bluuebunny/crossref_metadata_2025_split huggingface dataset:
Polymerization and gelation of fibronogen in D2O
Authors: LARSSON Ulf 
Published: March 2005

The solution properties of fibrinogen and the thrombin‐induced activation and gelation of fibrinogen in 95% D2O at pH 7.4 were compared to those in H2O under similar conditions.The initial release rates of fibrinopeptides A and B in D2O were slightly slower than those in H2O. However, the values of the Michaelis‐Menten parameters Km and V for the release of the two peptides in D2O and H2O in the presence of 0.5 M NaCl were about the same.From turbidity measurements at 450 nm it is obvious that fibrinogen is soluble in a slightly more narrow range of NaCl concentration and that the fibrin gels have a higher degree of lateral aggregation in D2O than in H2O.The variation of fibrinogen concentration, thrombin concentration, pH and ionic a strength have a similar dependence on the final gel structure and clotting time in D2O and H2O.SDS‐gel electrophoresis on fibrin samples, which were cross‐linked by factor XIII, yielded results where the cross‐linking of the γ‐chain appeared to be the same in D2O and H2O. The α‐chain cross‐linking was somewhat faster in D2O than in H2O.When fibrinogen solutions in 95% D2O were incubated at 20 mM CaCl2, a slow gelation of fibrinogen was observed, which was found to be induced by trace amounts of factor XIII. The final gel turbidity appeared to be about the same for this gelation as for that induced by thrombin.The differences in solubility for fibrinogen, kinetics for the enzyme reaction and optical properties for the fibrin gels in D2O and H2O may be explained by differences in electrostatic interactions, hydrogen bonding and hydration of fibrinogen in these two media.
Methanogenesis and ATP synthesis in methanogenic bacteria at low electrochemical proton potentials
Authors: KAESLER Bruno , SCHÖNHEIT Peter 
Published: March 2005

The rate of methane formation from H2 and CO2, the intracellular ATP content and the electrochemical proton potential (ΔμH+) were determined in cell suspensions of Methanobacterium thermoautotrophicum, which were permeabilized for K+ with valinomycin (1.2 μmol/mg protein). In the absence of extracellular K+ the cells formed methane at a rate of 4 μmol min−1 (mg protein)−1, the intracellular ATP content was 20 nmol/mg protein and the ΔμH+ was 200 mV (inside negative). When K+ was added to the suspensions the measured ΔμH+ decreased to the value calculated from the [K+]in/[K+]out ratio. Using this method of ΔμH+ adjustment, it was found that lowering ΔμH+ from 200 mV ([K+]in/[K+]out= 1000) to 100 mV ([K+]in/[K+]out= 40) had no effect on the rate of methane formation and on the intracellular ATP content. At ΔμH+ values below 100 mV ([K+]in/[K+]out < 40) both the rate of methanogenesis and the ATP content decreased. Methanogenesis completely ceased and the ATP content was 2 nmol/mg when ΔμH+ was adjusted to values lower 50 mV ([K+]in/[K+]out < 7). The data show that methanogenesis from H2 and CO2 and ATP synthesis in M. thermoautotrophicum are possible at relatively low electrochemical proton potentials. Similar results were obtained with Methanosarcina barkeri.Protonophoric uncouplers like 3,5,3′,4′‐tetrachlorosalicylanilide (TCS) or 3,5‐di‐tert‐butyl‐4‐hydroxy‐benzylidenemalononitrile (SF 6847) were found not to dissipate ΔμH+ below 100 mV in M. thermoautotrophicum even when used at high concentrations (400 nmol/mg protein). This finding explains the observed uncoupler insensitivity of methanogenesis and ATP synthesis in this organism.
Study of the coinduction by fatty acids of catalase A and acyl‐CoA oxidase in standard and mutant Saccharomyces cerevisiae strains
Authors: SKONECZNY Marek , CHELSTOWSKA Anna , RYTKA Joanna 
Published: March 2005

