bluuebunny/crossref_metadata_2025_split huggingface dataset:
Thermisches Schmelzen und optische Rotations‐Dispersion von DNA‐Hg2+‐Komplexen
Authors: Luck Gerhard , Zimmer Chritoph 
Published: March 2005

The effect of Hg2+ on the secondary structure of various DNA's with different (G + C) content is investigated by optical rotatory dispersion and melting temperature measurements.Marked deformations of the optical rotatory dispersion spectra can be observed already at 1 Hg2+ per 500 DNA‐P. The changes in specific rotations beyond 250 nm are more effective for AT‐rich than for GC‐rich DNA. Comparison to corresponding DNA‐Cu2+‐complexes indicates the greater tendency of Hg2+ to produce conformational changes in the DNA double‐helical structure due to its higher affinity to the bases. DNA from Proteus mirabilis (42 mole‐% G + C) shows an increase in its melting temperature around 0.02 and at 0.09 Hg2+/DNA‐P. At 6 nM Na+ the Tm increases up to 0.03 Hg2+/DNA‐P. Between 0.03 and 0.07 Hg2+/DNA‐P Tm decreases whereas awt higher Hg2+ concentrations melting tempeature increases agains. Similar behaviour was found with DNA's of varying GC content. The decrease of Tm is more effective for GC‐rich than for AT‐rich DNA's.The observed changes in the optical ratotory dispersion and melting profiles of DNA‐Hg2+ complexes are explained in terms of a specific binding of Hg2+ to adjacent thymine bases and AT‐pairs in accordance with previous results obtained by other authors. At 6 nM Na+ Hg2+ may combine with phosphate sites up to 0.03 Hg2+/DNA‐P. Within the decreasing Tm range the destabilization is attributed to a preferred formation of complexes of the type thymine‐Hg2+‐thymine whereas the stabilization effect at higher metal ion concentrations is presumably due to cross‐link formation between AT paris of opposite stands.
The Effect of Temperature and Ionic Strength on the Electrophoretic Mobility of Yeast Mitochondrial RNA
Authors: Grivell Leslie A. , Reijnders Lucas , Borst Piet 
Published: March 2005

The electrophoretic mobilities of mitochondrial and cell‐sap ribosomal RNAs from Saccharomyces carlsbergensis have been measured relative to the mobility of Escherichia coli rRNA in 2.4% polyacrylamide gels at various temperatures and ionic strengths. At 5° C and in 20 mM sodium acetate, 40 mM Tris‐acetate, 2 mM EDTA (pH 7.8), mitochondrial and cell‐sap rRNAs co‐migrated and were only partially resolved from E. coli rRNA. When the temperature of electrophoresis was increased the mobility of the mitochondrial rRNAs (and to a lesser extent of the cell‐sap rRNAs) decreased relative to the mobility of the E. coli rRNA species, reaching a minimum between 9 and 16° C. As a consequence the mitochondrial and cell‐sap rRNAs were completely resolved in this buffer at temperatures above 9° C. Similar effects of temperature on relative electrophoretic mobility were observed in 90 mM Tris‐borate buffer at pH 8.3 and the effect was even more pronounced at low ionic strength (10 mM NaCl, 5 mM Tris‐HCl, 2 mM EDTA, pH 7.5).At low ionic strength the decrease in the relative electrophoretic mobility of mitochondrial rRNA with an increase in temperature was accompanied by a decrease in sedimentation coefficient from 21–22 S (large component) and 14 S (small component) at 5° C to 16 and 11.5 S at 20° C (calculated in relation to assumed S‐values of 23 and 16 S for the rRNA species of E. coli).We conclude that the electrophoretic mobility of RNA in polyacrylamide gels is more dependent on secondary structure than previous work had suggested and that caution is needed to interpret the relative mobility of mitochondrial RNA on gels in terms of molecular weight. In addition, our results show that judicious variation of temperature and ionic strength may allow the separation on gels of RNA mixtures that cannot be resolved under standard conditions.
Thermodynamic and Kinetic Aspects of the Interconversion of Chemical and Osmotic Energies in Mitochondria
Authors: Azzone Giovanni Felice , Massari Stefano 
Published: March 2005

