bluuebunny/crossref_metadata_2025_split huggingface dataset:
A major nucleolar protein, nucleolin, induces chromatin decondensation by binding to histone Hl
Authors: ERARD Monique S. , BELENGUER P. , CAIZERGUES‐FERRER M. , PANTALONI A. , AMALRIC F. 
Published: March 2005

Using circular dichroism to probe the extent of DNA condensation in chromatin, we have demonstrated that a major nucleolar protein, nucleolin, can decondense chromatin. By means of various binding assays we show that nucleolin has a strong affinity for histone H1 and that the phosphorylated N‐terminal domain, rich in lengthy stretches of acidic amino acids, is responsible for this ionic interaction. Additional experiments clearly demonstrate that nucleolin is unable to act as a nucleosome core assembly or disassembly factor and hence has little affinity for the core histone octamer.We propose that this nucleolar protein induces chromatin decondensation by binding to histone H1, and that nucleolin can therefore be regarded as a protein of the high‐mobility‐group type.
Structural characterization of a murine myeloid leukaemia inhibitory factor
Authors: SIMPSON Richard J. , HILTON Douglas J. , NICE Edouard C. , RUBIRA Michael R. , METCALF Donald , GEARING David P. , GOUGH Nicholas M. , NICOLA Nicos A. 
Published: March 2005

A leukaemia inhibitory factor (LIF) which induces macrophage differentiation in M1 murine myeloid leukaemia cells and suppresses their proliferation in vitro has been isolated in sufficient quantities (30 μg) from Krebs ascites tumour cell conditioned medium to permit its partial characterization by amino acid sequence analysis. The combination of sensitive microbore column (1.0 and 2.1 mm internal diameter) HPLC technology and microsequence analysis has enabled the positive identification of 125 of the total 179 amino acid residues (70%) in the molecule. The amino acid sequence data reported here permitted the isolation of a partial cDNA clone encoding LIF [Gearing et al. (1987) EMBO J. 6, 3995–40021. A candidate C‐terminus of the LIF molecule predicted from the amino acid sequence was confirmed by subsequent isolation of a cDNA clone corresponding to the C‐terminus of the protein.No strong similarity was revealed when the amino acid sequence of LIF was compared with other haemopoietic growth factors, in particular granulocyte‐macrophage colony‐stimulating factor, granulocyte colony‐stimulating factor and tumour necrosis factor‐α or interleukins. The protein sequence data reported here indicate three sites of post‐translational modification (N‐linked glycosylation).
Hyper‐regulation of pyr gene expression in Escherichia coli cells with slow ribosomes
Authors: JENSEN Kaj Frank 
Published: March 2005

UTP‐modulated attenuation of transcription is involved in regulating the synthesis of pyrimidine nucleotides in Escherichia coli. Thus, expression of two genes, pyrBI and pyrE, was shown to be under this type of control. The genes encode the two subunits of aspartate transcarbamylase and orotate phosphoribosyltransferase respectively. The levels of these enzymes are inversely correlated with the intracellular concentration of UTP. Modulation of attenuation seems to be a consequence of the effect of UTP concentration on the mRNA chain growth rate. Reducing the UTP pool retards RNA polymerase movement. Mechanistically this will couple the ribosomes translating a leader peptide gene more tightly to the elongating RNA polymerase. The ribosomes will then be more prone to prevent the folding of the mRNA chains into terminating hairpin structures when RNA polymerase is at the attenuator and has to decide whether transcription should terminate or continue into the structural genes. This paper described a study of pyrBI and pyrE gene regulation in cells where the ribosomes move slowly as a result of mutation in rpsL. It appears that expression of the two genes is hyper‐regulated by the UTP pool in this type of cells.Furthermore, the attenuator model can only account for the results if it is assumed that UTP‐concentration‐dependent pausing of transcription occurs in vivo in the two pyr gene leaders such that RNA polymerase waits for the coupled ribosomes before transcribing into the attenuator regions.
Isolation, molecular properties and kinetic studies of a strict β‐fucosidase from Lactuca sativa latex
Authors: GIORDANI Roger , NOAT Georges 
Published: March 2005

