bluuebunny/crossref_metadata_2025_split huggingface dataset:
Sequence‐specific assignment of the 1H and 15N nuclear magnetic resonance spectra of the reduced recombinant high‐potential iron‐sulfur protein I from Ectothiorhodospira halophila
Authors: Bertini Ivano , Felli Isabella Caterina , Kastrau Dieter H. W. , Luchinat Claudio , Piccioli Mario , Viezzoli Maria Silvia 
Published: March 2005

A 1H and 15N NMR investigation through two‐dimensional and three‐dimensional spectroscopy has been performed on the reduced form ([Fe4S4]2+) of the recombinant high‐potential iron‐sulfur protein (HiPIP) I from Ectothiorhodospira halophila expressed in Escherichia coli. [Fe4S4]2+ clusters in proteins are paramagnetic with a relatively low μeff of about 0.8 μB/iron ion, but the paramagnetic effects on nuclear relaxation are so strong as to yield T1 values of a few milliseconds and linewidths of hundreds of hertz for the nuclei closest to the paramagnetic center. Despite these features, 71 out of 73 residues were identified, most of which were assigned completely as far as proton resonances are concerned; as many as 68 residues could be assigned without any reference to the existing X‐ray structure. A total of 88% of all protein protons and 58 out of 69 peptide HN nitrogen signals were assigned. To the best of our knowledge, this is the most extensive 1H assignment of a paramagnetic protein to date.Protons sensitive to the proximity of the cluster were assigned through suitable NOE spectroscopy experiments. Three out of the four coordinated cysteines were assigned, and two residues have been identified whose peptide HN protons give rise to H bonds with coordinated sulfur atoms. The inter‐residue NOE cross peaks are in qualitative agreement with the secondary and tertiary structure as obtained from the available X‐ray crystallographic analysis of the wild‐type protein at 250‐pm resolution. It is therefore shown that the expressed protein is properly folded and that it is a reliable model for the wild‐type protein. These data are meaningful for the detection of structural differences among mutants in future studies.
Solvent Hydrogen Isotope Effects and Anion Inhibition of CO2 Hydration Catalysed by Carbonic Anhydrase from Pisum sativum
Authors: Johansson Inga‐Maj , Forsman Cecilia 
Published: March 2005

Chloroplast carbonic anhydrase from Pisum sativum has been studied to elucidate the catalytic mechanism and to test if the mechanism proposed for human carbonic anhydrase II is also valid for pea carbonic anhydrase. The catalytic activity was found to depend on the chemical nature of the buffer. Barbital buffer gives the highest turnover number at infinite buffer concentration and the lowest Km value with respect to the buffer, while the kinetic parameters obtained in the imidazole‐type buffer, 1‐methylimidazole, do not differ from those obtained using the biological‐type buffer Mops. The anion inhibition of CO2 hydration was investigated using SCN− at pH 6–9. The binding of the anion was found to be pH dependent with the strongest interaction at low pH. We obtained an uncompetitive inhibition pattern at high pH and noncompetitive inhibition patterns at pH 7 and low pH. The catalytic mechanism was further tested by measurements of the solvent hydrogen isotope effects on the kinetic parameters for CO2 hydration. The observed effects were comparatively small with a kcat value of approximately 2 irrespective of the pH. The effect on kcat/Km and on Km changes when going from high pH to pH 7 and low pH. At high pH, the solvent isotope effect in Km is at least 3, giving a value below 1 for kcat/Km, while at pH 7 and low pH the major effect is found in kcat/Km with values of 2.6 and 2.9.The dependence of the CO2‐hydration activity on the buffer concentration is in agreement with a ping‐pong mechanism with buffer acting as a second substrate. This is analogous to the behaviour of human carbonic anhydrase II. The inhibition patterns and the observed isotope effects at high pH can also be explained within the framework of the catalytic mechanism for human carbonic anhydrase II, with a rate‐determining and buffer‐dependent part. The results are consistent with a mechanism involving a proton transfer that contributes to rate limitation. However, the isotope effects found at pH 7 and low pH indicate that some part of the mechanism has changed. Moreover, we cannot decide whether the mechanism for pea carbonic anhydrase involves an internal proton‐shuttle group, or if the buffer molecule acts in a direct proton transfer from the zinc‐coordinated water.
Identification of Functional Domains in the Cytoskeletal Protein Talin
Authors: Niggli Verena , Kaufmann Stephan , Goldmann Wolfgang H. , Weber Thomas , Isenberg Gerhard 
Published: March 2005

