bluuebunny/crossref_metadata_2025_split huggingface dataset:
Transcription of High‐Molecular‐Weight RNA from Hen‐Oviduct Chromatin by Bacterial and Endogenous Form‐B RNA Polymerases
Authors: Cox Ronald F. 
Published: March 2005

With the aid of the ribonuclease inhibitor, heparin, techniques were developed to isolate high‐molecular‐weight RNA products synthesised by Micrococcus and homologous form B RNA polymerases using hen oviduct chromatin as template. These methods are suitable for analysis of RNA transcripts synthesised in vitro by a variety of enzymes and templates.
A highly sensitive assay for ribonuclease, based on the ability of the latter to degrade ovalbumin mRNA, confirmed that chromatin and enzyme samples contained significant amounts of ribonuclease activity.
Heparin completely inhibits initiation of both bacterial and homologous enzymes, but has no effect on elongation. The average rates of elongation of RNA polymerases on chromatin DNA could therefore be determined. These values were 3–6 nucleotides per second for bacterial enzyme and 1–2 nucleotides per second for homologous enzyme.
By allowing initiation of bacterial enzyme prior to the addition of heparin, RNA chains approaching 2000 nucleotides in length (molecular weight 0.7 X 106) were synthesised after 1 min; at later times, these chains exceeded 5000 nucleotides in length.
Hen oviduct chromatin contains endogenous RNA polymerase activity which represents predominantly the elongation of form B enzymes. RNA chains ranging from 100 to 2000 nucleotides long are still attached to these enzymes in isolated chromatin. By allowing elongation of these chains in vitro, products ranging from 2000 to 10000 nucleotides in length (molecular weight 0.7 to 3.0 X 106) were synthesised.
Estimates of the number of endogenous RNA polymerases bound to chromatin DNA suggest that approximately 10% of the total form B RNA polymerase molecules per cell are retained in the isolated chromatin fraction.
Unsaturated Fatty Acids as Reactivators of the Calcium‐Dependent ATPase of Delipidated Sarcoplasmic Membranes
Authors: The Rudy , Hasselbach Wilhelm 
Published: March 2005

The reconstituting effect of unsaturated fatty acids whose chain length varied between C6–C24 on delipidated sarcoplasmic membranes has been analyzed.
The reconstituted sarcoplasmic ATPase requires 70–106 mol fatty acid per 100000 dalton for optimal activity.
Half‐maximal activity of the reconstituted sarcoplasmic ATPase is reached between 0.2 μM and 0.6 μM ionized calcium.
The ATPase activities of the reconstituted vesicular preparations show an identical steep decline at pH values > 7, whereas the native preparation still exhibits a considerable activity at pH 8.0.
The most effective reactivating unsaturated fatty acids are those with a chain length of C16–C18 and whose double bonds are in the middle of the chain.
Characterization of Cd, Zn‐Thionein (Metallothionein) Isolated from Rat and Chicken Liver
Authors: Weser Ulrich , Rupp Heinz , Donay Falk , Linnemann Frithjof , Voelter Wolfgang , Voetsch Walter , Jung Günther 
Published: March 2005

