bluuebunny/crossref_metadata_2025_split huggingface dataset:
Étude du rameau dégradatif commun des hexuronates chez Escherichia coli K 12
Authors: Pouysségur Jacques M. , Stoeber François R. 
Published: March 2005

The investigation of hexuronate (d‐glucuronate and d‐galacturonate) metabolism in Escherichia coli K 12 has been completed by studying the biochemical properties of the 2‐keto‐3‐deoxy‐6‐phosphogluconate aldolase.The enzyme was obtained from a culture of E. coli K 12 grown on galacturonate followed by a 175‐fold purification which included these steps: sonic extraction of the enzyme, protamine sulfate precipitation, ammonium sulfate fractionation, heat step and DEAE‐Sephadex chromatography. The richest fraction of the chromatographic peak represented 5% of the initial proteins, and has been purified 600‐fold.The basic biochemical properties of the enzyme have been established using the DEAE‐Sephadex purified extract. The enzyme is stable in a 0.2 M Tris‐HCl buffer, pH 7.6; its pH optimum in this buffer is about 7.6. The aldolase seems to function in the absence of coenzyme. In the above‐mentioned properties it appears to be related to the enzyme which has been studied in Pseudomonas fluorescens. The Michaelis constant for the substrate 2‐keto‐3‐deoxy‐6‐phospho‐gluconate is about 2 × 10−4 M. Numerous compounds tested have no affinity for this aldolase: only the glyceraldehyde‐3‐phosphate (substrate of the reverse reaction) and the gluconate‐6‐phosphate competitively inhibit the aldolase with Ki constants of respectively 4 × 10−4 M and 8 × 10−4 M. The narrow specificity of this aldolase is demonstrated by comparing the compounds with and without affinity for the enzyme; the simultaneous presence of a carboxyl function in position 1 and of a phosphate group in position 6 is necessary for ligand fixation on the enzyme site in the cleavage reaction. The study of denaturation by heat illustrates the thermostability of this enzyme and suggets strongly that at least 99% of the aldolase activity is carried by a homogeneous protein. Half‐denaturation is obtained in 10 min at 78°.From the point of view of the physiological regulation, we have shown that the aldolase, which is induced by either of the two hexuronates or by gluconate (the metabolism of which involves the same kind of enzymatic cleavage), is controlled by a single structural gene. Extracts of this aldolase which have been induced separately by each of the three compounds, were purified, mixed and chromatographed on DEAE‐Sephadex: the mixture is not dissociable. The kinetics of thermal denaturation of each of these crude extracts are absolutely identical. Finally, the isolation of mutants deficient in aldolase has contributed genetic evidence. These mutants have always simultaneously lost the faculty of metabolizing the three compounds. Spontaneous reversion of these mutants, allowing growth on any of the three substrates, or transduction of any of the three growth phenotypes always leads to the recovery of growth on either of the other two compounds. These different and independent arguments bear out the uniqueness of the aldolase regardless of the inducer employed.
Studies on Ferricytochrome c
Authors: Greenwood Colin , Wilson Michael T. 
Published: March 2005

Titration of the 695 nm band of ferricytochrome c has been fitted by a model involving four ionic species over the pH range 0–11.
A co‐operative effect between protein denaturants and pH has been observed in the bleaching of the 695 nm band. This effect is discussed in terms of a conformational change and protein titration data suggest this to be of a limited nature.
Circular dichroism and magnetic circular dichroism studies have revealed that the 695 nm absorption band has an associated, well defined dichroism. Magnetic circular dichroic spectra are discussed in terms of haem symmetry.
Wechselwirkungen der Hefe‐Phosphofructokinase mit Dextranblau 2000
Authors: Kopperschläger Gerhard , Diezel Wolfgang , Freyer Renate , Liebe Stefan , Hofmann Eberhard 
Published: March 2005

