bluuebunny/crossref_metadata_2025_split huggingface dataset:
Kinetic and Thermodynamic Properties of the Ternary Complex between F‐actin, Myosin Subfragment 1 and Adenosine 5′‐[β,γ‐imido]triphosphate
Authors: KONRAD Manfred , GOODY Roger S. 
Published: July 2010

Equilibrium constants for the formation of a ternary complex between actin, myosin subfragment 1 (S1) and the non‐hydrolyzable ATP analog adenosine 5′‐[β,γ‐imido]triphosphate (AdoPP[NH]P) were determined from light‐scattering titrations under a variety of conditions. The affinities of S1 (binding constant K1) and acto · S1 (K4) for AdoPP[NH]P have relatively low dependencies on temperature (ΔH°∼ ‐15–30 kJ mol−1) and ionic strength, in contrast to the affinities of S1 (K2 and S1 · AdoPP[NH]P (K3) for actin which are influenced quite strongly by temperature (ΔH°∼ 50–65 kJ mo1−1) and ionic strength, K2 decreasing by a factor of 10–15 between I= 0.05 M and I= 0.2 M and K3 decreasing by a factor of 5. K1, and by detailed balance K2 as well, were found to be about 10‐times higher than hitherto reported values (K1= 3.4 × 107 M−1, K2= 6 × 108 M−1, at 24°C, I= 0.09 M, pH 8.0). The binding of ADP to S1 is about 10‐fold weaker than that of AdoPP[NH]P, being however much more exothermic (ΔH°= ‐70 kJ mol−1 at I= 0.1 M) and having a negative standard entropy change (ΔS°= ‐125 J mol−1 K−1), in contrast to AdoPP[NH]P binding for which the calculated ΔS° had positive values.The observed rate constant of dissociation of acto · S1 by AdoPP[NH]P showed an almost hyperbolic dependence on the nucleotide concentration, reaching a maximum of 15 s −1 at I= 0.055 M and 5 s −1 at I= 0.275 M, pH 8.0, 23°C; at 5°C this value was somewhat higher. The rate constant of dissociation of AdoPP[NH]P from its complex with acto · S1 was estimated to exceed 400 s−1 at 23°C, and to be of the order of 150 s−1 at 4°C. The observed rate constant for the association of the S1 middot; nucleotide complex and actin was proportional to actin concentrations up to 60 μM, thus defining an apparent second‐order rate constant of 2 × 104 M−1 s−1 at I= 0.125 M and 23°C. A reaction scheme is proposed in which isomerizations of the acto · S1 and acto · S1 · nucleotide complexes can occur.
Synthesis in vitro of Novel Nucleic‐Acid‐Like Polymers of Ehrlich Ascites Tumor Cells
Authors: KAGAWA Kazuko , KAGAWA Haruo 
Published: July 2010

When cytoplasmic particles isolated from Ehrlich ascites tumor cells were incubated with [14C]ribose, es and inorganic phosphate in the presence of MgCl2 and Tris/HCl (pH 7.0), radioactivity was incorporated into trichloroacetic‐acid‐insoluble materials.
Radioactive products, extracted and purified by DEAE‐cellulose column chromatography, were hydrolyzed in i M HCl at 100°C for 1 h and the resultant radioactive neutral sugar components were purified and identified as ribose, glucose and rhamnose by gas chromatography and mass spectrometry.
When [14C]glucose was incubated in the same reaction system, radioactivity was also incorporated into trichloroacetic‐acid‐insoluble materials. Purified radioactive products were hydrolyzed in 1 M HCl at 100°C for 1 h and the resultant radioactive neutral sugar components were also shown to be ribose, glucose and a little rhamnose, as was the case with the products synthesized from [14C]ribose.
When [14C]deoxyribose was incubated in the same reaction system, little incorporation was observed.
The radioactive products were incubated with venom phosphodiesterase and the digests were analyzed by Dowex lX4 column chromatography. The results suggest that the radioactive products contained ribo‐nuclcotides and ‘nucleotides’ containing glucose (glucose ‘nucleotides’).
When the radioactive products were incubated with both venom phosphodiesterase and alkaline phosphatase, ribonucleosides and glucose ‘nucleosides’ were detected. In Dowex 1X4 (borate form) chromatography, tibonuclcosides were eluted coincidentalhy with ribonucleoside markers (Cyd Ado Urd and Guo), whereas the elution profiles of glucose ‘nucleosides’ were totallydifferent fromfacto of ribonucleosides.
Activation and Deactivation of the Glucocorticoid Hormone‐Receptor Complex
Authors: ARÁNYI Péter 
Published: July 2010