Evidence is presented that Saccharomyces cerevisiae can metabolize fatty acids via the inducible peroxisomal β‐oxidation pathway even when these acids are not the sole carbon source.The fatty acids of chain length of C10–C18 induce acyl‐CoA oxidase simultaneously with catalase A but have no effect on catalase T and acyl‐CoA dehydrogenase.The coinduction of both acyl‐CoA oxidase and catalase A is recorded in strains with both active catalase A and T or displaying only catalase A activity.In mutants lacking catalase A, the induction of acyl‐CoA oxidase is observed without a concomitant increase in catalase activity.After centrifugation in a linear Ficoll gradient of the particulate fraction from the cells grown on ethanol and oleate the activity of acyl‐CoA oxidase cosediments with catalase A.The relationship of catalase A to acyl‐CoA oxidase is discussed.
Ricin and α‐sarcin alter the conformation of 60S ribosomal subunits at neighboring but different sites
Authors: TERAO Kazuo , UCHIUMI Toshio , ENDO Yacta , OGATA Kikuo 
Published: March 2005

The effects of ricin and α‐sarcin separately or in combination on the conformation of rat liver ribosomes were investigated by measuring the relative accessibility of individual ribosomal proteins to N‐ethylmaleimide after 80s ribosomes were treated with these toxins.By using a double‐labelling technique in which ribosomes were incubated with the toxins and then treated with 3H‐labelled or 14C‐labelled N‐ethylmaleimide, it was found that labelling of protein L14 was specifically reduced by treatment with ricin, and that of proteins L3 and L4 by treatment wit α‐sarcin, suggesting that the toxins alter the conformation of ribosomes in the vicinity of these proteins. When ribosomes were treated with both ricin and α‐sarcin, the extent of labelling of protein L3 was reduced compared to that observed after treatment with α‐sarcin alone.These results are discussed in relation to previous observations showing that these three proteins are neighbours in the 60S ribosomal subunit and probably play important roles in protein biosynthesis, and in the actions of ricin and α‐sarcin on 28s rRNA.
Control analysis of metabolic networks
Authors: GIERSCH Christoph 
Published: March 2005

The summation theorem C1J+…+ CnJ= 1 for flux control coefficients CiJ is shown to be equivalent to the assumption that flux J is a homogeneous function of degree 1 of enzyme concentrations E1,…,En, that is to the assumption J (tE1,…,tEn) =tJ (E1,…,En) for any t≠ 0. Likewise, the summation theorem C1Xj+…+ CnXj= 0 for concentration control coefficients CiXj is equivalent to homogeneity of degree 0 of steady‐state metabolite concentrations Xj, or to Xj (tE1,…,tEn) =Xj (E1,…,En). From this equivalence it is obvious that metabolic control analysis applies only to homogeneous systems.
The summation theorem for flux control coefficients is shown to be equivalent to that for concentration control coefficients, provided all reaction rates vi are homogeneous functions of enzyme concentrations Ei.
The equivalence between homogeneity of flux J and the summation theorem for flux control coefficients is used to analyse branching of fluxes in metabolic pathways in terms of flux control coefficients.
Insulin‐like growth factor I receptors on mouse neuroblastoma cells
Authors: OTA Akira , WILSON Gaye Lynn , LEROITH Derek 
Published: March 2005

We have characterized receptors for the insulin‐like growth factor (IGF‐I) on the mouse neuroblastoma cell line N18 as well as NG108, the hybrid cell line of N18 and rat glioma (C6). In this cell‐free system, IGF‐I and insulin stimulated the phosphorylation of 95‐kDa and 105‐kDa proteins. Using appropriate antibodies we were able to demonstrate that the IGF‐I receptor β subunit has two subtypes of 95 kDa and 105 kDa. On the other hand, insulin receptor β subunit is a separate single 95‐kDa protein. Enzymatic digestion of IGF‐I receptor β subunit subtypes by glycopeptidase F resulted in similar molecular masses (84 kDa and 86 kDa) on SDS‐PAGE, which suggests that the difference in molecular masses between two subtypes is attributable to the differences in N‐linked complex‐type carbohydrate chains on the extracellular domain of β subunits. This conclusion is further supported by peptides of similar molecular mass following staphylococcal V8 protease digestion.Analysis of IGF‐I receptor β subunit subtypes in these cells may provide insights into the mechanism of action of IGF‐I on neural tissues.
Biosynthesis of vitamin B‐12 in anaerobic bacteria
Authors: VOGT Joachim R. A. , LAMM‐KOLONKO Lydia , RENZ Paul 
Published: March 2005