A β‐fucosidase located in the latex serum of Lactuca sativa has been isolated and purified to homogeneity. This enzyme greatly differs from the other fucosidases already known in that it is strictly specific for the fucosyl residue and for the anomeric β carbon. It is the first time that such an enzyme is shown to exist.The enzyme is a monomer and its molecular mass is close to 37 kDa. Its sedimentation constant is equal to 2.8 S. It is very stable at pH 5.5 in citrate/phosphate buffer but extensive denaturation occurs up to pH 7.5. Kinetic studies have shown that two ionization steps probably control the rate of the enzymatic hydrolysis.No precise information could be obtained about the possible in vivo role of this β‐fucosidase. However, the pure enzyme can release fucose from the cell walls obtained from hypocotyls of L. sativa. This result may be taken as evidence for the presence of β‐fucosidic links in these walls so that the enzyme could be involved in the differentiation of articulated laticifers.
Purification and caracterization of DNA polymerase α from plasmodia of Physarum polycephalum
Authors: WEBER Cornelia , FISCHER Heinrich , HOLLER Eggehard 
Published: March 2005

DNA polymerase α from Physarum polycephalum has been purified fromfreshly harvested microplasmodia. An inhibitory activity was removed by precippitation with poly(ethyleneimine) and interfering type‐β‐like DNA polymerase by chromatography on phosphocellulose. The preparation was free of endonucleases and exonucleases.The DNA‐polymerizing polypeptide had a molecular mass of 140kDa by polyacrylamide gel electrophoresis under denaturing conditions. It was contained in purified samples and in crude cell extracts. Peptides of smaller size that reacted with antibodies against this protein were generated during purification and prolonged standing. Molecular sizing under non‐denaturing conditions resulted inhigh‐molecular‐mass forms.The isoelectric point (pI) was 6.7 ± 0.2, the pH optimum at pH 6.8, and the temperature optimum at 40°C. Monovalent salts, Li+, Na+, NH4+, K+, were inhibitory except for small activation maxima at 10mM, 75 mM and 100 mM in the case of Na+, NH4+ and K+ respectively. The bivalent cations Mg2+ and Mn2+ had broad activity maxima at 3‐20 mM concentrations, which were shifted to 0.05‐0.1 mM in the case of Mn2+ and synthetic DNA homopolymers.The numbers of molecules of DNA polymerases in Physarum nuclei were calculated and compared with the established number of molecules of DNA polymerasesinhigher eucaryotes
Fluorinated analogues of spermidine as substrates of spermine synthase
Authors: BAILLON Jean G. , MAMONT Pierre S. , WAGNER Joseph , GERHART Fritz , LUX Philippe 
Published: March 2005

A series of mono‐and geminal difluorinated analogues of spermidine (4‐azaoctane‐1, 8‐diamine) have been tested as potential substrates of partially purified rat hepatoma (HTC) cell or pure bovine spleen spermine synthase (EC 2.5.1.22).Substitution of the hydrogen atoms of the methylene group at position 7 by one or two fluorine atoms decreases 8‐fold and 160‐fold the apparent Km values for the HTC cell enzyme respectively. Similarly, the Km values of 7‐monofluoro and 7, 7‐difluorospermidine for the pure bovine enzyme are reduced 8‐fold and 100‐fold respectively, incomparison with spermidine. Di‐, but not monofluoro substitution, in the 6‐position causes a 5‐fold reduction in the affinity for the HTC cell enzyme. Gem‐fluorine substitution in the 2‐position a abolishes substrate capability.In addition to their high affinity for spermine synthase, 7‐monoflurospermidine and 7, 7‐difluorospermidine cause substrate inhibition. This phenomenom, which is more pronounced in the case of the difluorinated analogues is pH‐dependent.These enzymatic findings are discussed with regard to the protonation sites of the spermidine analogues, determined by potentiometric titration, which vary as a function of the number and position of the fluorine substituents relative to the basic amino groups.
Effect of solvent, pressure and temperature on reaction rates of the multiheme hydroxylamine oxidoreductase
Authors: BALNY Claude , HOOPER Alan B. 
Published: March 2005