The conversion of osmotic energy into ATP has the following properties:a) K+, as effluent ion, can be replaced by Rb+, whereas when H+, as influent, is replaced by other cations, the synthesis is abolished.b) Both the effluent cation and the influent H+ seem to provide energy for the synthesis. The minimal sum of ΔpH +ΔpK, compatible with the synthesis, is 2.5 units, corresponding to an osmotic potential of 3.45 kcalories per gram‐ion equivalent−1. The number of ion equivalents exchanged per ATP synthesized is dependent on the dimension of the osmotic potential.c) The osmotic potential compatible with the ATP synthesis is partially affected by the phosphate potential.
The conversion of redox into osmotic energy has the following properties:a) The number of K+ ion equivalents exchanged with protons per energy rich bond is dependent on the concentrations of H+ and K+ in the inner and outer aqueous phases, increasing up to a maximal value of 4.b) The number of K+ ion equivalents exchanged with protons per energy rich bond increases parallel to the increase of the amount of valinomycin, up to the value of 4.
On the basis of available data the following thermodynamic parameters are evaluated:a) The relationship between osmotic potential and ATP synthesis.b) The relationship between redox and osmotic energy.c) The osmotic potential barrier for ion translocation.
A molecular mechanism for the interconversion of osmotic and chemical energies is discussed.
Isoleucyl‐tRNA Synthetase
Authors: Penzer Geoffrey R. , Bennett Edward L. , Calvin Melvin 
Published: March 2005

Fluorescence properties of purified isoleucyl‐tRNA synthetase isolated from Escherichia coli B have been studied. No changes in the quantum yield, energy or polarisation of the emission were detected in the presence (either individually or in combinations) of the substrates and cofactors required for activation of L‐isoleucine.In 2.5 M urea enzyme activity and intrinsic fluorescence intensity (at 340 nm) each decrease with time, showing similar kinetics and rate constants. The rate of this decay is reduced in the presence of ligands which can bind to the enzyme and the effect has been used to measure dissociation constants for enzyme‐ligand complexes. Values have been obtained for the complexes between enzyme and l‐isoleucine (Kdiss= 25 μM), l‐valine (Kdiss=300μM), ATP(Kdiss= 150 μM) and PPi (Kdiss= 200 μM) at 25°. The effects of ionic strength, and the temperature dependence and urea concentration dependence of l‐isoleucine binding have also been studied. Magnesium ions, which are required for catalysis, do not greatly affect the binding of single substrates, but changes are seen in the presence of ATP and l‐isoleucine together. The magnesium ion concentration dependence of this effect (half‐point about 200 μM) and the equilibrium constant for l‐isoleucine activation (2 μM) have both been measured.The reliability of the methods has been discussed. Results have been interpreted in terms of current theories of amino acid activation. The binding parameters are sufficient to explain the stability of enzyme bound l‐isoleucyladenylate without invoking conformation changes. This is consistent with the absence of substrate induced fluorescence changes. Magnesium effects are explained in terms of reduced electrostatic repulsion between reactants bearing like charges.
The Respiratory System of Azotobacter vinelandii
Authors: Ackrell Brian A. C. , Jones Colin W. 
Published: March 2005

The growth and respiration of Azotobacter vinelandii, together with the composition and activity of its membrance‐bound respiratory system are compared during nitrogen‐fixing batch growth at high and low aeration.
The aeration rate determines the length of the lag period, the extent of logarithmic growth and the respiratory activity of the culture; logarithmic growth ceases when the dissolved oxygen concentration drops to approximately 32 μM. Aconsiderable increase in whole cell respiratory activity is observed on changing from low to high aeration conditions.
During logarithmic, non‐oxygen‐limited growth, oxidase activities and respiratory carrier concentrations, with the exception of cytochrome o, remain low; the concentration of cytochrome o starts to rise when the ambient oxygen concentration falls to approximately 120 μM. Phosphorylation efficiencies, particularly at site I, are very low at high ambient oxygen concentrations but increase as the oxygen concentration decreases. Site I P/2e ratio is maximal (approx. 0.5) only when the oxygen concentration becomes growth limiting.
At the turnover point into non‐logarithmic, oxygen‐limited growth, respiratory carrier concentrations increase considerably; primary dehydrogenase activities increase by 2‐fold whereas the activity of the minor phosphorylating terminal pathway increases by 9–13 fold.
A working hypothesis is proposed to account for the ability of the ambient oxygen concentration to control both the activity and the energy conservation efficiency of A. vinelandii respiration.
Optical Rotatory Dispersion of Alkali‐Denatured Cytochrome Oxidase and Heme a‐Polylysine Complexes
Authors: Yong Fook Choy , King Tsoo E. 
Published: March 2005