Cytosol glucocorticoid receptors acquired the ability to bind to DNA‐cellulose on incubation at 25–37°C and/or in media of high ionic strength (0.3 M K Cl). However, this activation was transient only. It was followed by deactivation whose rate was also dependent on incubation temperature and on the presence of potassium chloride. Deactivation resulted in a decreased but non‐zero binding to DNA‐cellulose. Specific triamcinolone acetonide binding to the receptor protein was not lost under the same conditions.Deactivation commenced before it became apparent, probably together with activation. Preactivated complex underwent deactivation even in conditions that would not allow significant decrease in DNA‐cellulose binding without previous incubation at 37°C.In contrast to previous reports it was found that fast activation/deactivation took place in the presence of 4 mM Ca2+.Molybdate ions slow down both activation and deactivation, but do not prevent activation by heat.Heat activated/deactivated complex differed in size from both non‐activated and Ca2+‐deactivated complexes. This finding suggests that heat and Ca2+‐induced deactivation follow different mechanisms.
Rapid Induction of the Synthesis of Phenylalanine Ammonia‐Lyase and of Chalcone Synthase in Elicitor‐Treated Plant Cells
Authors: LAWTON Michael A. , DIXON Richard A. , HAHLBROCK Klaus , LAMB Chris 
Published: July 2010

Changes in the rate of synthesis of phenylalanine ammonia‐lyase and chalcone synthase, two characteristic enzymes of phenylpropanoid biosynthesis, have been investigated by direct immunoprecipitation of in vivo [35S]methionine‐labelled enzyme subunits in elicitor‐treated cells of dwarf French bean (Phaseolus vulgaris). Elicitor, heat‐released from cell walls of Colletotrichum lindemuthianum, the causal agent of anthracnose disease of bean, causes marked but transient increases in the rates of synthesis of both enzymes concomitant with the phase of rapid increase in enzyme activity at the onset of phaseollin accumulation during the phytoalexin defence response.Increased rates of synthesis of both enzymes can be observed 20 min after elicitor treatment and the pattern of induction of synthesis of phenylalanine ammonia‐lyase and chalcone synthase are broadly similar with respect to elicitor concentration and time, maximum rates of synthesis being attained between 2.5 h and 3.0 h after elicitor treatment. Within this overall co‐ordination small but distinct differences between the enzymes were observed in: (a) the elicitor concentrations giving maximum enzyme synthesis, and (b) the precise timing of maximum enzyme synthesis, with that for chalcone synthase occurring 20–30 min earlier than that for phenylalanine ammonia‐lyase. However, for a given rate of enzyme synthesis, induction of the activities of phenyl‐alanine ammonia‐lyase and chalcone synthase is more efficient at high elicitor concentrations. This may reflect the operation under certain circumstances of post‐translational control of the activity levels of these enzymes as implicated for phenylalanine ammonia‐lyase by previous density‐labelling experiments [Lawton et al. (1980) Biochim. Biophys. Acta, 633, 162–175]. The same pattern of induction of enzyme synthesis is observed with elicitor preparations from a variety of sources.
The Complete Amino‐Acid Sequence of the Low‐Spin Class II Cytochrome c‐556 from Agrobacterium tumefaciens Strain B2a
Authors: TEMPST Paul , VAN BEEUMEN Jozef 
Published: July 2010

The amino acid sequence of the soluble monohaem cytochrome c‐556 from Agrobacterium tumefaciens, strain B2a, has been determined. The sequence was derived from peptides obtained by digestion of the apoprotein with trypsin and chymotrypsin, and by subdigestion of some of the peptides with Staphylococcus aureus protease and thermolysin. Sequencing of the various peptides was achieved by a combination of manual dansyl‐Edman degradation and automatic liquid‐phase sequence analysis.The main characteristic of this cytochrome is that the haem‐binding sequence Cys‐Xaa‐Yaa‐Cys‐His occurs in the C‐terminal region of the polypeptide chain, the first cysteine being located 11 residues ahead of the C‐terminal lysine‐122. As such, the protein belongs to cytochrome c sequence class II (sensu Ambler). The cytochrome c‐556 is the first example known of a class II cytochrome of the low‐spin type isolated from an obligate aerobic organism.
The Inhibition Pattern of Antibiotics on the Extent and Accuracy of tRNA Binding to the Ribosome, and Their Effect on the Subsequent Steps in Chain Elongation
Authors: WURMBACH Peter , NIERHAUS Knud H. 
Published: July 2010

Ac[14C]Phe‐tRNA (cognate) and Ac[3H]Leu‐tRNA (miscognate) were bound as competing peptidyl‐tRNA analogues to tightly coupled ribosomes programmed with poly(U). The test system allows the determination of the accuracy of tRNA selection at the A and P site, as well as the measurement of the peptidyltransferase and translocation reactions in the presence of various antibiotics. The following results were obtained.