In anaerobic microorganisms the origin of C atoms 2 and 4–7 of the 5,6‐dimethylbenzimidazole moiety of vitamin B‐12 is still unknown. In order to tackle this problem we added several 14C‐labeled putative precursors to Eubacterium limosum fermentations. The degradation of the isolated vitamin B‐12 revealed that only d‐erythrose, 14C‐labeled in different positions, was efficiently incorporated into the 5,6‐dimethylbenzimidazole part.The 5,6‐dimethylbenzimidazole obtained from an experiment with d‐[U‐4C]erythrose was further degraded. It was found that C‐2 was unlabeled, whereas half of the label was located in C‐5 plus C‐6, and the other half in C‐4 plus C‐7.These results demonstrate that in E. limosumd‐erythrose is a precursor of C‐atoms 4, 5, 6 and 7 of the 5,6‐dimethylbenzimidazole part of vitamin B‐12.
Importance of polysomal mRNA‐associated polypeptides for protein synthesis initiation in yeast
Authors: HERRERA Flor , TRIANA Ledia , BOSCH Irene 
Published: March 2005

The polysomal mRNA from the cell‐free system of the yeast Saccharomyces cerevisiae, in the absence of exogenous energy, binds to the 40s ribosomal subunit thus forming a 48 s preinitiation complex which, with energy added, is converted into 80s initiation complex. By using ribosomes with a high affinity to polysomal mRNA (pmRNA) from an edeine‐resistant mutant of S. cerevisiae in place of wild‐type ribosomes, increased quantities of the 48s preinitiation complex are obtained.The pmRNA is found associated with several polypeptides having molecular masses of 115–98 kDa, 72 kDa, 60 kDa and 51 kDa. These polypeptides, labelled with 125I, interact with 40s and 80s ribosomes and are essential for the formation of the 48s and 80s initiation complexes inasmuch as deproteinized pmRNA alone cannot initiate the process. In addition, other polypeptides present in the cytosol are required to carry out the above‐mentioned steps of protein synthesis.
Effect of amino acid substitutions and deletions on the thermal stability, the pH stability and unfolding by urea of bovine calbindin D9k
Authors: WENDT Boel , HOFMANN Theo , MARTIN Stephen R. , BAYLEY Peter , BRODIN Peter , GRUNDSTRÖM Thomas , THULIN Eva , LINSE Sara , FORSÉN Sture 
Published: March 2005

The influence of amino acid substitutions and deletions on the stability of bovine calbindin D9k, the smallest protein known with a pair of EF‐hand calcium‐binding sites, has been studied using circular dichroism and ultraviolet absorption spectroscopy. The five modifications are confined to one of the two Ca2+ ‐binding sites. The Ca2+‐loaded forms of the wild‐type and mutant calbindins are too stable to be significantly denatured by heating at 90° C or by adding 8 M urea. For the Ca2+ ‐free (apo) forms thermal unfolding appears to be only half complete at 90° C, while denaturation is complete in 7–8 M urea. Four of the mutant proteins show reduced resistance towards unfolding by urea, but one of the modified proteins (Glu‐17 → Gln) shows an increased stability, presumably because of a reduced electrostatic repulsion in the native state.According to X‐ray crystallographic data the OH group of the single tyrosine of calbindin (Tyr‐13) is hydrogen‐bonded to the carboxyl group of Glu‐35, thus linking the two α helices flanking the N‐terminal Ca2+ site. The pKof ionization of the Tyr‐13 hydroxyl group was over 13 for calcium forms of the wild‐type protein, between 12.3 and 12.8 for the calcium form of three mutants and between 11.5 and 11.7 for the apoproteins. Significant differences in pH stability between wild type and mutants were observed in the calcium forms, but were not apparent in the apo forms.
An EPR study of the rotational dynamics of actins from striated and smooth muscle and their complexes with heavy meromyosin
Authors: MOSSAKOWSKA Malgorzata , BELÁGYI Jozef , STRZELECKA‐GOLASZEWSKA Hanna 
Published: March 2005