The optical rotatory dispersion of alkali denatured cytochrome oxidase, the heme a‐poly‐l‐lysine complex in the presence and absence of Emasol‐1130 (polyoxyethylene‐sorbitan monolaurate), the heme a‐poly‐d‐lysine complex and the protoheme‐poly‐l‐lysine complex were studied in the Soret region at pH 11.6–12.0. No anomalous rotations were observed in the oxidized form of either the denatured oxidase or any of the synthetic heme a complexes. The reduced form of the denatured oxidase exhibited a coupled‐type dispersion curve having a prominent trough at 437.5 nm flanked by two small peaks. Carbon monoxide shifted the trough to 435 nm. These optical rotatory dispersion profiles which differed from those given by the native enzyme and heme a‐poly‐l‐lysine complexes were similar at least qualitatively to those exhibited by heme a‐poly‐d‐lysine complex. The amplitude of rotations for both the denatured enzyme and the heme a‐poly‐d‐lysine complex was several fold smaller than those of heme a‐poly‐l‐lysine complexes. The optical rotatory dispersion spectra of the heme a‐poly‐d‐lysine complex in the reduced and carbon monoxide forms were found not to be mirror images of those of the corresponding l complex.These results are interpreted to mean that alkali causes drastic conformational changes in the enzyme molecule and bring about effective heme‐heme interactions, that the orientation of the heme a molecule in the denatured enzyme differs from that observed in the synthetic complex, and that at least one of the ligands coordinated to the heme iron in the denatured enzyme is a lysyl residue.Optical rotatory dispersion titrations of the heme a‐poly‐l‐lysine complex and its carbon monoxide derivative at constant heme a concentration showed a linear increase in rotation with increase of polylysine concentration but the rotation then decreased in excess polylysine. Similar anomalous titration behavior was observed for heme a‐poly‐l‐lysine in Emasol. In contrast, protoheme‐poly‐l‐lysine and the carbon monoxide treated heme a‐poly‐l‐lysine in Emasol gave the conventional rectangular hyperbolic titration curve. These results suggest that the observed optical rotation of heme a‐polylysine complexes are complicated and are affected to a great extent by the aggregative properties of heme a molecules. Protoheme which lacks the large hydrophobic side chain and the formyl group, exhibits no such anomaly when complexed with polylysine.
Incorporation in vitro of [14C]Leucine into the Mitochondrial Protein of Lucilia cuprina
Authors: Williams Keith L. , Birt L. Michael 
Published: March 2005

The energy requirements for incorporation in vitro of [14C]leucine into sterile mitochondria isolated from thoracic flight muscles of newly‐emerged Lucilia cuprina have been studied.There is an absolute requirement for addition of either ADP or ATP. Oxidisable substrates (pyruvate, DL‐α‐glycerophosphate, proline), or an ATP‐generating system increased incorporation above that observed with added ADP or ATP alone. Both the rate and duration of incorporation are strongly dependent on the concentration of adenine nucleotides used. Experiments with oligomycin and 2,4‐dinitrophenol suggest that energy for incorporation is provided as ATP.With non‐sterile preparations, incorporation was, independent of added adenine nucleotides, and almost constant for up to four hours with no added substrate or adenine nucleotide. It was inhibited by added substrate and also by added amino acids, especially isoleucine and valine.
Optical Rotatory Studies on Nucleic Acid Derivatives n–π* Transitions and the Relation of the B2u Cotton Effect to Conformation in Pyrimidine and Azapyrimidine Nucleosides
Authors: Rogers Gordon T. , Ulbricht's Tilo L. V. 
Published: March 2005

It is shown by solvent studies that O2:2′‐ and O2:5′‐cyclouridine derivatives give a small long wavelength Cotton effect which is due to an n–π* transition. The sign of the B2u Cotton effect (corresponding to the main ultraviolet absorption band) is as predicted by the previously formulated rule for pyrimidine nucleosides. Cotton effects due to n–π* transitions have also been observed in a number of 6‐azauridine derivatives.Optical rotatory dispersion and circular dichroic data for a variety of azapyrimidine nucleosides are presented, and discussed in relation to corresponding data for pyrimidine nucleosides. The sign of the B2u Cotton effect in azapyrimidine nucleosides (which is negative in 6‐β‐d‐azauridine) can be explained on the basis of conformational considerations, or by assuming that the direction of the transition moment is different from that in pyrimidine nucleosides, owing to the presence of an additional nitrogen in the ring. Both of these possible explanations are illustrated by appropriate projection diagrams relating the base‐sugar torsion angle ΨCN to the B2u Cotton effect.
Ribosomal Proteins
Authors: Hindennach Ingrid , Kaltschmidt Eberhard , Wittmann Heinz‐Günter 
Published: March 2005