Cognate tRNA could be bound to either the A or P site with nearly 10% specificity. In contrast, miscognate tRNA could be exclusively bound to the P site but poorly to the A site.
The accuracy of tRNA selection (extent of binding) was sixfold higher at the A site than at the P site.
The extent of A site binding for both cognate and miscognate tRNA was severely inhibited by viomycin, tetracycline and neomycin, and to a lesser extent by streptomycin. No inhibition of binding at the P site was found with all antibiotics tested. This finding, and that of the preceding point, is taken as evidence that acetylated aminoacyl‐tRNA (an analogue for a peptidyl‐tRNA) has direct access to the P site and does not reach this site via a transient A site binding.
In addition to the known effects of streptomycin and neomycin on tRNA selection at the A site, viomycin was found to enhance drastically the error fraction at this site.
The peptidyltransferase is inhibited by the drugs in the following order: chloramphenicol > viomycin, neomycin > streptomycin. Equivalent results were found with cognate and miscognate tRNA. The translocation reaction was severely inhibited by viomycin and neomycin and to a lesser extent by streptomycin.
A striking correspondence was found between the error‐inducing capacity (distortion of codon‐anticodon interaction at the A site) and the inhibition of the translocation reaction by various drugs. This finding suggests that codon‐anticodon interaction at the ribosomal A site is an important if not essential prerequisite for the translocation reaction.
Kinetics of calcium binding to calbindin mutants
Authors: FORSÉN Sture , LINSE Sara , THULIN Eva , LINDEGÅRD Boel , MARTIN Stephen R. , BAYLEY Peter M. , BRODIN Peter , GRUNDSTRÖM Thomas 
Published: July 2010

The kinetics of calcium dissociation from wild‐type bovine calbindin D9k (the smallest protein known with a pair of EF‐hand calcium‐binding sites) and five mutants with single amino‐acid substitutions and/or deletions has been studied by stopped‐flow fluorescence methods, using the calcium chelator Quin 2. The modifications are confined to the N‐terminal half of the molecule, at or near the first calcium‐binding site (I). Substitutions and deletions of amino acids in the calcium‐binding loop of site I primarily affect the rate of Ca2+ dissociation from this site with only minor effects on the dynamic properties of the C‐terminal calcium‐binding site (II). This finding corroborates and extends previous kinetic results obtained from 43Ca‐NMR studies on the same set of mutants. By contrast, removal of the hydrogen bond between Tyr‐13 and Glu‐35, an interaction linking the two α‐helices flanking site I, through replacement of Tyr‐13 with Phe, has no observable effect on the rate of Ca2+ dissociation from the protein.Comparison of this kinetic data with binding‐constant data, previously obtained in our laboratories, shows that the decrease in Ca2+‐affinity of site I, observed in most mutants, is predominantly due to an increased off‐rate from this site. At low ionic strengths the second‐order rate constants for Ca2+‐binding to both Ca2+ sites of calbindin D9k are calculated to be of the order of 109 M−1 s−1 for all proteins studied. At higher ionic strengths (0.1 M KCl) the rates of Ca2+ dissociation from both sites are increased by a factor of three or more, suggesting a transition state which is ionic in nature.
Mutants with base changes at the 3′‐end of the 16S RNA from Escherichia coli
Authors: ROTTMANN Norbert , KLEUVERS Barbara , ATMADJA Johannes , WAGNER Rolf 
Published: July 2010