The preparation of pure metallothionein from Cd‐pretreated rats and chicken was performed using short‐time treatment with chloroform/ethanol followed by ion‐exchange and gel chromatography. The protein contained 7 g‐atoms metal ions per 12000 g protein. The Zn to Cd ratio was 1:2.4 ± 0.1. The Cd, Zn‐thionein remained homogeneous during Sephadex G‐25, G‐50 and G‐75 gel filtration, during polyacrylamide disc‐gel electrophoresis and in the analytical ultracentrifuge. The high content of cysteine residues (approx. one‐third of the total residues) was consistent with the cysteine content of metallothionein isolated from human or equine tissues. The Cd, Zn‐thionein was of considerable temperature stability. The physicochemical properties were examined by ultraviolet spectroscopy, circular dichroism (CD) measurements and X‐ray photoelectron spectroscopy. The millimolar absorption coefficient of the native protein was at 250 nm ɛ250= 80.6 mM−1· cm−1. Virtually no aromatic amino acids were present. The ɛ250 value of the apoprotein prepared by metal displacement using HCl was ɛ250= 13.2 mM−1· cm−1. When the ultraviolet spectrum of the apoprotein was recorded at pH 6.6 a distinct peak was detectable at 255 nm (ɛ250= 18.7 m M−1· cm−1). The existence of inter‐ or intramolecular disulphide formation was confirmed by CD measurements employing the polyethyleneglycol esters of di‐tert‐butyloxy‐carbonyl‐l‐cystine and tert‐butyloxycarbonyl‐l‐cysteine as model compounds for high‐molecular‐weight and water‐soluble cysteine compounds, respectively.From CD data it was concluded that Cd, Zn‐thionein exists mainly as a random‐coil peptide. Some indication of the binding of Cd and Zn in the native protein with ⊖S–R moieties was obtained from ultraviolet data. Final proof of the exclusive coordination of these metal ions with cysteine sulphur was successful using both circular dichroism measurements and especially X‐ray photo‐electron spectroscopy. The last method proved most convenient for determining the binding energies of the core electrons of Cd (Cd 3d3/2= 411.0 eV and Cd 3d5/2= 404.4 eV), Zn (2 p3/2= 1021.0 eV) and S (2p = 161.7 eV). The corresponding binding energies for S in the cystine complexes with Zn2+ and Cd2+ were 163.0 eV and 162.8 eV, respectively.
Glutamate déshydrogénase
Authors: Dessen Philippe , Pantaloni Dominique 
Published: March 2005

The object of this paper is to analyse the effects of the coenzymes NAD(P)+ and NAD(P)H, and the effectors ADP and GTP, on the polyhexameric structure of pig‐liver dehydrogenase. The linear polymerisation model proposed by Eisenberg for the native quaternary structure of this protein is valid with any effector; the observed variations of the degree of polymerization are explained by the modification of the apparent association constant of the hexamers. The appendices I and II define the free and associated areas and give, the theoretical foundations of the variation of the association constant of hexamers in terms of the binding of the ligands to the protomers.The increase in the degree of polymerization of the glutamate dehydrogenase with the binding of NAD(P)H is explained by a higher affinity of the coenzymes for the protomers which have an associated are compared to the protomers which have a free area. No variation is observed with NAD(P)+, ADP, or GTP alone. The formation of the protein · GTP · NAD(P)H ternary complex leads to a complete depolymerization when the two ligands are in saturating concentrations.The systematic study of the variations of polymerization in terms of increasing concentration of NAD(P)H at constant concentration of GTP, or in terms of increasing concentration of GTP at constant concentration of NAD(P)H shows that the interaction between the two opposite protomers of two consecutive hexamers is responsible for the sigmoidal shape of the depolymerization curves. The reversibility of this effect by ADP is assigned to a competition between the binding of ADP and the binding of GTP.
A Non‐Intercalating Proflavine Derivative
Authors: Müller Werner , Crothers Donald M. , Waring Michael J. 
Published: March 2005

A proflavine derivative with bulky substituents, 2,7‐di‐t‐butyl‐proflavine, binds to DNA but apparently not by intercalation. The methods used to study this problem include equilibrium dialysis, relaxation kinetics and hydroxdynamic studies on low‐molecular‐weight and closed‐circular DNA. These show that binding produces no detectable lengthening or unwinding of the double helix, and that the binding reaction is much faster than is the case with unsubstituted proflavine. We conclude that an outside‐bound complex is formed.We also measured equilibrium binding isotherms for DNA samples of varying G · C content, and for several synthetic polynucleotides. These results are consistent with a model in which a bound 2,7‐di‐t‐butyl‐proflavine molecule occupies three base pairs, and strong binding requires that two of these three be A · T pairs. Comparison of poly(dA) · poly(dT) with poly(A) · poly(U) shows a preference of at least 100‐fold for binding to the former over the latter. Also, we found that the thymine methyl groups are probably not responsible for the A · T specificity.The optical properties of the outside complex include a red shift of the absorbance maximum, with an increase in maximum absorbance instead of the more common decrease. The fluorescence of the dye is enhanced on binding, especially to an A · T‐rich‐DNA.The kinetic results show that two complex forms are present, differing in intrinsic affinity by about an order of magnitude. Both are formed with second‐order rate constants in excess of 108 M−1 s−1 and it is therefore very unlikely that either is an intercalated form. The absorbance spectrum of the bound dye changes as the degree of binding increases, due, at least in part, to the changing contribution of the two binding mechanisms. The conclusions concerning binding specificity refer to the dominant complex form; we have no firm information about the specificity of the weaker complex.
On the Specificity of Interactions between Transfer Ribonucleic Acids and Aminoacyl‐tRNA Synthetases
Authors: Pachmann Ulrich , Cronvall Eva , Rigler Rudolf , Hirsch Reinhard , Wintermeyer Wolfgang , Zachau Hans G. 
Published: March 2005