50 S ribosomal subunits of Escherichia coli obtained by zonal centrifugation in B XV rotors were treated with various concentrations of LiCl in the absence and presence of urea. This treatment resulted in three protein fractions, each of which was subjected to CM‐cellulose chromatography. Gel filtration in Sephadex G‐100 (and preparative polyacrylamide electrophoresis) were used for further separation. The purity of the isolated proteins was better than 95% as shown by four methods. Proteins were isolated in yields of 6–90 mg depending mainly on the number and kind of purification steps.Instead of fractionation with LiCl prior to column chromatography, precipitation of extracted proteins at various concentrations of ammonium sulfate was tested. This method is useful if a given protein, e.g. from mutants is wanted in large quantity.
Studies on the Mechanism and Stereospecificity of the Urocanase Reaction
Authors: Kaeppeli Franz , Rétey Jänos 
Published: March 2005

Nuclear‐magnetic‐resonance spectroscopy was used to follow the course of the urocanase reaction. It was shown that 3‐(4′‐hydroxyimidazol‐5′‐yl)‐propionate is the true enzymic product and that it spontaneously tautomerises to recemic 3‐(imidazol‐4′‐one‐5′‐yl)‐propionate.
Conversion of urocanate in deuterium oxide by a crude‐enzyme preparation from Pseudomonas putida led to a product containing two adjacent CH2H‐groups. Oxidative degradation of this product afforded optically pur (RR)‐[2H2]succinic acid. This finding defines the sterochmistry of proton addition to the double bond of urocate.
Substrate Complexes of Phenylalanyl‐tRNA Synthetase from Escherichia coli
Authors: Kosakowski Heinz M. , Böck August 
Published: March 2005

The stoichiometry of substrate binding to phenylalanyl‐tRNA synthetase from Escherichia coli K10 was investigated. Isolation of the phenylalanyl · adenylate · enzyme complex by means of gel filtration revealed that phenylalanine and the nucleotide are bound in about equimolar amounts and in a stoichiometric ratio. Stable complexes between tRNAPhe and phenylalanyl‐tRNA synthetase could be separated from free tRNAPhevia sucrose‐gradient zone‐centrifugation or gel filtration. Determination of the amount of tRNAPhe bound to phenylalanyl‐tRNA synthetase (molecular weight 181000) by absorption at 260 nm or by the amino‐acid‐acceptance activity yielded ratios close to one. tRNAPhe· enzyme complexes were most stable at pH values below 6.5; complex formation between phenylalanyl‐tRNA synthetase and non‐cognate tRNA species was not observed. For determination of the affinity and ratio of binding of phenylalanine the non‐equilibrium dialysis method was employed: a Kd of 30 μM was obtained; by the criteria of this method one binding site for the amino acid could be demonstrated.Phenylalanyl‐tRNA synthetase of E. coli is protected against heat inactivation by Mg, ATP, ATP and phenylalanine or tRNAPhe. By means of the protection constant, the apparent affinity constant for tRNAPhe was determined to be 3 × 107 M−1 at pH 6.0 and 37 °C. The dependence of the π‐values obtained on pH indicates that the affinity of phenylalanyl‐tRNA synthetase for tRNA drastically increases at more acid pH. The same tendency was obtained upon determination of the pH‐dependence of the Km values for tRNAPhe.Out of the 24 SH‐groups of phenylalanyl‐tRNA synthetase which are reacting with 5,5′‐dithio‐bis(2‐nitrobenzoic acid) in the presence of 8 M urea only less than one half of them are oxidized on the native enzyme. The kinetics of titration of the sulfhydryl is grossly changed in the presence of ATP, ATP and phenylalanine or tRNAPhe. In addition, in the presence of ATP and phenylalanine or of tRNAPhe 2‐3 SH‐groups are completely protected from oxidation. Under this condition loss of activity which accompanies the SH‐group oxidation is greatly diminished.
Stereochemistry of the Citrate‐Lyase Reaction
Authors: Buckel Wolfgang , Lenz Helmut , Wunderwald Peter , Buschmeier Verena , Eggerer Hermann , Gottschalk Gerhard 
Published: March 2005

Using a medium of tritiated water for the conversion of fumarate to malate and using a medium of deuterium oxide for the cleavage of citrate, chiral acetate was generated enzymically in the sequence fumarate → malate → oxaloacetate → citrate → acetate.
Acetate of opposite chirality was produced in the same sequence by replacing fumarate and tritiated water by [2,3‐3H2]fumarate and ordinary water, respectively.
The configuration of acetate generated in (1) was shown to be R, and the configuration of acetate generated in (2) was shown in to be S.
It was concluded that citrate lyase cleaves citrate with inversion of configuration at the methylene group which becomes methyl.
Response of Phosphopyruvate Carboxylase to Tryptophan Metabolites and Metal Ions
Authors: Snoke Roy E. , Johnston James B. , Lardy Henry A. 
Published: March 2005