The functionally important 3′ domain of the ribosomal 16S RNA was altered by in vitro DNA manipulations of a plasmid‐encoded 16S RNA gene. By in vitro DNA manipulations two double mutants were constructed in which C1399 was converted to A and G1401 was changed to either U or C and a single point mutant was made wherein G1416 was changed to U.Only one of the mutated rRNA genes could be cloned in a plasmid under the control of the natural rrnB promoters (U1416) whereas all three mutations were cloned in a plasmid under the control of the λ PL promoter. In a strain coding for the temperature‐sensitive λ repressor cI857 the mutant RNAs could be expressed conditionally.We could show that all three mutant rRNAs were efficiently incorporated into 30S ribosomes. However, all three mutants inhibited the formation of stable 70S particles to various degrees.The amounts of mutated rRNAs were quantified by primer extension analysis which enabled us to assess the proportion of the mutated ribosomes which are actively engaged in in vivo protein biosynthesis. While ribosomes carrying the U1416 mutation in the 16S RNA were active in vivo a strong selection against ribosomes with the A1399/U1401 mutation in the 16S RNA from the polysome fraction is apparent. Ribosomes with 16S RNA bearing the A1399/C1401 mutation did not show a measurable protein biosynthesis activity in vivo. The growth rate of cells harbouring the different mutations reflected the in vivo translation capacities of the mutant ribosomes. The results underline the importance of the highly conserved nucleotides in the 3′ domain of the 16S RNA for ribosomal function.
Viscosity‐dependent energy barriers and equilibrium conformational fluctuations in oxygen recombination with hemerythrin
Authors: LAVALETTE Daniel , TETREAU Catherine 
Published: July 2010

The recombination kinetics of photo‐dissociated oxyhemerythrin (Sipunculus nudus) have been investigated between 298 K and 90 K. Fast geminate recombinations compete with oxygen escape into the solvent, from which a subsequent slower bimolecular rebinding takes place. In phosphate buffer (pH 7.7) at 278 K, the fast and slow processes are exponential and have comparable amplitudes. Whereas the oxygen escape rate rapidly decreases upon increasing the viscosity, the inward rate from the solvent is found to be independent of viscosity, up to about 50 cP (50 mPa · s). The data suggest that a Brownian‐motion‐driven displacement of one or several side‐chain residues is implied in oxygen escape from within the protein and also that hemerythrin undergoes a conformational change in the deoxy state.At higher viscosities and lower temperature only the geminate phase is observed and the kinetics progressively depart from an exponential. Below about 130 K, the kinetics resemble those reported in the literature for heme proteins. They are consistent with a temperature‐independent non‐equilibrium frozen distribution of conformational substates. However, between 190 K and 130 K, the profile of the kinetics is invariant on a log/log plot and the results simply differ by a translation along the log t axis. It is shown that this property is expected only for a temperature‐dependent distribution of substates in a Boltzmann equilibrium. From room temperature, where rebinding is exponential, down to the ‘freezing’ temperature, the geminate recombinations display a variety of kinetic laws. It can be shown, however, that for a broad class of substate distributions, the initial slope of the kinetic plot follows an Arrhenius relationship. The activation energy is equal to that of the exponential rate constant measured at high temperature. This result establishes the conditions under which protein data obtained from low‐temperature kinetics can be extrapolated to physiological temperature.
Purification and Characterization of Coenzyme F390 Synthetase from Methanobacterium thermoautotrophicum (strain H)
Authors: Vermeij Paul , Detmers Frank J. M. , Broers Frank J. M. , Keltjens Jan T. , Van Der Drift Chris 
Published: July 2010

Coenzyme F390 synthetase catalyzes the formation of 8‐hydroxyadenylylated‐coenzyme F420 (coenzyme F390‐A) from coenzyme F420 and ATP in some methanogenic Archaea. The presence of coenzyme F390 was found when these organisms were exposed to oxygen. To get more insight into the defined function of coenzyme F390, the coenzyme F390 synthetase from Methanobacterium thermoautrophicum was purified from a cell‐free extract and its catalytic properties were determined. The synthetase was purified 150‐fold to a specific activity of 0.45 μmol · mim−1· mg protein−1. The enzyme consisted of one polypeptide of approximately 51 kDa. The isolated enzyme showed a tendency to aggregate into dimers and tetramers upon concentration. Co‐elution during purification of GTP‐dependent coenzyme F390 synthetase activity suggested that the synthetase is also capable of 8‐hydroxyguanylylated‐coenzyme F420, (coenzyme F390‐G) formation.Initial‐velocity measurements of the two‐substrate reaction showed that the enzyme kinetics for the coenzyme F390 synthetase reaction proceeded by a ternary‐complex mechanism. The coenzyme F390 synthetase displayed a Km for coenzyme F420 of 39 μM and a Km for ATP of 1.7 mM.In contrast to the enzyme in the cell‐free extract, the isolated enzyme was active under aerobic and anaerobic conditions. Treatment with air was not required to obtain the enzyme in an active form. However, 1,5‐dihydro‐coenzyme F420 (coenzyme F420H2) appeared to be a potent competitive inhibitor (Ki 3 μM) with respect to coenzyme F420. The latter findings may explain why the enzyme could only be detected in crude extracts that had been exposed to air, i.e. treatment with air causes the oxidation of reduced coenzyme F420 present in anaerobic extracts. The results of this study are discussed in view of the proposed role for coenzyme F390 in methanogenic metabolism.
Cloning and Expression of Glucosidase I from Human Hippocampus
Authors: Kalz‐Füller Burga , Bieberich Erhard , Bause Ernst 
Published: July 2010