Yeast phosphofructokinase binds to Blue Dextran 2000® with remarkable high affinity. Whereas phosphofructokinase from muscle and erythrocytes also have high affinity to Blue Dextran, aldolase, hexokinase, pyruvate kinase from muscle and glyceraldehyde‐3‐phosphate dehydrogenase show no affinity to this chromophore.The chromophoric component of Blue Dextran seems to be responsible for these interactions, because chromophore‐free dextran 2000 does not show any affinity to phosphofructokinases.Blue Dextran immobilized by fixation in cross‐linked polyacrylamide gel can be used as a valuable tool for affinity chromatography of phosphofructokinase. The conditions of binding, namely pH dependence, effects of ionic strength and of enzyme effectors have been studied using gel‐fixed phosphofructokinase. ATP (2–3 mM) but not ITP (up to 5 mM) specifically splits the enzyme chromophore complex.With non‐inhibitory concentrations of ATP Blue Dextran inhibits the enzyme very strongly showing competition with respect to ATP. No correlation between inhibition by Blue Dextran and fructose‐6‐phosphate concentration could be detected.A model is proposed for the interactions of Blue Dextran with phosphofructokinase, postulating binding of the chromophore to the ATP binding sites of the enzyme.
Kinetic Study of the Dicarboxylate Carrier in Rat Liver Mitochondria
Authors: Palmieri Ferdinando , Prezioso Girolamo , Quagliariello Ernesto , Klingenberg Martin 
Published: March 2005

The rate of uptake in mitochondria as catalyzed by the dicarboxylate carrier of dicarboxylates and, in the presence of N‐ethylmaleimide, of inorganic phosphate, has been analyzed by using the inhibitor stop method.

By comparing the dependence on substrate concentration of the rate of succinate, malonate and malate uptake, it is found that the Km (malate) = 0.23 < Km (malonate) = 0.37 < Km (succinate) = 1.17 mM. The V values, however, are not significantly different for all three dicarboxylates (V∼ 70 μmoles/g protein × min at 9°C).
Succinate, malate and malonate are shown to be competitive with each other in the kinetics of uptake.
The inhibition of the rate of malate uptake by 2–phenylsuccinate is found to be competitive.
Several “permeant” anionic substrates such as acetate, pyruvate, 3‐hydroxybutyrate, glutamate and aspartate have no effect on the rate of dicarboxylate uptake, even when added in large excess.
The rate of malonate uptake is inhibited by 2‐oxoglutarate but unaffected by citrate, while the rates of succinate and malate uptake are decreased by both 2‐oxoglutarate and citrate. Malate is inhibited more than succinate. The inhibition of the rate of malate uptake by citrate and 2‐oxoglutarate is shown to be non‐competitive. This is explained due to a re‐exchange of accumulated malate by citrate or 2‐oxoglutarate.
In the presence of N‐ethylmaleimide, externally added Pi inhibits the rate of dicarboxylate uptake. The Pi inhibition of malate uptake is found to be mixed with a larger share of non‐competitive than competitive inhibition.
The V of the rate of P1 uptake is approximately equal to that of malate uptake in the presence of N‐ethylmaleimide. The K1 is, however, higher for Pi (Ki=Kp= 1.95 mM) than for malate.
It is concluded firstly that the dicarboxylate carrier specifically transports either a dicarboxylate or Pi (but not both together), and secondly that the carrier has two separate binding sites, one specific for Pi and the other specific for the dicarboxylates.
Spin Labeling Studies of d‐Glyceraldehyde‐3‐phosphate Dehydrogenase
Authors: Balthasar Wolff 
Published: March 2005

d‐Glyceraldehyde‐3‐phosphate dehydrogenase has been reacted with spin labels, which were designed to elucidate the geometry of the active site of the enzyme. Three series of spin labels were used, which represented either substrate and coenzyme analogs or irreversible inhibitors, the separation of the active site directing part from the nitroxide radical being readily adjusted through the length of the intervening methylene chain.The effect of this variation on the electron paramagnetic resonance spectrum of the labeled protein as well as the effect of coenzyme binding on these spectra have been investigated. All spectra are complex and represent the superposition of at least three different types of spectra. These three types of spectra are correlated to nitroxide radicals in three different types of motion: (a) very fast rotation in a fluid region, (b) medium fast rotation of a hindered radical and (c) slow rotation of an almost immobilized radical. The spin labels differ only in the relative population in the three states related to the three types of motion. Also addition of the coenzyme NAD only alters this relative population. A spin labeled coenzyme analog, which has the nitroxide in the region of the sugar‐nicotinamide bond, is bound to the protein and displays an electron paramagnetic resonance spectrum typical for an immobilized radical. The geometry of the active of d‐glyceraldehyde‐3‐phosphate dehydrogenase is complex and cannot be described as a simple cleft, but instead as a system of narrow and wider spaces, which is changed by the binding of the coenzyme.
Co‐operative Non‐Enzymic
Authors: Podder Sunil K. 
Published: March 2005

