotide Sequence of Three Isoaccepting Lysine tRNAs from Rabbit Liver and SV40‐Transformed Mouse Fibroblasts
Authors: RABA Manfred , LIMBURG Klaus , BURGHAGEN Margaret , KATZE Jon R. , SIMSEK Mehmet , HECKMAN Joyce E. , RAJBHANDARY Uttam L. , GROSS Hans J. 
Published: March 2005

The lysine isoacceptor tRNAs differ in two aspects from the majority of the other mammalian tRNA species: they do not contain ribosylthymine (T) in loop IV, and a ‘new’ lysine tRNA, which is practically absent in non‐dividing tissue, appears at elevated levels in proliferating cells. We have therefore purified the three major isoaccepting lysine tRNAs from rabbit liver and the ‘new’ lysine tRNA isolated from SV40‐transformed mouse fibroblasts, and determined their nucleotide sequences. Our basic findings are as follows.

The three major lysine tRNAs (species 1, 2 and 3) from rabbit liver contain 2′‐O‐methyl‐ribosylthymine (Tm) in place of T. tRNA1Lys and tRNA2Lys differ only by a single base pair in the middle of the anticodon stem; the anticodon sequence C‐U‐U is followed by N‐threonyl‐adenosine (t6A). tRNA3Lys has the anticodon S‐U‐U and contains two highly modified thionucleosides, S (shown to be 2‐thio‐5‐carboxymethyl‐uridine methyl ester) and a further modified derivative of t6A (2‐methyl‐thio‐MN6‐threonyl‐adenosine) on the 3′ side of the anticodon. tRNA3Lys differs in 14 and 16 positions, respectively, from the other two isoacceptors.
Protein synthesis in vitro, using synthetic polynucleotides of defined sequence, showed that tRN2Lys with anticodon C‐U‐U recognized A‐A‐G only, whereas tRNA3Lys, which contains thionucleotides in and next to the anticodon, decodes both lysine codons A‐A‐G and A‐A‐A, but with a preference for A‐A‐A. In a globin‐mRNA‐translating cell‐free system from ascites cells, both lysine tRNAs donated lysine into globin. The rate and extent of lysine incorporation, however, was higher with tRNA2Lys than with tRNA3Lys, in agreement with the fact that α‐globin and β‐globin mRNAs contain more A‐A‐G than A‐A‐A codons for lysine.
A comparison of the nucleotide sequences of lysine tRNA species 1, 2 and 3 from rabbit liver, with that of the ‘new’ tRNA4Lys from transformed and rapidly dividing cells showed that this tRNA is not the product of a new gene or group of genes, but is an undermodified tRNA derived exclusively from tRNA2Lys. Of the two dihydrouridines present in tRNA2Lys, one is found as U in tRNA4Lys; the purine next to the anticodon is as yet unidentified but is known not to be t6A. In addition we have found U, T and Ψ besides Tm as the first nucleoside in loop IV.
Enzymic Synthesis of Lignin Precursors
Authors: WYRAMBIK Dorothea , GRISEBACH Hans 
Published: March 2005

Isoenzyme 2 of cinnamyl‐alcohol dehydrogenase from soybean suspension cultures was purified about 3800‐fold to apparent homogeneity by an improved purification procedure involving biospecific elution of the enzyme from a NADP+‐agarose column. On sodium dodecylsulfate gels the dehydrogenase showed only one protein band with Mr 40000 ± 500.The enzyme is strongly inhibited by thiol reagents. Various metal chelators as well as the non‐chelating 7,8‐benzoquinoline also inhibited enzyme activity. Inhibition by 10 mM 1,10‐phenanthroline could be partially reversed by addition of Zn2+. 1,10‐Phenanthroline and 7,8‐benzoquinoline are non‐competitive inhibitors with respect to NADP+. The presence of zinc in the dehydrogenase was proved by atomic absorption spectroscopy and by specific incorporation of 65Zn into the enzyme.In steady‐state kinetics inhibition patterns were obtained which are consistent with an ordered bi‐bi mechanism in which NADP(H) is the first substrate to bind and the last product released.The cinnamyl‐alcohol dehydrogenase belongs to the A‐specific dehydrogenases and removes the pro‐R hydrogen from coniferyl alcohol. The enzyme shows many similarities with alcohol dehydrogenases from horse and rat liver and from yeast.
Properties of the F0F1 ATPase Complex from Rhodospirillum rubrum Chromatophores, Solubilized by Triton X‐100
Authors: SCHNEIDER Erwin , MÜLLER Hans‐Werner , RITTINGHAUS Klaus , THIELE Volker , SCHWULÉRA Udo , DOSE Klaus 
Published: March 2005