Glucosidase I, the first enzyme in the N‐linked oligosaccharide processing pathway, cleaves the distal α1,2‐linked glucose residue from the Glc3‐Man9‐GlcNAc2 oligosaccharide precursor highly specifically. A human hippocampus cDNA library was screened against oligonucleotide probes, generated by PCR using primers derived from the amino acid sequences of tryptic peptides of pig liver glucosidase I. Two independent λ clones were isolated which allowed the construction of a full‐length glucosidase I cDNA of 2881 bp. This cDNA construct encodes, in a single open reading frame, a polypeptide of 834 amino acids corresponding to a molecular mass of 92 kDa. The 92‐kDa protein contains a single N‐glycosylation site of the Asn‐Xaa‐Thr/Ser type at Asn655, as well as a strongly hydrophobic sequence close to its N‐terminus (amino acids 38–58) which, most likely, functions as a transmembrane anchor. The amino acid sequences of all tryptic peptides of the pig liver enzyme were found, with little deviation, within the coding sequence. This demonstrates the authenticity of the cDNA construct and the close evolutionary relationship between the enzymes from human hippocampus and pig liver. In contrast, the nucleotide and amino acid sequence revealed no homology with other processing enzymes cloned so far.Transfection of COS 1 cells with the glucosidase I cDNA construct resulted in overexpression (about fourfold) of enzymic activity, which was inhibited strongly by 1‐deoxynojirimycin or N,N‐dimethyl‐deoxynojirimycin. The expressed enzyme, with a molecular mass of 95 kDa, is degraded by endoglycosidase H to a 93‐kDa form, indicating that it carries a high‐mannose oligosaccharide chain at Asn655. The presence of this glycan is in line with the localization of glucosidase I in the lumen of the endoplasmic reticulum, shown by immunofluorescence microscopy. The hydrophobicity profile as well as the removal by trypsin of an ≈4‐kDa polypeptide from the membrane‐associated glucosidase I in intact microsomal structures, supports the view that the enzyme is a type‐II transmembrane glycoprotein, which contains a short cytosolic tail of ≈37 amino acids, followed by a single transmembrane domain and a large C‐terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane.
Role of Amino Acid Sequences Flanking Dibasic Cleavage Sites in Precursor Proteolytic Processing
Authors: Rholam Mohamed , Brakch Noureddine , Germain Doris , Thomas David Y. , Fahy Christine , Boussetta Hamadi , Boileau Guy , Cohen Paul 
Published: July 2010

The amino acid sequences flanking 352 dibasic moieties contained in 83 prohormones and pro‐proteins listed in a database were examined. Frequency calculations on the occurrence of given residues at positions P6 to P′4 allowed us to delineate a number of features which might be in part responsible for the in vivo discrimination between cleaved and uncleaved dibasic sites. These include the following: amino acids at these positions were characterized by a large variability in composition and properties; no major contribution of a given precursor subsite to endoprotease specificity was observed; some amino acid residues appeared to occupy preferentially certain precursor subsites (for instance, Met in P6 and P3, Asp and Ala in P′1, Pro in P6, Gly in P3 and P′2 etc.) whereas some others appeared to be excluded.Most amino acid residues occupying the P′1 position in these precursor cleavage sites were tolerated. But the β‐carbon branched side chain residues (Thr, Val, Leu, Ile) and Pro, Cys, Met and Trp were either totally excluded or poorly represented, suggesting that they might be unfavourable to cleavage. The biological relevance of these observations to the efficacy of dibasic cleavage by model propeptide convertases was in vitro tested using both pro‐ocytocin convertase and Kex2 protease action on a series of pro‐ocytocin related synthetic substrates reproducing the Pro7→Leu15 sequence of the precursor in which the Ala13 residue (P′1 in the LysArg‐Ala motif) was replaced by various amino acid residues. A good correlation was obtained on this model system indicating that P′1 residue of precursor dibasic processing sites is an important feature and may play the role of anchoring motif to S′1 convertase subsite.We tentatively propose that the present database, and the corresponding model, may be used for further investigation of dibasic endoproteolytic processing of propeptides and pro‐proteins.