The rotational motions of the actin from rabbit skeletal muscle and from chicken gizzard smooth muscle were measured by conventional and saturation transfer electron paramagnetic resonance (EPR) spectroscopy using maleimide spin‐label rigidly bound at Cys‐374. The conventional EPR spectra indicate a slight difference in the polarity of the environment of the label and in the rotational mobility of the monomeric gizzard actin compared to its skeletal muscle counterpart. These differences disappear upon polymerization. The EPR spectra of the two actins in their F form and in their complexes with heavy meromyosin (HMM) did not reveal any difference in the rotational dynamic properties that might be correlated with the known differences in the activation of myosin ATPase activity by smooth and skeletal muscle actin.Our results agree with earlier EPR studies on skeletal muscle actin in showing that polymerization stops the nanosecond rotational motion of actin monomers and that F‐actin undergoes rotational motion having an effective correlation time of the order of 0.1 ms. However, our measurements show that complete elimination of the nanosecond motions requires prolonged incubation of F‐actin, suggesting that the slow formation of interfilamental cross‐links in concentrated F‐actin solutions contributes to this process.We have also used the EPR spectroscopy to study the interaction between HMM and actin in the F and G form. Our results show that in the absence of salt one HMM molecule can cooperatively interact with eight monomers to produce a polymer which closely resembles F‐actin in its rotational mobility but differs from the complex of F‐actin with HMM. The results indicate that salt is necessary for further slowing down, in a cooperative manner, the sub‐millisecond internal motion in actin polymer and for a non‐cooperative change in the intramonomer conformation around Cys‐374 on the binding of HMM.
Purification and comparative properties of the cytosolic isocitrate dehydrogenases (NADP) from pea (Pisum sativum) roots and green leaves
Authors: CHEN Ridong , LE MARÉCHAL Pierre , VIDAL Jean , JACQUOT Jean‐Pierre , GADAL Pierre 
Published: March 2005

The cytosolic isocitrate dehydrogenases (NADP‐IDH) were purified to homogeneity from pea roots and green leaves with a high yield by ammonium sulfate precipitation, DEAE‐cellulose chromatography, Sephacryl S‐200 gel filtration, Matrex red‐A affinity chromatography and phenyl‐Superose HR 5/5 HPLC.Both isoenzymes were dimeric proteins, consisting of two apparently identical 41‐kDa subunits, having similar secondary structures with an α‐helical content of 20% and a β‐pleated sheet content of 43%. Similarly immunoassays suggested that the two isoenzymes were closely related in terms of antigenic determinants. However, the two proteins were distinguishable by their electrophoretic mobilities and amino acid compositions.The profiles of the two isoenzymes as a function of pH were similar and exhibited a broad pH optimum from 8.5 to 9.0 with Mg2+ as cofactor and 8.0 to 8.5 when Mn2+ was used. Compared to the root isoenzyme, the leaf NADP‐IDH appeared to be more heat‐labile. However, these isoenzymes exhibited similar behavior for thermal denaturation in the presence of bovine serum albumin and were stabilized upon addition of substrate, metal and coenzyme. Their values of activation energy were estimated as 47 kJ/mol. When using Mg2+ as cofactor, the two isoenzymes displayed identical KMn2+m, KDL‐isocitratem and KNADPm values, which were calculated to be 2.1 μM, 5.7 μM and 2.7 μM respectively. With Mg2+ as cofactor, their KMg2+mKDL‐isocitratem and KNADPm values were also not statistically different, being 34.0 μM, 15.2 μM and 2.6 μM for the root NADP‐IDH, and 29.0 μM, 20.3 μM and 3.1 μM for the leaf isoenzyme.From the above data it can be concluded that although the cytosolic NADP‐IDH in pea roots and leaves are organ‐specific isozymes, their similar physicochemical and kinetic properties suggest that the two isozymes might be involved in identical metabolic functions.
Enhancing effect of a phorbol ester and of retinoic acid on glucocorticoid induction of chenodeoxycholate hydroxylation in hepatoma cultures
Authors: LAMBIOTTE Maurice , CRESTEIL Thierry , THIERRY Nicole 
Published: March 2005