The cytoskeletal protein talin potentially plays a key role in actin–membrane linkage. It is able to nucleate actin filament growth in vitro while binding simultaneously to lipid bilayers. Thrombin digestion of human platelet talin yields two polypeptide domains of 200 kDa and 47 kDa. We have purified these fragments and analyzed their functional properties: the 200‐kDa fragment was active in nucleating actin filament formation and reduced the viscosity of filamentous actin, comparable to the effects of the intact protein. The 47‐kDa fragment was inactive in this respect.However, the 47‐kDa polypeptide, but not the 200‐kDa fragment, interacted specifically with large liposomes containing acidic phospholipids. This is demonstrated by selective, hydrophobic photolabeling of the 47‐kDa fragment using phosphatidylserine liposomes containing trace amounts of a photoactivatable phospholipid analogue and by selective co‐sedimentation of this domain with the liposomes. The 200‐kDa fragment, whether alone or in conjunction with the small fragment, neither incorporated significant amounts of label nor co‐sedimented with the liposomes.We thus are able to attribute specialized functions to distinct domains on the talin molecule. These enable the protein to interact simultaneously with actin filaments and lipid membranes.
Pre‐Steady‐State MgATP‐Dependent Proton Production and Electron Transfer by Nitrogenase from Azotobacter vinelandii
Authors: Duyvis Martina G. , Wassink Hans , Haaker Huub 
Published: March 2005

MgATP‐dependent pre‐steady‐state proton production by nitrogenase from Azotobacter vinelandii was studied by monitoring the absorbance changes at 572 nm of the pH indicator o ‐cresolsulphonphtalein in a weakly buffered solution. The absorbance changes are characterized by a constant phase, a single exponential decrease and a linear decrease. The observed rate constant for the single exponential MgATP‐dependent proton production by reduced nitrogenase proteins at 20.0°C is 14±24 s−1. No proton production with a rate constant comparable to the observed rate constant of electron transfer (kobs≈ 100 s−1) was detected. The extent of the observed MgATP‐dependent proton production is determined by the redox state of the nitrogenase proteins before mixing with MgATP; less protons are produced when more electrons are transferred from the Fe protein to the MoFe protein. Values of 2.7±0.3 mol H+produced/mol MoFe protein with oxidized Fe protein, and 1.1±0.1 mol H+produced/mol MoFe protein with reduced Fe protein, were found. The data are interpreted to mean that protons are taken up after electron transfer from the Fe protein to the MoFe protein; the ratio electrons transferred/H+uptake was calculated to be 1.2±0.2.After mixing the nitrogenase proteins with MgADP, proton production takes place as well. The proton‐production curve did not have a constant phase and the observed rate constant of the single exponential reaction is higher, compared to MgATP‐dependent proton production (kobs≈ 35 s−1). The amount of protons produced depends also on the redox state of the Fe protein; no proton production was observed with the oxidized Fe protein; with dithionite‐reduced Fe protein a value of 3.1±0.4 mol H+produced/mol MoFe protein was found (or 0.5±0.1 mol H+/mol Fe protein). Similar results were obtained when only the Fe protein was mixed with MgADP, but the observed absorbance changes were smaller; mixing of dithionite‐reduced Fe protein with MgADP resulted in the production of 0.17±0.05 mol H+/mol Fe protein.All reported absorbance changes were absent when the experiments were performed in a buffered solution.The series of events that occur after mixing of the nitrogenase proteins with MgATP will be presented and discussed. In the case of the reduced Fe protein, electron transfer takes place at a rate of 100 s−1 which is followed by H+ production (kobs≈ 14 s−1). When there is no electron transfer (oxidized Fe protein) the rate constant of the MgATP‐induced proton production decreases. When electrons are transferred, stoichiometrically less protons are produced.
Electron Paramagnetic Resonance Studies of the Methylmalonyl‐CoA Mutase Reaction
Authors: Zhao Ymin , Abend Andreas , Kunz Meinrad , Such Peter , Rétey János 
Published: March 2005