The interactions of yeast seryl‐tRNA and phenylalanyl‐tRNA synthetases with the cognate and the noncognate tRNA have been studied by fluorescence spectroscopy. The binding process was followed by observing the changes in tryptophan fluorescence of the synthetases as well as the changes in the polarized fluorescence of the tRNAs covalently labeled with ethidium bromide, proflavine, or 1, N6‐etheno‐ATP. The degree of polarization of the fluorescence labels is particularly sensitive to changes in rotational diffusion of the tRNA. Both methods can be regarded as complementary and gave comparable results. There exist, however, cases such as the interaction between seryl‐tRNA synthetase and tRNASer in the absence of Mg2+, where the binding process could only be detected by measurement of the fluorescence polarization.Unspecific interactions between synthetases and tRNAs (or tRNA‐dye compounds) could be detected and were found to be highly dependent on pH, salt concentration and on the buffer used. In potassium phosphate buffer pH 7.3 discrimination between cognate and noncognate tRNA by seryl‐tRNA synthetase increases with addition of salt. The tryptophan fluorescence of phenylalanyl‐tRNA synthetase was influenced differently by the cognate and the noncognate tRNA while the stabilities of the complexes between this synthetase and the ethidium‐labeled tRNAPhe and tRNASer were rather similar. Seryl‐tRNA synthetase apparently discriminates more efficiently than phenylalanyl‐tRNA synthetase between the cognate and the noncognate tRNAs.The presence of two binding sites on seryl‐tRNA synthetase and the presence of one binding site on phenylalanyl‐tRNA synthetase for cognate as well as noncognate tRNA could be deduced. For the specific interaction between tRNASer and seryl‐tRNA synthetase in the presence of Mg2+ indications for cooperative behavior have been found. Experiments on competition between noncognate and cognate tRNAs as well as tRNA half molecules for the binding to the synthetase indicate an overlap of specific and unspecific binding sites.We conclude that the unspecific interaction may be an important initial step preceding the specific binding and recognition of the tRNA by the synthetase.
The Time Dependence of the Activity of δ‐Chymotrypsin at High pH
Authors: Garel Jean‐Renaud , Labouesse Bernard 
Published: March 2005

When δ‐chymotrypsin at a high initial pH is mixed with a specific ester substrate, the hydrolytic reaction exhibits a lag phase that can be observed from the release of both products, the alcohol or the amino acid. This lag phase is under the dependence of the ionization of a group of pK 9, at 15°C, which has been previously shown to be the α‐amino group of isoleucine‐16. This lag corresponds to the time needed for the enzyme to isomerize from its “high‐pH” to its “neutral” conformation, and fluorescence measurements have been separately used to monitor this isomerization in the presence of substrate. The initial activity observed when the enzyme is mixed with the substrate is proportional to the concentration of enzyme present in its neutral conformation and is not significantly measurable when the enzyme is in its high‐pH conformation.It is concluded that the high‐pH form of δ‐chymotrypsin is inactive and cannot be involved in the observed activity of the enzyme at high pH. Therefore the activity of δ‐chymotrypsin exhibited at high pH is linked to a substrate‐promoted isomerization of the protein, that is, to a displacement of the conformational equilibrium presented by the protein towards its active neutral conformation.
Analysis of the Methylation Sites in Yeast Ribosomal RNA
Authors: Klootwijk Jacobus , Planta Rudi J. 
Published: March 2005