Tryptophan, administered to rats, inhibits gluconeogenesis, but increases the activity of hepatic phosphopyruvate carboxylase when assayed in vitro. Quinolinate, a metabolite of tryptophan, inhibits gluconeogenesis in perfused livers, and inhibits phosphopyruvate carboxylase in liver cytosol from tryptophan‐treated rats but not from normal rats.Metal‐ion activation and quinolinate inhibition of hepatic cytosol phosphopyruvate carboxylase has been investigated in vitro. Of the cations tested, only Fe2+, Co2+, Cd2+ and Mn2+ appreciably increased this enzymic activity. Quinolinate inhibited the activity of divalent cation‐treated enzyme in the order Co2+ Fe2+ Cd2+ and did not inhibit the Mn2+‐treated phosphopyruvate carboxylase. Other tryptophan metabolites, metal chelators, and structural analogs of quinolinate have been tested, but only quinolinate lowered the activity of the Fe2+‐treated enzyme substantially.The elevated phosphopyruvate carboxylase activity in cytosol of tryptophan‐treated rats was lowered in vitro by quinolinate to the same extent as was the activity of the Fe2+‐treated enzyme from normal rats. Ferrous iron is proposed as the physiologically important cation and quinolinate the important metabolite responsible for altering the activity of phoshopyruvate carboxylase in vivo following tryptophan administration.
Studies of Glutamate Dehydrogenase
Authors: Markau Klaus , Schneider Jens , Sund Horst 
Published: March 2005

The investigation of the association‐dissociation equilibrium of beef liver glutamate dehydrogenase by light‐scattering measurements over a wide range of protein concentration (0.02 to 50 mg/ml) has shown that this equilibrium can be described as an open equilibrium (Mi+n Mh⇋ Mi+h, i and h≥ 1, M = oligomer) with identical equilibrium constants for all steps and without any limit. The dependence of the apparent weight‐average molecular weight on the protein concentration reaches a maximum at about 9 mg/ml. The decrease of the molecular weight at higher protein concentrations must be attributed to effects of nonideality (as expressed in relation to a first approximation by the second virial coefficient). At 20°C and in M/15 sodium phosphate buffer the equilibrium constant was found to be 9.0 × 105 M−1 (corresponding to a free energy change of 7.8 kcal/mol for each consecutive step in the association reaction) and the second virial coefficient to be 9 nmol × 1 × g−2. The calculation of the dependence of the reduced viscosity on the protein concentration based on these results conforms with the experimental data. However, it was found that the length of the oligomer obtained from light‐scattering measurements was dependent on the wavelength. The reason for this phenomenon is not quite obvious.Low pH and low ionic strength promote the association of the oligomers. It was found that the sedimentation coefficient at pH 6.2 and an ionic strength of 0.04 is 31 S (34 S in the presence of 4.4 mM NAD+) compared to 27 S at an ionic strength of 0.115. The pH dependence of the sedimentation coefficient at low ionic strength indicates the participation of a group in the association with a pK near 7, possibly an imidazole group. At high ionic strength the sedimentation coefficient is almost pH independent.From the results it may be concluded that, in the interaction between the oligomers at low ionic strength, ionic effects are involved that are shielded at higher ionic strength.GTP and NADH cause the dissociation into the oligomer characterized by an s020,w value of 13.0 ± 0.3. The dissociation is complete in the presence of 20 mM GTP and NADH. The result that the s020,w values of the oligomer are the same in the absence and in the presence of effectors, GTP/NADH, conforms to our X‐ray small‐angle measurements.
Inactivation, Stabilization and Some Properties of ATP: Glutamine Synthetase Adenylyltransferase from Escherichia coli B
Authors: Wolf Dieter , Ebner Eberhard , Hinze Helga 
Published: March 2005

ATP: glutamine synthetase adenylyltransferase loses activity after purification, forming aggregates with high molecular weight. This inactivation can be prevented by bovine serum albumin. Freshly prepared adenylyltransferase of low specific activity is reactivated by bovine serum albumin. It is shown that the effect of bovine serum albumin on adenylyltransferase is due to a protein—protein interaction. A mechanism for the inactivation, reactivation and stabilization is proposed.The molecular weight of adenylyltransferase was estimated by ultracentrifugation to be 115000. The isoelectric point is 4.2 to 4.3. Adenylyltransferase was found to be specific for native glutamine synthetase.