A cold‐stable oligomycin‐sensitive F0F1 ATPase complex from chromatophores of Rhodospirillum rubrum FR 1 was solubilized by Triton X‐100 and purified by gel filtration.
The F0F1 complex is resolved by sodium dodecyl sulfate electrophoresis into 14 polypeptides with approximate molecular weights in the range of 58000–6800; five of these polypeptides are derived from the F1 moiety of the complex which carries the catalytic centers of the enzyme.
The purified F0F1 complex is homogeneous according to analytical ultracentrifugation and isoelectric focusing.
The molecular weight as determined by gel filtration is about 480000 ± 30000. so20,w is 1.45 ± 0.1 S and the pI is 5.4.
The amino acid composition of the F0F1 complex is compared with the data obtained for the F1 moiety of the enzyme.
Quantitative data on the sensitivity to N,N′‐dicyclohexyl‐carbodiimide as well as kinetic parameters, regarding substrate specificity and dependence of ATPase activity on divalent cations, are reported.
Monomer‐Micelle Transition of the Ganglioside GM1 and the Hydrolysis by Clostridium perfringens Neuraminidase
Authors: RAUVALA Heikki 
Published: March 2005

The action of Clostridium perfringens neuraminidase on the ganglioside GM1 tritiated in the ceramide moiety was studied. The rates of hydrolysis of the GM1 ganglioside were determined from radioactivity in the neutral glycolipid product, which was separated from the substrate on DEAE‐Sephadex columns.In order to study the physical state of the substrate in the conditions used in the neuraminidase treatment, the critical micelle concentrations of the GM1 ganglioside were determined using formation of the triiodide anion in aqueous iodine solution as an indicator. The critical micelle concentrations were also obtained by determining the non‐sedimenting radioactivity at different concentrations of the labeled ganglioside per total volume used in ultracentrifugation experiments. In addition, the concentrations of the monomeric ganglioside were concluded from the results of the ultracentrifugation studies.The increase in the reaction rate of the GM1 hydrolysis as the function of the substrate concentration was leveled off at 25–28 μM ganglioside. The abrupt change at this concentration is interpreted as reflecting the monomer‐micelle transition of the ganglioside in the conditions used (50 mM sodium acetate buffer, pH 4.6). The critical micelle concentration was 29 μM on the basis of the triiodide test, and ultracentrifugation revealed the critical micelle concentration 28 μM. The reaction velocity of the hydrolysis was decreased immediately above the critical micelle concentration, and became constant at higher concentrations of the ganglioside. A close correlation to these changes in the reaction rate is suggested to exist in the concentrations of the monomeric GM1 ganglioside.Saturation of the buffer used in the neuraminidase assays with butanol effected a striking change in the plot of reaction rate versus ganglioside concentration. The reaction rate increased up to 100–110 μM GM1 ganglioside. The shift of the inflexion point in the rate plot from 25–28 μM to 100–110 μM ganglioside concentration is suggested to be due to a respective change in the critical micelle concentration effected by butanol. N‐Acetylneuraminyllactosyl ceramide, lactosyl ceramide and asialo‐GM1 ganglioside had an inhibitory effect on the reaction. In contrast, N‐acetylneuraminyllactose, lactose and some other free saccharides were not inhibitory. The results demonstrate that factors other than the saccharide structure must be taken into account when substrate specificity of a glycosidase is studied using competition experiments. It is suggested that the inhibition effected by the glycolipids is due to an increase in the micellar state of the GM1 ganglioside.
Enzyme Memory
Authors: MEUNIER Jean‐Claude , BUC Jean , RICARD Jacques 
Published: March 2005