The substrate‐dependent homolysis of the cobalt‐carbon bond and generation of organic radicals in the coenzyme‐B12–methylmalonyl‐CoA‐mutase complex have been demonstrated by EPR measurements. Both the natural substrate methylmalonyl‐CoA, its 13C‐substituted analogue and the non‐hydrolysable synthetic substrates succinyl‐dethia(carba)‐CoA, succinyl‐dethia(dicarba)‐CoA and 4‐carboxy‐2‐oxo‐butyl‐CoA induced similar but not identical EPR signals. 3‐Carboxypropyl‐CoA, a novel competitive inhibitor, has been synthesised. Its Ki, value of 89 ± 6 μM was in the same range as the Km, of succinyl‐CoA. Using [5′‐3H]adenosylcobalamin, an enzyme‐dependent tritium transfer to the inhibitor has been shown. The enzyme‐coenzyme‐inhibitor complex also exhibited EPR signals that were less structured and less intensive than the corresponding signals with active substrates.These results prove that the inhibitor also induces cobalt‐carbon bond homolysis and undergoes reversible hydrogen transfer but not rearrangement.
Characterization of a Rat C6 Glioma‐Secreted Follistatin‐Related Protein (FRP)
Authors: Zwijsen An , Blockx Herman , Van Arnhem Wouter , Willems Jean , Fransen Lucie , Devos Kathleen , Raymackers Jos , Van De Voorde André , Slegers Herman 
Published: March 2005

A protein was isolated from rat C6 glioma‐conditioned medium and was biochemically characterized. The heparin‐binding protein has a native molecular mass of 55–75000Da, a molecular mass of 40–48000 Da under denaturing conditions, and a pI of 5.0–6.0. Based on the determined partial amino acid sequences, the full lenght cDNA encoding the rat and human proteins were cloned. The cDNA sequences identified the isolated rat and human protein as the homologue of a recently reported mouse osteoblast‐transforming‐growth‐factor‐β1‐inducible protein, encoded by the TSC‐36 gene [Shibanuma, M., Mashimo, J., Mita, A., Kuroki, T. & Nose, K. (1993) Eur. J. Biochem. 217, 13–19].Analysis of the human, rat and mouse amino acid sequences indicates that these proteins are highly conserved (>92% sequence identity). Sequence similarities with follistatin and the follistatin‐like domain of agrin are revealed. The relationship with follistatin and agrin points to possible common functions for the cloned follistatin‐related proteins (FRP).The protein has no effect on the inhibitory action of transforming growth factor‐β1, on CCl‐64 cell growth.
Isolation and Structural Analysis of Oligosaccharide Phosphates Containing the Complete Carbohydrate Chain of the Lipopolysaccharide from Vibrio cholerae Strain H11 (Non‐O1)
Authors: Bock Klaus , Vinogradov Evgeny V. , Holst Otto , Brade Helmut 
Published: March 2005