Hydroxylamine oxidoreductase (HAO) of the ammonia‐oxidizing bacterium Nitrosomonas catalyzes the oxidation: NH2OH + H2O · HNO2+ 2e−+ 2 H+. The heme‐like chromophore P460 is part of a site which binds substrate, extracts electrons and then passes them to the many c hemes of the enzyme. Reduction of the c hemes by hydroxylamine is biphasic with apparent first‐order rate constants k1, and k2,. CO binds to ferrous P460 with apparent first‐order rate constants, k1,CO. In this work we have measured the binding of CO to ferrous P460 of hydroxylamine oxidoreductase and the reduction by substrate of some of the 24 c hemes of the ferric enzyme. These reactions have been studied in water and 40% ethylene glycol, at temperatures ranging from‐15°C to 20.7°C and at hydrostatic pressures ranging over 0.1‐80 MPa. From the measurements, thermodynamic parameters ΔV‡ (activation volume), ΔG‡, ΔH‡ and ‡‡S‡, have been calculated. CO binding. Binding of CO to ferrous P460 was similar to the binding of CO to ferrous horseradish peroxidase. The change of solvent had only a limited effect on ΔV# (‐30 ml Δ mol−1), ΔG‡, ΔH‡ or S‡ and did not cause an inflection in the Arrhenius plot or downward displacement of the linear relationship between In k1,CO and P at a critical temperature. Binding was exothermic at high temperatures. The response of the binding of CO to solvent, temperature and pressure suggested that the CO binding site had little access to solvent and was not susceptible to change in protein conformation. Fast phase of reduction of c hemes. Changing the solvent from water to 40% ethylene gh col resulted in a decrease from 90% to 50% in the relative number of c hemes reduced during the fast phase, an increase in activation volume from −3.6 ml ± mol−1 to 57 ml · mol−1 and changes in other thermodynamic parameters. The activation volume increased with decreasing temperature. The Arrhenius plot had a downward inflection at about 0°C and, in water or ethylene glycol, the linear dependence of In k1 on P was displaced downwards as the temperature changed from 3.5°C to ‐15°C. Slow phase of reduction of c hemes. Changing the solvent from water to 40% ethylene glycol resulted in an increase in the relative number of c hemes reduced during the slow phase from 10% to 50%. The activation volume, which was not measurable in water because of the low absorbance change, was ‐30 ml & mol−1 in ethylene glycol. The activation volume increased with increasing temperature. The Arrhenius plot had a downward inflection at about 0°C yet the linear dependence of In k2 on P had no downward displacement in this temperature range. At high pressure and low temperatures the rate of heme reduction increased with decreasing temperature. In contrast to the reaction of CO with the substrate site, the results suggested that changes in enzyme conformation imposed by changes in solvent, temperature or pressure affected the rates of intramolecular electron transfer from the substrate site to c hemes.
Histidyls in lobster arginine kinase
Authors: ROUX‐FROMY Madeleine , PHO Dang Ba , KASSAB Rhida 
Published: March 2005

The role of histidyls in lobster arginine kinase (EC 2.7.3.3) has been studied by 1H‐NMR spectroscopy of the enzyme and its complexes with substrates or their analogues and 31P‐NMR spectroscopy of complexes with ADP.Five histidyls were detected by 1H‐NMR in native enzyme (His 1 to His 5). Three of them appeared possibly to be implicated in catalysis: His 3, whose pH/titration was affected by arginine binding, and His 1 and 4, show from paramagnetic relaxation by Mn2+ to be close (≤ 1.2 and ≤ 1.27 nm respectively) to the metal cofactor. His 4 was broadened beyond detection in the presence of any adenine nucleotide. In the enzyme reversibly inactivated by histidine ethoxyformylation, the modified histidyl was His 1. In the transition state analogue complex (in which NO‐3 mimics the transferred phosphoryl), Hill plots of histidyl pH/titration curves showed that His 1 and His 3 were both interacting with the same set of three titratable groups and hence spatially close. 31P‐NMR demonstrated that ADP binding in this complex was unaffected by the chemical modification of His 1.It is concluded that His‐ethoxyformyl‐enzyme is inactive because ethoxyformyl‐His 1 is unable to titrate. This is consistent with His 1 acting as the acid‐base catalyst. However our results, which do not indicate any catalytic role of His 3, exclude any H‐bonding of His 1 on either substrate. Involvement is needed of at least one other titratable residue for the proton evolved in the catalysis to exchange directly with the guanidino substrate.
Activation of phosphofructokinase 2 by insulin in cultured hepatocytes without accompanying changes of effector levels or cAMP‐stimulated protein kinase activity ratios
Authors: MÜLLER Andreas , UNTHAN‐FECHNER Kirsten , PROBST Irmelin 
Published: March 2005

Activation of glycolysis by insulin in cultured adult rat hepatocytes is accompanied by an activation of phosphofructokinase 2 (PFK 2). PFK 2 activation might be caused by insulin‐dependent changes of (a) metabolite levels, (b) basal and (c) Br8cAMP‐stimulated cAMP‐dependent protein kinase activity; this problem was investigated.