A theory is presented that associates burst (or lag) kinetics with the respective concentrations of enzyme initial states X1 and X6 and with the cooperation of a mnemonical enzyme. The theory predicts that for an enzyme with a negative cooperation, decreasing the initial concentration of X1 (or increasing that of X6) tends to increase the induction time. This increase may correspond to a reversal of a burst into a lag. Similarly, if the enzyme has a positive cooperation, decreasing the initial concentration of X6 (or increasing that of X1) increases the induction time.The first case above is expected to apply to wheat germ hexokinase LI, X1 being the form that binds glucose preferentially, and X6 the one that binds glucose 6‐phosphate. By changing solely the respective concentrations of the two initial forms, one may expect to modify the pre‐steady‐state phase but not the steady‐state kinetics of the reaction.By jumping the temperature of the enzyme solution from 4°C to 30°C and letting the trans‐conformation equilibrium relax for various periods of time before mixing enzyme with the substrates, one can analyse the effect of the relative concentrations of X1 and X6 on the induction time. One can estimate in that way one of the rate constants of the transconformation between the two free enzyme forms. The shorter the incubation time at 30°C then the smaller is the negative induction time (in absolute values).Another possibility of controlling the ratio between the two initial concentrations of the enzyme, is to pre‐mix hexokinase with glucose 6‐phosphate and to arrange that glucose‐6‐phosphate concentration, after mixing enzyme and substrates, is held constant whatever the pre‐mixing conditions.When wheat germ hexokinase LI is pre‐mixed 30 min at 30°C with glucose 6‐phosphate before the reaction starts, the burst does not disappear. If, on the other hand, pre‐mixing is effected at 4°C the burst is reversed into a lag. This result is taken to mean that the equilibrium constant between the two free enzyme forms (the ‘circle’ and the ‘rhombus’) is strongly dependent on temperature.A direct study of the effect of glucose 6‐phosphate on the conformational equilibrium of wheat germ hexokinase, gives support to this interpretation. If hexokinase is mixed at 4°C with glucose 6‐phosphate a slow increase in fluorescence of tryptophanyl residues is observed, which indicates that the ‘rhombus’ conformation accumulates under these conditions. On the other hand, at 30°C, glucose 6‐phosphate does not produce any significant change in the fluorescence of the protein. As expected, these results imply that the equilibrium between the two free enzymes species is freely reversible at 4°C and nearly irreversible at 30°C.The equations derived from the mnemonical model allow fitting or simulation of the experimental results.
Regions of the Lipopolysaccharide of Pseudomonas aeruginosa Essential for Antitumor and Interferon‐Inducing Activities
Authors: TANAMOTO Ken‐ichi , ABE Chiyoji , HOMMA J. Yuzuru , KOJIMA Yasuhiko 
Published: March 2005

Resistance against ascites tumor development and interferon‐inducing activity were demonstrated in lipopolysaccharide derived from the protein‐lipopolysaccharide complex obtained from an autolysate of Pseudomonas aeruginosa. Lipid A obtained from the lipopolysaccharide was sufficient to induce interferon in vitro but no antitumor activity was found if lipid A or the polysaccharide derived from lipopolysaccharide was injected into the animal.Chemical modification of the polysaccharide portion or deacylation of the lipopolysaccharide also diminished antitumor activity. In contrast, interferon was induced by these incomplete lipopolysaccharides. These results indicate that both the lipid A portion and covalently linked polysaccharide are necessary for the inhibition of ascites tumor development, whereas incomplete lipid A with amide‐linked fatty acids is sufficient to induce interferon in vitro.
Properties of Succinylated Wheat‐Germ Agglutinin
Authors: MONSIGNY Michel , SENE Claude , OBRENOVITCH Angèle , ROCHE Annie‐Claude , DELMOTTE Francis , BOSCHETTI Egisto 
Published: March 2005