In cultures of the differentiated clones Faza 967, Fao and HF1‐4, derived from Reuber hepatoma, physiological doses of glucocorticoid induce chenodeoxycholate 6β‐hydroxylation, a microsomal cytochrome‐P‐450‐mediated activity (enhanced in liver by phenobarbital and not by benzo[α]anthracene). Whereas 12‐O‐tetradecanoylphorbol 13‐acetate (TPA) alone has no effect, the tumor promoter, when added to dexamethasone, enhances this induction. This enhancement, half‐maximum with 10 ng/ml TPA, is a function of the dose between 1 ng/ml and 50 ng/ml; 50 ng/ml (80 nM) increase 4–7‐fold the induction rate (as measured in cultures by the amount of bile acid hydroxylated per 106 cells in 24 h, and in homogenates from treated cells) and 2.5–fold the maximum activity attained by the third day of induction. When added to cultures of the dedifferentiated clone H5, treated with benzo[α]anthracene, TPA does not influence benzo[α]pyrene hydroxylase induction, as shown by the total and relative amounts of the various hydrosoluble benzo[α]pyrene metabolites. TPA does not affect tyrosine aminotransferase induction in dexamethasone‐treated Fao cultures. The enhancement is not suppressed by indomethacin, an inhibitor of prostaglandin synthesis. After dexamethasone removal from induced Faza 967 cultures, addition of TPA to the medium does not affect the decay rate of the chenodeoxycholate‐hydroxylating activity.Retinoic acid similarly enhances the induction by dexamethasone of chenodeoxycholate hydroxylation, both in treated Faza 967 cultures and in homogenates from treated cultures. The effects of TPA and retinoic acid are additive.These results suggest a possible cooperation at the transcriptional level between transactive factors, involving TPA‐mediated alterations, retinoic acid and glucocorticoid receptors. The system described might provide a convenient experimental approach in the study of its mechanism.
Purification and characterization of rat‐liver cytosolic epoxide hydrolase
Authors: SCHLADT Ludwig , HARTMANN Renate , WÖRNER Walter , THOMAS Helmut , OESCH Franz 
Published: March 2005

Rat liver cytosolic epoxide hydrolase has been purified and characterized.The enzyme was purified from tiadenol‐induced rat liver 540‐fold with respect to trans‐stilbene oxide as a substrate. Similar purification was obtained with the substrates trans‐β‐ethyl styrene oxide and styrene 7,8‐oxide, the specific activities decreasing in the order trans‐β‐ethyl styrene oxide > styrene 7,8‐oxide > trans‐stilbene oxide. The enzyme exerts highest activity at pH 7.4. Km and Vmax of the pure enzyme for trans‐stilbene oxide were 1.7 μ and 205 nmol × min−1× mg protein−1 respectively. With trans‐stilbene oxide as a substrate, the inhibition by organic solvents (2.5% by vol.) increased in the order ethanol < methanol < acetone < isopropanol = N,N‐dimethyl formamide < acetonitrile < tetrahydrofuran. The native enzyme, with a molecutar mass of 120 kDa, consists of two 61‐kDa subunits.Digestion of rat liver cytosolic and microsonal epoxide hydrolase by. three proteases resulted in markedly different peptide maps.Western‐blot analysis with antiserum agnnst rat liver cystosolic epoxide hydrolase revealed a single band with the purified enzyme, and with liver cytosol from control and clofiberate‐iduced rats. No cross‐reactivity was observed with purified rat microsomal epoxide hydrolase or microsomes.A positive reaction at the same molecular mass was obtained with liver cytosol of mouse, guinea pig. Syrian hamster and New Zealand white rabbit but not with that of green monkey.
A rapid method of epitope mapping
Authors: LORENZO Fredéric , JOLIVET André , LOOSFELT Hugues , THU VU HAI Mai , BRAILLY Sylvie , PERROT‐APPLANAT Marline , MILGROM Edwin 
Published: March 2005