The absorbance and the optical rotatory dispersion of solutions of GpGpGpC and GpCpCpC individually and of the mixture of these two were measured in 0.1 M phosphate buffer (pH 7.2) as a function of temperature and concentration. The data indicate that below room temperature GpGpGpC self‐associates into large aggregates which presumably involve both G · C and G · G base pairs. It interacts as well with GpGpCpC to form a 1:1 GpGpGpC: GpCpCpC complex containing 4 G · C base pairs. The kinetics of formation of the 1:1 complex, as well as of self‐associated complexes, were studied by temperature jump relaxation method. The transition temperature Tmof the 1:1 complex was determined from the variation of the relaxation amplitude with temperature. From the concentration dependence of Tm, the values of – 21.6 kcal/mole and –50 entropy units were calculated for ΔH and ΔS respectively for the formation of the 1:1 complex.Temperature‐jump relaxation experiments on the individual components over the temperature range 20–40°C indicate a single process which was too rapid to follow (t0.5≪ 5 μsec), corresponding to the melting of a single stranded helix. In experiments with mixed solutions a slower measurable process was observed in the millisecond time range, which was attributed to 1:1 complex formation with a recombination rate constant kr= 5.4 106M−1× sec−1 and a dissociation rate constant kd= 40 sec−1 (at 21°C), ΔE‡r= 4.5 kcal/mole and ΔE‡d=+ 28.7 kcal/mole. Analysis of thermodynamic and kinetic data in terms of the “all or none” model suggests that the formation of the second base pair is the rate limiting step in the G · C helix formation. A single base pair adjacent to a pre‐existing helical sequence can be formed in 0.1 μsec. In contrast, the apparent formation and dissociation rate constants of GpGpGpC aggregates are smaller than those of the 1:1 complex by several orders of magnitude. The existence of a syn‐anti type equilibrium below room temperature is postulated to explain this observation.
An Efficient Procedure for the Isolation of Polyribosomes from Tissue Culture
Authors: Gielkens Arno L. J. , Berns Ton J. M. , Bloemendal Hans 
Published: March 2005

In this paper an efficient procedure is described for the isolation of polysomes from tissue culture cells. Optimal yields, especially of the heavier classes of polysomes can only be obtained at a rather high KC1 concentration in the presence of nonionic detergent. Evidence is provided that the lower polysomal yield after extraction at low salt concentration is not due to breakdown of the polysomes by RNAase.Furthermore, the yield of polysomes is pH dependent only at low salt concentration, while at high salt concentration no influence of the pH is observed. Refreshing of growth medium several hours before harvesting the cells, increases the ratio of polysomes to 80 S monomers.
Recherches sur les acides désoxyribonucléiques d'Acetabularia mediterranea
Authors: Heilporn Viviane , Limbosch Sylvie 
Published: March 2005

The density of the DNA present in the cytoplasm of Acetabularia mediterranea has been determined by centrifugation in a CsCl gradient. Electron microscope examination has demonstrated that a unique DNA having a density of 1.704 g/cm3 is extracted from a pure fraction of chloro‐plasts. The method elaborated by Suyama and Bonner for the isolation of the mitochondria has enabled us to isolate a sub‐cellular fraction with cytochrome oxidase activity, from which we were able to extract a small amount of pure DNA which is probably mitochondrial DNA, having only one density peak at 1.715 g/cm3; unlike mitochondrial DNA, chloroplast DNA is sensitive to prolonged maintenance of the algae in the dark, as well as to treatment of a suspension of chloroplasts and mitochondria with deoxyribonuclease. Under our experimental conditions, centrifugation of cytoplasmic DNA in CsCl to which ethidium bromide is added seems to exclude the possibility that circular molecules of DNA occur whether of chloroplastic or of mitochondrial origin. Both cytoplasmic DNA readily undergo thermal denaturation. Renaturation of chloroplast DNA is incomplete, whereas mitochondrial DNA renatures readily completely. During the process of cyst formation and nuclear multiplication, it is possible to extract newly formed DNA which is probably nuclear: it has a density of 1.696 g/cm3. All three DNA, with a boyant density of 1.696, 1.704, 1.715 g/cm3, respectively, disappear after treatment with deoxyribonuclease, but remain intact after treatment with a mixture of α‐ and β‐amylases.We would like to suggest that following many years of laboratory culture, Acetabularia mediterranea may have given rise to new genetically distinct species. Such an hypothesis would explain the apparent lack of agreement between our own findings and those of Gibor and of Schweiger.
Comparative Structural Studies of the Active Site of ATP: Guanidine Phosphotransferases
Authors: der Terrossian Elisabeth , Desvages Gisèle , Pradel Louise‐Anne , Kassab Rhida , van Thoai Nguyen 
Published: March 2005