The physicochemical and binding properties of succinylated wheat germ agglutinin are described in comparison with these of unmodified wheat germ agglutinin. Succinylated wheat germ agglutinin is an acidic protein with a pI of 4.0 ± 0.2 while the native lectin is basic, pI of 8.5. The solubility of succinylated wheat germ agglutinin is about 100 times higher than that of the unmodified lectin at neutral pH. Both lectins are dimeric at pH down to 5, and the dissociation occurs at pH lower than 4.5. The binding of oligosaccharides of N‐acetylglucosamine to both lectins is very similar on the basis of fluorescence and phosphorescence studies. The minimal concentration required to agglutinate rabbit red blood cells is about 2 μg/ml with both lectins and the concentrations of N‐acetylglucosamine and di‐N‐acetylchitobiose which inhibit agglutination are similar with both lectins.The number of succinylated wheat germ agglutinin molecules bound to the surface of mouse thymocytes was ten times lower than that of the unmodified lectin although the apparent binding constant was only slightly different between the two lectins.The dramatic decrease of the apparent number of cell surface receptors upon succinylation of the lectin is discussed on the basis of the decrease of the isoelectric point and of the acidic properties of the cell surface.
Biochemical Research on Oogenesis
Authors: WEGNEZ Maurice , DENIS Herman 
Published: March 2005

Transfer RNA makes up 30–40% of total RNA in previtellogenic oocytes of Xenopus laevis. The bulk of tRNA is associated with 5‐S RNA and two proteins in a high‐molecular‐weight complex sedimenting at 42 S.
We show here that all kinds of tRNA are present in the 42–S particles and all of them sediment coincidently. Particle tRNA is fully charged in vivo. During purification of the 42‐S particles tRNA becomes partially uncharged. When purified particles are incubated in vitro with amino acids and ATP a charging reaction occurs without disruption of the nucleoprotein complex. Many aminoacyl‐tRNA synthetases can be shown to co‐sediment with the 42‐S particles. We conclude that complete aminoacylation of tRNA within the storage particles results from the activity of particle‐bound aminoacyl‐tRNA synthetases.
Structural Studies on the Immunodominant Group of Lipid A from Lipopolysaccharide of Yersinia pseudotuberculosis
Authors: GORBACH Vladimir I. , KRASIKOVA Inna N. , LUKYANOV Pavel A. , RAZMAKHNINA Olga Yu. , SOLOV'EVA Tamara F. , OVODOV Yury S. 
Published: March 2005

Lipid A isolated from lipopolysaccharide of Yersinia pseudotuberculosis was used for immunization of rabbits to afford antisera to lipid A with titers of 1:640 in the passive hemolysis test. Exhaustion of immune serum with sheep erythrocytes decreased antibody titers up to 1:160.Authentic samples of 2‐(dl‐3‐hydroxytetradecanoyl)amino‐2‐deoxy‐d‐glucose 6‐phosphate, 2‐tetradecanoylamino‐2‐deoxy‐d‐glucose 6‐phosphate and 2‐acetamido‐2‐deoxy‐d‐glucose 6‐phosphate have been synthesized in order to carry out a comparative study of inhibitory activity of these compounds and lipid A using a system of lipid A and antiserum to lipid A. As a result, the immunodominant moiety of the lipid A of Y. pseudotuberculosis proved to contain a d‐glucosamine residue acylated with 3‐hydroxytetradecanoic acid at the amino group. The nature of the fatty acid acylating the amino group of glucosamine does not play an important role in the structure of immunodominant moiety of lipid A.
Binding of Thiostrepton to a Complex of 23‐S rRNA with Ribosomal Protein L11
Authors: THOMPSON Jill , CUNDLIFFE Eric , STARK Michael 
Published: March 2005