Cells cultured with 0.1 nM insulin for 48 h exhibited a low glycolytic rate and low fructose 2,6‐bisphosphate [Fru(2,6)P2] levels. Addition of insulin increased Fru(2,6)P2 and Fru(l,6)P2 levels sequentially which points to PFK 2 as first target enzyme of insulin action.
Concentrations of Glc6P, Fru6P, phosphoenolpyruvate, glycerol 3‐phosphate and citrate, which modulate PFK 2/fructose 2,6‐bisphosphatase 2 activity, were not altered by insulin.
Activation of PFK 2 by insulin occurred without changes in the levels of total and protein‐bound cAMP. Bound cAMP amounted to about 14% of total cAMP.
Insulin neither decreased the basal dissociation state of the cAMP‐dependent protein kinase nor lowered the sensitivity of the kinase towards cAMP in cell extracts.
Addition of the phosphodiesterase‐resistant Br8cAMP to the cultures increased cAMP levels 3–4‐fold, elevated the protein kinase activity ratio from 0.14 to 0.6 and decreased the Fru(2,6)P2 level and the rate of glycolysis. When Br8cAMP and insulin were given together, insulin was capable of counteracting Br8cAMP in that it activated glycolysis and PFK 2 and elevated the level; however, it did not decrease the elevated protein kinase activity ratio.

It is concluded that insulin presumably does not activate PFK 2 through changes in cAMP and effector levels or through inhibition of cAMP‐dependent protein kinase dissociation. The data support the hypothesis that insulin may act via activation of PFK 2 phosphatase.
Chemical modification of tyrosine‐38 in P‐Hydroxybenzoate hydroxylase from Pseudomonas fluorescens by 5′‐P‐fluorosulfonylbenzoyladenosine: A probe for the elucidation of the NADPH binding site?
Authors: Van BERKEL Willem J. , MÜLLER Franz , JEKEL Peter A. , WEIJER Wicher J. , SCHREUDER Herman A. , WIERENGA Rik K. 
Published: March 2005

P‐Hydroxybenzoate Hydorxylase from Pseudomonas Fluorescens was covalently modified by the nucleotide analog 5′‐(P‐fluorosulfonylbenzoyl)‐adenosine in the presence of 20% dimethylsulfoxide, The inactivation reaction is pH‐dependent and does not obey pseudo‐first‐order kinetics, due to spontaneous hydrolysis of the reagent. The kinetic data further indicate that a weak, reversible enzyme‐inhibitor complex is an intermediate in the inactivation reaction and that only one amino acid residue is responsible for the loss of activityThe inactivation is strongly inhibited by NADPH and 2′, 5′ADP. Steady‐state kinetics and 2′,5′ADP bioaffinity chromatography of the modified enxzyme suggest that the enssential residue is not directly involved in NADPH binding.Sequence studies show that Tyr‐38 is the main residue protected form modicfication in the presence of NADPH. From crystallorgraphic studies it is known that the hydroxyl group of Tyr‐38 is 1.84 nm away form the active site. Model‐Building studies using computer graphics show that this distance can be accommodated when FSO2BzAdo binds in an extended comformation with the sulfonylbenzoyl protion in an orientaiton different from the nicotinamide ring of NADPH.
Long‐term ingestion of ammonium increases acetylglutamate and urea levels without affecting the amount of carbamoyl‐phosphate synthase
Authors: FELIPO Vicente , MIÑANA María‐Dolores , GRISOLÍA Santiago 
Published: March 2005

Rats were fed the following diets: standard (20% protein), high‐protein (80%), protein‐free, standard plus ammonium and protein‐free plus ammonium for six weeks. The standard plus ammonium diet was prepared to contain ammonia equivalent to that supplied by the high‐protein diet. Addition of ammonium acetate 20% by mass) to the 20% protein or protein‐free diets results in 2.3‐ and 10‐fold increases of urea excretion respectively, without increase of carbamoyl‐phosphate synthase.Supplementation of the standard diet with ammonium increases the mitochondrial content of acetylglutamate from 830 to 1590 pmol/mg protein, and of the protein‐free diet from 130 to 1040 pmol/mg. However, ingestion of ammonium did not increase the activity of acetylglutamate synthase. Therefore the efflux of acetylglutamate from mitochondria was determined. After 30 min at 37°C liver mitochondria from rats on standard diet released 61% of the initial acetylglutamate while mitochondria from animals on standard plus ammonium diet released only 20%. These results indicate that ingestion of ammonium increases the content of acetylglutamate in rat liver by decreasing its efflux from mitochondria. This effect is similar to that produced in mice by a high protein diet [Morita et al. (1982) J. Biochem. (Tokyo) 91, 563‐569]. However while the high‐protein diet increases carbamoyl‐phosphate synthase content, the ammonium diet does not.
DNA‐binding properties of gene‐5 protein encoded by bacteriophage M 13
Authors: BULSINK Henk , HARMSEN Ben J. M. , HILBERS Cornelis W. 
Published: March 2005