The active thiol group of lombricine kinase from Lumbricus terrestris muscle was labelled with N‐ethyl‐[1‐14C]maleimide. The resulting inactivated N‐ethyl‐[1‐14C]succinimido enzyme was then subjected to tryptic hydrolysis.The peptide containing the labelled essential thiol group was isolated and found to contain: Leu‐Gly‐Tyr‐Ile‐Thr‐[14C]Cys‐Pro‐Gly‐Ser‐Asn‐Leu‐Gly‐Thr‐Leu‐Arg. The amino acid sequence around this thiol group was very similar with that of homologous ATP: guanidine phosphotransferases previously studied, arginine kinase from Homarus vulgaris muscle, creatine kinases from ox brain and ox muscle and from rabbit muscle.In addition among the other enzymes of this group, lombricine kinase is of special interest since it is the only dimeric enzyme of molecular weight ≃ 80000 which possesses only one essential thiol group and one nucleotide binding site per two subunits.
Ribosomal Proteins
Authors: Hindennach Ingrid , StÖFFLER Georg , Wittmann Heinz‐Günter 
Published: March 2005

All 21 ribosomal proteins from 30 S subunits of Escherichia coliwere isolated in a pure state by the following methods: separation of subunits by zonal centrifugation in B X V rotors, extraction of proteins, CM‐cellulose chromatography with pyridine formate gradients, gel filtration on Sephadex G‐100 and, in some cases, preparative polyacrylamide block electrophoresis. The purity of isolated proteins was 97–100% shown by disc electrophoresis at pH 4.5 in urea, by two‐dimensional polyacrylamide electrophoresis, by dodecylsulfate gel electrophoresis and by cellulose acetate gel electrophoresis. The yield of the isolated proteins was 7–110 mg depending on the protein and the number of purification steps.Fractionation of total 30 S proteins with ammonium sulfate is recommended for quick isolation of proteins S15 and S20 in large quantities.
Studies on Adenosine Deaminase
Authors: Maguire M. Helen , Sim Meng K. 
Published: March 2005

The substrate specificity of adenosine deaminase purified from the maternal component of the bovine placenta has been studied in detail, and the effects of different types of inhibitors have been examined. The enzyme catalysed the hydrolytic removal of amino, chloro, hydroxylamino, methoxy and methoxyamino substituents from the 6 position of purine ribonuclesides; 2‐substitution by amino, chloro or fluoro groups reduced the V of adenosine and 6‐chloropurineriboside but 2‐amino substitution increased the V of 6‐methoxypurineriboside. The results indicate that both a bond‐forming component dependent on steric factors, and a bond‐stretching component dependent on the electronegativity of the leaving group are involved in the rate‐determining formation of the transition complex. 2‐Alkylamino‐, 2‐alkylthio‐, and 2‐halogenoadenosines are competitive inhibitors with Ki values which conform the importance of the basicity of N1 of substrates and inhibitors in their binding to the active site of the enzyme.The enzyme was shown to have dissociating groups with pKa values of 9.8, 8.9 and 5.7. Enzyme activity was inhibited by p‐chloromercuribenzote (PCMB), and titration of the native enzyme with this mercurial showed that there are two sulphydryl groups per mole of enzyme. There was no reaction of the enzyme with the sulphydryl reagent, 5,5′‐dithiobis‐(2‐nitrobenzoic acid). 2‐Mercaptoethanol and glutathione in high concentrations inhibited the enzyme; inhibition by glutathione was irreversible.
Biochemische Differenzierung der Pr1‐/Pr2‐determinierenden von der MN‐determinierenden N‐Acetyl‐Neuraminsäure durch Acetylierungsversuche mit Erythrocytenglykoproteinen
Authors: Merz Wolfgang , Roelcke Dieter 
Published: March 2005

In contrast to MN receptors, Pr1 and Pr2 antigens of isolated glycoprotein of erythrocytes are not inactivated by acylation of free amino groups.By introduction of easily leaving groups, e.g. trifluoroactic residues, inactivation of the MN activity can be made reversible.No differences between Pr1 and Pr2 antigens could be found with the reactions described in this paper.Acylation in aqueous phase (buffer/anhydrous acetic acid) and in organic phase (pyridine/anhydrous acetic acid) gave identical results qualitatively and quantitatively. It was not possible to split the N‐acetic binding neither under mild alkaline conditions nor with aminoacylase. 4–5 amino groups out of 11 could be acylated, whereas succinylation in aquous phase (buffer/anhydrous succinic acid) resulted in modification of only one free amino group, the calculations being based on a molecular weight of 30000. Following succinylation MN activity was also lost, whereas the antigenic activity of Pr1 and Pr2 remained unchanged. The appearance of a negative charge instead of the positive charge of the free amino group on the glycoprotein did not affect Pr1 or Pr2 activity in any way.If glycoprotein was esterified under conditions which cause esterification of free sialic acid to a wide extent neither MN activity nor Pr1/Pr2 activity was altered.
Mechanism of Action of Specific Antiserum on Pasteurella septica
Authors: Griffiths Elwyn 
Published: March 2005