Thiostrepton binds with high affinity and with a 1:1 stoichiometry to a complex formed between Escherichia coli 23‐S ribosomal RNA and ribosomal protein L11 of E. coli or the homologous protein BM‐L11 of Bacillus megaterium.In the presence of T1 ribonuclease, protein BM‐L11 and thiostrepton protect from degradation a fragment of E. coli 23‐S RNA estimated to be about 50 nucleotides in length.
Transcription Activity of a DNA‐Protein Complex Isolated from Spinach Plastids
Authors: BRIAT Jean‐François , LAULHERE Jean‐Pierre , MACHE Régis 
Published: March 2005

A DNA · protein complex of about 150 S is isolated from purified spinach chloroplasts by Sepharose 4B gel filtration.A DNA‐dependent RNA polymerase activity is found associated with the complex. This DNA · protein complex is able to initiate RNA chains in vitro. The RNA synthesis is more dependent on CTP than other nucleoside triphosphates. 50% of the activity is still present with 0.6 M KCl. The temperature optimum occurs between 30°C and 35°C. Rifampicin and rifamycin SV have no inhibitory effect.RNA products have been characterized by gel filtration and by hybridization with chloroplast DNA (ctDNA). At the beginning of transcription RNA products are linked to the transcription complex and are later detached. The molecular weight of the product ranges between 0.07 × 106 and 2 × 106. A part of the product (3–4%) has a molecular weight higher than 2 × 106. No endogenous RNase activity was present during the molecular weight determination experiments.Hybridization experiments show that at least 75% of the RNA products are hybridizable with ctDNA and that 40% of these products are composed of chloroplast ribosomal RNA, showing that rDNA is preferentially transcribed.
Somatic Antigens of Shigella
Authors: DMITRIEV Boris A. , KNIREL Yurily A. , SHEREMET Olga K. , SHASHKOV Alexander A. , KOCHETKOV Nikolay K. , HOFMAN Irihna L. 
Published: March 2005

The specific polysaccharide was obtained from the lipopolysaccharide of Shigella newcastle by mild acid hydrolysis and further purified by permeation chromatography on Sephadex G‐50. It was found to consist of l‐rhamnose, 2‐acetamido‐2‐deoxy‐d‐galactose, d‐galacturonic acid residues and O‐acetyl groups in the molar ratios of 2:1:1:1.On the basis of 1H and 13C nuclear magnetic resonance spectroscopy, methylation analysis, partial acid hydrolysis, Smith degradation, and chromium trioxide oxidation, the following structure can be assigned to the repeating oligosaccharide unit of the polysaccharide:‐4)dGalA(β1‐3)dGalNAc‐(β1‐2)lAc3Rha(α1‐2)lRha(α1‐, where Gal A = galacturonic acid, GalNAc =N‐acetylgalactosamine, Ac3Rha = 3‐O‐acetylrhamnose.The structural and immunochemical data presented prove that Sh. newcastle lipopolysaccharide belongs to a ‘non‐classical’ type of somatic antigens with acidic O‐specific polysaccharide chains.
A Comparison of the Initiation Factors of Eukaryotic Protein Synthesis from Ribosomes and from the Postribosomal Supernatant
Authors: THOMAS Adri , GOUMANS Hans , AMESZ Hans , BENNE Rob , VOORMA Harry O. 
Published: March 2005

A purification procedure is described for the initiation factors of protein synthesis from rabbit reticulocytes: (a) from the ribosomal wash and (b) from the postribosomal supernatant.A comparison is made between these preparations with respect to yield and specific activity. eIF‐4A and eIF‐4D occur mainly in the postribosomal supernatant; eIF‐2, eIF‐4C and eIF‐5 are more evenly divided over both fractions, whereas eIF‐1, eIF‐3 and eIF‐4B are found almost exclusively in the ribosomal wash. No significant difference in specific activity could be detected when factors from both sources were compared, with a possible exception of eIF‐4A and eIF‐4D.