The methylation sites and the number of methyl groups of 26‐S and 17‐S ribosomal RNA (rRNA) of Saccharomyces carlsbergensis were investigated.28‐S and 17‐S rRNA contain 43 and 24 methyl groups per molecule, respectively, of which in both molecules 6 are attached to the heterocyclic bases. In 26‐S rRNA the base‐methyl groups are present as two copies of m3U, m5C as well as m1A, whereas the methylated bases in 17‐S rRNA are one m7G, one methylated cytidine and two of m26A.In 17‐S rRNA there are 11 different alkali‐stable (i.e. ribose‐methylated) dinucleotides, while 26‐S rRNA contains 15 different alkali‐stable dinucleotides, and in addition 4 alkali‐stable trinucleotides (Am‐Cm‐Gp, Am‐Gm‐Cp, Am‐Am‐Up and Um‐Gm‐Ψp).Most of the methylated oligonucleotides found in a ribonuclease T1 digest of 26‐S or 17‐S rRNA occur in approximately one‐molar amounts. The combined data suggest that yeast ribosomal RNA is essentially homogeneous in its methylated sequences and in addition that the methylation is a highly specific process.
Influence of Buffers, Protein Concentration and Ionic Strength upon the Dissociation of Pila Haemocyanin
Authors: Cox Jos A. , Elliott Francis G. 
Published: March 2005

The study of the dissociation of Pila leopoldvillensis haemocyanin by means of light scattering has shown that the protein concentration has no influence upon the dissociation into halves and tenths.Different buffer ions were shown to have specific effects upon the dissociation of Pila haemocyanin: with maleate and phosphate the dissociation curves are shifted towards lower pH values; with imidazole and Tris the shift is in the opposite direction.Takening into account the high molecular weight of the protein, the binding of chloride ions, as determined by the ΔHp method, is very small. Therefore pure KCl solutions rather than buffers were used in further work on the dissociation of haemocyanin.In view of these observations an attempt is made to interpret the influence of the pH and ionic strength upon the dissociation‐association reaction according to a sequential loop model: the forward and back reaction proceed along opposite sequential pathways, each starting with a particular change of the ionisation state of the wholes, halves or tenths. According to this reaction scheme the ionisation constant of the sites responsible for the dissociation into halves is 6.96; and that for the dissociation of halves into tenths is 9.65.
Heterogeneity and Complexity of Chlorella Chloroplastic DNA
Authors: Bayen Marcel , Rode André 
Published: March 2005

The heterogeneity and complexity of Chlorella chloroplastic DNA has been investigated by means of thermal denaturation and renaturation kinetics.The chloroplastic DNA presents a Tm of 82.2°C, a maximal hyperchromicity of 40.2% and a dispersion coefficient Δ2/3 of 6°C. The absorbance melting profiles display irregularities indicating an intramolecular heterogeneity of the DNA. The first derivated curves of the melting profiles show the presence in the chloroplastic DNA of two main components of Tm 81 °C and 84.7 °C (which represent together about 80% of the total DNA) and possibly three other minor ones.Denatured and sheared DNAs renature rapidly as a fast and a slow‐renaturing class with a kinetic complexity of 3 · 107 daltons and 2.3 · 108 daltons respectively (assuming a value of 2.5 · 109 daltons for Escherichia coli DNA). If correction is applied to account for the low d(G + C) content of the chloroplastic DNA, the kinetic complexity of the slow‐renaturing component is found to be in the range of 1.2‐1.3 · 108 daltons.Our results confirm and extend the fact that chloroplastic DNAs are similar in terms of kinetic complexity and heterogeneity. This is in good agreement with a possible common origin for all the chloroplastic DNAs.From the discrepancy between analytical and kinetic complexities of Chlorella chloroplastic DNA, one can assume that this DNA is extensively reiterated and that the unique nucleotide sequence is present in about 20 copies in the chloroplastic genome.
Stimulation of ATP Synthesis by a Membrane Potential in Chloroplasts
Authors: Schuldiner Shimon , Rottenberg Hagai , Avron Mordhay 
Published: March 2005

Chloroplasts preloaded with protons by light‐induced proton uptake do not synthesize ATP in a following dark period if the proton concentration gradient (ΔpH) is lower than 2.5–3.0 pH units. At such suboptimal ΔpH values synthesis of ATP can be obtained by imposing a diffusion potential across the chloroplast membrane. This potential was realized by providing a high external concentration of either KCl in the presence of valinomycin or of NaCl in the presence of monactin. Diffusion‐potential‐induced stimulation of ATP synthesis was observed both in chloroplasts and in bacterial chromatophores.The stimulation could be abolished by the addition of a highly permeant anion, like picrate. It is proposed that the effect of the diffusion potential indicates that an increase in the electrochemical gradient of protons can be coupled to the synthesis of ATP.
Structure‐Activity Relationship in Tryptophanyl‐Transfer Ribonucleic Acid Synthetase from Beef Pancreas
Authors: Iborra François , Mourgeon Gérard , Labouesse Bernard , Labouesse Julie 
Published: March 2005