Circumstantial evidence suggests that the ability of the host to prevent the uptake of iron by pathogenic bacteria may constitute an important means of defence. A detailed biochemical study of the way antisera exert their antibacterial effects and the possible involvement of iron has now been initiated.
Work with Pasteurella septica antiserum has revealed a totally unsuspected way in which antiserum and complement exert their antibacterial effects. The process is very quick and specific and results in the cessation of bacterial multiplication; cells in stasis then appear to be killed by the operation of a secondary event. The inhibitory process operates by affecting the biochemistry of the bacterial cell leading first to an inhibition of net RNA synthesis and then to an inhibition of all macromolecular synthesis.
The presence of free iron in the serum did not prevent the initiation of the inhibition but it did allow macromolecular synthesis, cell multiplication and rapid growth to be resumed later. In contrast, the presence of haematin in the serum allowed the cells to continue multiplying in the presence of antiserum without delay. Addition of haematin to bacteria which had been inhibited by antiserum restored net RNA synthesis immediately. Net protein synthesis and multiplication were restarted within 15–20 min and DNA synthesis in 40 min.
Dissociation of Pila Haemocyanin at Low Ionic Strength
Authors: Hoebeke Johan J. , Elliott Francis G. 
Published: March 2005

The study of the haemocyanin of Pila leopoldivillensis by means of zone electrophoresis on cellulose acetate has shown that its dissociation is favoured by lowering of the ionic strength.These results were confirmed and extended over a larger range of pH and ionic strength values by means of light scattering measurements. An interpretation of the results is given in terms of ionic equilibria. The type and the number of charged groups involved in the dissociation into halves and into tenths are proposed.
Substrate‐and Antibiotic‐Binding Sites at the Peptidyl‐Transferase Centre of Escherichia coli Ribosomes
Authors: Fernandez‐Muñoz Rafael , Monro Robin E. , Vazquez David , Torres‐Pinedo Ramon 
Published: March 2005

In Escherichia coli there is one binding site per ribosome for lincomycin, chloramphenicol and erythromycin.These antibiotics act at closely related sites on the 50‐S subunit. There are, however, some interesting differences in the properties of the three sites, such as: (a) the apparent possibility of obstructing the lincomycin site without closing the chloramphenicol site, (b) asymmetry of competition effects between chloramphenicol and erythromycin, (c) differential responses to ethanol and (d) differential responses to other antibiotics (puromycin, macrolides, streptogramins A and B and others also acting on the larger ribosome subunit).
Isolierung und partielle Charakterisierung eines 8Sα3‐Glykoproteins aus Humanserum
Authors: Haupt Heinz , Baudner Siegfried , Kranz Theodor , Heimburger Norbert 
Published: March 2005

Isolation and partial characterization of a still‐unknown 8Sα3‐glycoprotein from human serum is described. Supernatant II is used as the starting material; it is a side fraction from the Rivanol®‐ammonium‐sulphate procedure of preparing albumin and gamma‐globulin on a large scale.8Sα3‐glycoprotein proves to be homogeneous according to free‐boundary electrophoresis and to molecular‐sieve electrophoresis. During electrofocusing several bands can be observed in the rangs of pH 4.2–4.6. In the characterization of this protein by ultracentrifugation a slight inhomogeneity becomes evident which is not observed when gel filtration is applied. The sedimentation constant is 7.87 S and the molecular weight is calculated to be 220000. The sedimentation coefficient decreases by treatment with propionic acid or an acid solution of urea. A partial dissociation of the molecule is achieved by means of reducing agents. It is also possible to dissociate this protein completely with 2‐mercaptoethanol followed by alkylation and treatment with an acid‐urea solution.8Sα3‐glycoprotein contains approximately 30% carbohydrate, from which 9.2% is sialic acid. The protein moiety accounts for 70% of the total molecule. The isolated protein is characterized by high solubility in solutions containing protein‐precipitating agents. The concentration in normal human serum is in the range of 3.1–4.5 mg/100 ml.