A method is described to map contiguous epitopes recognized by monoclonal antibodies in the case when the cDNA for a protein has been cloned. The cDNA is inserted into an expression vector allowing its acellular transcription, followed by the translation of the resulting messenger RNA. C‐terminally truncated species of the protein are either generated by cutting the cDNA with restriction enzymes or arise spontaneously through stops occurring during translation of the mRNA. If necessary, progressive digestion by Bal31 of the cDNA can be used to produce an array of polypeptides having different C‐terminal lengths. Immunoprecipitation then allows determination of the shortest protein recognized by the monoclonal antibody and thus to define its site of action.This method has been applied to the study of a group of selected monoclonal antibodies among the 59 that have been prepared against the rabbit progesterone receptor. Four immunogenic domains were identified lying between amino acids 1‐60, 101‐110, 295‐325 and 370‐396. There were no antibodies directed against the DNA‐binding or the steroid‐binding regions of the receptor. This is probably due to the high degree of amino acid sequence conservation in these domains, observed when comparing receptors from different species. The antibodies cross‐reacting with highest affinity for the human receptor interact with the first immunogenic domain (amino acids 1‐60).The 79‐kDa form (‘subunit A’) of the receptor was shown to lack the two more N‐terminally localized immunogenic domains (amino acids 1‐60 and 101‐110). The 65‐kDa form lacked, in addition, the domain localized between amino acids 295 and 325. These two forms of the receptor thus correspond to deletions of the N‐terminal part of the protein.
Enzymes as biosensors
Authors: RICARD Jacques , BUC Jean 
Published: March 2005

When a free enzyme exists under different conformations that ‘slowly’ isomerize during the conversion of a substrate into a product, the corresponding ‘slow’ relaxation component may interfere with the steady‐state component. The apparent steady‐state rate that may be measured under these conditions is called the meta‐steadystate rate for it refers to the existence of meastable states of the enzyme during the reaction. By contrast to the real steady‐state rate, the meta‐steady‐state rate is dependent upon the initial state of the enzyme, that isonthe respective concentrations of the free enzyme forms.The simplest model that may display this type of behaviour is the mnemonical model. For a fixed concentrationof the last product of the reaction sequence the meta‐steady state is different depending on that concentration being reached by an increase or a decrease of a previous concentration. This means that the meta‐steady‐state rate describes a hysteresis loop as the product concentrations is increased and decreased. Owing to the existence ofmetastable states, the enzyme system behaves as a biosensor that is able to detect both a concentration andthe direction of a concentration change.The existence of the hysteresis loop of the meta‐steady‐state rate implies that the two free enzyme forms display hysteresis as well. A chemical potential, called the sensing potential, is specifically associated with the ‘perception’of the direction of the thermodynamic force generated by the decrease or the increase of the concentration of the ligand that binds to one of the enzyme conformations. The sensing potential of the enzyme conformer that does not bind the product increase and reaches a plateau as the chemical potential of that product is raised. Alternatively the sensing potential of the other conformer vanishes at low and high chemical potentials of the product and is significant for intermediate chemical potentials.Enzymes that display very slow conformation changesmay thus be viewed as elementary sensor devices.