The titration of the – SH groups of beef pancreas tryptophanyl‐tRNA synthetase by N‐ethylmaleimide and the consequent effect on the exchange and transfer reactions of the enzyme were examined.The 16 sulfhydryl groups of the native dimeric enzyme do not react homogeneously at pH 7, with 1 mM reagent. They can be divided into three classes with decreasing reactivities. The reaction of the first class (6 – SH groups) has no effect on activity. The reaction of the second class (4 – SH groups) brings about an increase of Km(app) for tryptophan both in the exchange and in the transfer reactions of the enzyme. The activity of the enzyme is lost during reaction of the third class (6 ‐ SH groups). This is evidenced by the decrease of the V of the [32P]PPi‐ATP isotope exchange and tRNA aminoacylation reactions and by the decrease in the amount of the tryptophanyladenylate · enzyme complex isolatable on Biogel P30. The destruction of the active site by alkylation of the sulfhydryl groups of class III can be the consequence of a structural change of the enzyme induced by the chemical modification. During the alkylation of the first two classes of ‐ SH groups the Km(app) for ATP, PPi and tRNA are not altered significantly.The presence of tryptophan or tryptophanyladenylate reduces the rate of titration of the ‐SH groups of class II and class III and in a parallel fashion the rates of increase of Km(app) for tryptophan and of decrease of V. The titration of the –SH groups of class I is not affected by the presence of substrates. The alkylation in the presence of tryptophanyladenylate demonstrates that class II is not homogeneous and that the two molecules of adenylate bound to the dimeric enzyme specifically protect two –SH groups out of the four of this class.
Studies on the Properties of Fish Hemoglobins Molecular Properties and Interaction with Third Components of the Isolated Hemoglobins from Trout (Salmo irideus)
Authors: Brunori Maurizio , Giardina Bruno , Chiancone Emilia , Spagnuolo Carla , Binotti Ines , Antonini Eraldo 
Published: March 2005

This paper reports some physico‐chemical and functional properties of two of the isolated hemoglobin components from trout (Salmo irideus) blood (Hb trout I and Hb trout IV).Both hemoglobins in the oxygenated state are tetrameric at pH between 7 and 7.8 and protein concentration above ∼ 0.1 mg/ml. The tetramer is extremely stable towards dissociation into subunits and the stability constants, estimated by differential gel filtration, are between 10 and 50 times larger than that of human hemoglobin in phosphate buffer. A very small degree of dissociation is also suggested by the very small extent of hybrid formation even in concentrated KI or triethanolamine.The dichroic properties of both Hb trout I and Hb trout IV show distinct features in the wavelength region between 300 and 200 nm. Hb trout IV resembles human hemoglobin in the shape of the CO spectrum and in the values of the reduced ellipticity (θ); furthermore upon deoxygenation the value ofθ at 222 nm becomes more negative by about 5%. On the other hand Hb trout I shows a different circular dichroism spectrum and in particular the ellipticity at 222 nm is lower than that of Hb trout IV. Moreover the spectrum is insensitive to the presence of heme ligands.Hb trout IV binds human haptoglobin (Hp) as shown by quenching of the fluorescence of the tryptophanyl residues of Hp upon addition of hemoglobin. The apparent stoichiometry of complex formation corresponds to two Hb tetramers per Hp molecule (while for human hemoglobin the stoichiometry corresponds to ∼ one tetramer per Hp). A clear cut effect of Hp binding is observed on the kinetics of O2 and especially CO combination as studied by flash photolysis.The effect of several third components on the O2 equilibrium of both Hb trout I and IV has been investigated. In general the effect of adding ions or other molecules to a solution of Hb trout I is very minor. On the other hand Hb trout IV is more susceptible to changes in solvent composition. Of particular importance is the action of ATP, the physiological organic phosphate, which is without effect on Hb trout I, while it has a clear effect on Hb trout IV. The addition of ATP shifts the Root effect, the characteristic property of Hb trout IV, to higher pH values, thereby acting as one of the allosteric effectors.