The irreversible dissociation kinetics of complexes of M13‐encoded gene‐5 protein with the polynucleotides poly(dA) and M13 DNA was studied by means of stopped‐flow experiments. A linear decay was found for all gene‐5‐protein · poly(dA) complexes and for the gene‐5‐protein · M13 DNA complexes for which the DNA lattice was completely saturated at the beginning of the dissociation experiments. Only at the end of the dissociation curve was a deviation from linearity observed. A single‐exponential decay was found for the dissociation of gene‐5‐protein · M13 DNA complexes when the DNA was not completely saturated initially. These results could be interpreted by assuming that dissociation of bound protein is only possible from isolated binding sites, while during the dissociation, rearrangement of bound protein clusters takes place continuously, including the formation of newly isolated bound protein. This redistribution results from a translocation of the protein along the lattice, which, for the poly(dA) complex, is fast with respect to the dissociation step, but which is slow for the M13 DNA complex. During this process the equilibrium cluster distribution predicted by the theory of McGhee and Von Hippel [1] is not maintained.The binding of gene‐5 protein to poly(dA) or poly(dT) does not result in a broadening of the nucleotide resonances in the NMR spectra of these polynucleotides, as had been observed for E. coli DNA‐binding protein and interpreted as an indication for a high rate of translocation of the protein on the polynucleotide [2]. The absence of line broadening for gene‐5‐protein · polynucleotide complexes is caused by the high binding cooperativity. As a consequence the majority of the protein molecules are bound in a cluster which makes the concentration of isolated bound protein very low. This results in a decrease of the signal/noise ratio at higher degrees of binding, but does not lead to line broadening while fast translocation still occurs.
Tuna pepsinogens and pepsins
Authors: TANJI Masao , KAGEYAMA Takashi , TAKAHASHI Kenji 
Published: March 2005

Three pepsinogens (pepsinogens 1, 2, and 3) were purified from the gastric mucosa of the North Pacific bluefin tuna (Thunnus thynuus orientalis). Their molecular masses were determined to be 40.4 kDa, 37.8 kDa, and 40.1 kDa, respectively, by SDS/polyacrylamide gel electrophoresis. They contained relatively large numbers of basic residues when compared with mammalian pepsinogens. Upon activation at pH 2.0, pepsinogens 1 and 2 were converted to the corresponding pepsins, in a stepwise manner through intermediate forms, whereas pepsinogen 3 was converted to pepsin 3 directly. The optimal pH of each pepsin for hemoglobin digestion was around 2.5. N‐acetyl‐L‐phenylalanyl‐L‐diiodotyrosine was scarcely hydrolyzed be each pepsin. Pepstatin, diazoacetyl‐DL‐norleucine methyl ester in the presence of Cu2+, 1,2‐epoxy‐3‐(p‐nitrophenoxy)propane and p‐bromophenacyl bromide inhibited each pepsin, although the extent of inhibition by each reagent differed significantly among the three pepsins.The amino acid sequences of the activation segments of these pepsinogens were determined together with the sequences of the NH2‐terminal regions of pepsins. Similarities in the activation segment region among the three tuna pepsinogens were rather low, ranging over 28–56%. A phylogenetic tree for 16 aspartic proteinase zymogens including the three tuna pepsinogens was constructed based on the amino acid sequences of their activation segments. The tree indicates that each tuna pepsinogen diverged from a common ancestor of pepsinogens A and C and prochymosin in the early period of pepsinogen evolution.
Soaking in Cs2SO4 reveals a caesium‐aromatic interaction in bovine‐liver rhodanese
Authors: Ulco KOOYSTRA P. J. , KALK Kor H. , HOL Wim G. J. 
Published: March 2005

Soaking crystals of rhodanese (thiosulphate: cyanide sulphurtransferase) in 2 M caesium sulphate reveals three caesium binding sites of this enzyme. One of these had been described before as a binding site for sodium ions and is located in a cleft close to the active site. In this site the monovalent cation is coordinated by five oxygen atoms. The first additional binding site seems to be quite special. The caesium ion is bound to the phenyl ring of a tryptophan residue. It is further liganded by two oxygen atoms. The third binding site is a result of crystal packing effects: caesium is liganded by four oxygen atoms, provided by two rhodanese molecules and one sulphate ion.It is likely that the binding of caesium affects the fluorescence of the tryptophan residue with which it interacts. Such possible effects should also be kept in mind when caesium ions are used as a quencher in fluorescence studies of proteins in general.