For the first time, an oligosaccharide has been prepared comprising the lipid A backbone, the core oligosaccharide and one repeating unit of the O‐specific polysaccharide (O‐chain) of a lipopolysaccharide. Lipopolysaccharide from Vibrio cholerae strain H11 (non‐O1) was deacylated and the products were separated by high‐performance anion‐exchange chromatography. Major fractions were a hexadecasaccharide trisphosphate 1, representing the core‐lipid A oligosacccharide substituted by one modified repeating unit of the O‐antigenic polysaccharide, a dodecasaccharide trisphosphate 2 and an undecasaccharide trisphosphate 3, representing the core‐lipid A region. Oligosaccharide 1 originated from β‐elimination upon alkaline hydrolysis of α‐galacturonic acid of the O‐chain; oligosaccharides 2 and 3 were most likely obtained from naturally occurring lipopolysaccharide species carrying no O‐chain. The structures of these compounds were elucidated on the basis of monosaccharide composition, and NMR investigations comprising correlation spectroscopy, total correlation spectroscopy and nuclear Overhauser enhancement spectroscopy experiments, as well as heteronuclear 13C,1H correlation spectroscopy. The structures are as follows:
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where R is β L‐threo ‐hex‐4‐enuronopyranosyl‐(1–4)‐α‐Neu‐(2–3)‐β‐GalA‐(1–3)‐β‐QuiN‐(1–4)‐β‐Sedf ‐(2‐ in 1, β‐Sedf ‐(2‐ in 2, and H in 3. Where not stated otherwise, sugars are pyranoses of the D‐series. Hep is L‐glycero ‐D‐manno ‐heptose, QuiN is 2‐amino‐2,6‐dideoxy‐glucose, Kdo is 3‐deoxy‐D‐manno ‐2‐octulosonic acid, Sed is D‐altro ‐heptulose and GalA is galacturonic acid.
Structure of the Escherichia coli 024 and 056 O‐Specific Sialic‐Acid‐Containing Polysaccharides and Linkage of these Structures to the Core Region in Lipopolysaccharides
Authors: Gamian Andrzej , Kenne Lennart , Mieszała Małgorzata , Ulrich Jacques , Defaye Jacques 
Published: March 2005

The lipopolysaccharides from Escherichia coli O24 and O56 could be separated into higher‐molecular‐mass and lower‐molecular‐mass fractions. Mild acid hydrolysis of lipopolysaccharides of both serotypes released an O‐specific polysaccharide and a tetrasaccharide repeating unit. Oligomers of the repeating unit, the core and the oligosaccharide that contains a fragment of the repeating unit linked to the core region were also obtained according to hydrolysis conditions. On the basis of sugar and methylation analyses, Smith degradation, fast‐atom‐bombardment mass spectrometry and NMR spectroscopy of the hydrolysis products, the biological repeating units of the 0‐specific polysaccharides were shown to be the following tetrasaccharides:
image

and
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The structures differ from the structures proposed previously by Kogan et al. [Kogan, G., Shashkov, A. S., Jann, B. & Jann, K. (1993) Carbohydr. Res. 238, 261–270; Kogan, G., Jann, B. & Jann, K. (1993) Carbohydr. Res. 238, 335–338].The O‐specific repeating unit in E. coli O24 lipopolysaccharide is linked to O6 of the terminal D‐galactose in the core region, whereas in O56 LPS the repeating unit is linked to O4 of a subterminal D‐glucose residue in an R2 type core.
Purification and Primary Structure of Metallothioneins Induced by Cadmium in the Protists Tetrahymena Pigmentosa and Tetrahymena Pyriformis
Authors: Piccinni Ester , Staudenmann Werner , Albergoni Vincenzo , De Gabrieli Raffaele , James Peter 
Published: March 2005

Tetrahymena pyriformis and Tetrahymena pigmentosa grown in the presence of a non‐toxic dose of cadmium, accumulate the metal in the cytosol. Purification by gel‐permeation, ion‐exchange and reverse‐phase high‐performance liquid chromatography showed that the metal is bound principally to newly formed proteins with ultraviolet spectra and cysteine contents similar to those of Cd2+‐metallothioneins from multicellular organisms. The isolated proteins revealed that the two species of ciliates each express two Cd2+‐isothioneins. The primary structures determined by both Edman degradation and mass spectrometry revealed that the equivalent proteins from T. pyriformis and T. pigmentosa have identical sequences and that the two isoforms in each species differ only by the presence or absence of a lysine residue at the N‐terminus. The development of automated mass spectrometric sequence analysis algorithms combined with an accurate determination of the molecular mass allowed the rapid confirmation of the sequences.The Tetrahymena metallothionein sequences are unusually long (105 and 104 amino acids) and show a unique internal homology which suggests that the proteins arose by gene duplication. The chains contain 31 cysteine residues, 15 of which are arranged in motifs characteristic of the mammalian metallothioneins; the remaining residues show several unique repeating motifs, which could have interesting consequences for the tertiary structure of the metal‐binding sites. Amino acid sequences of Tetrahymena metallothioneins have some similarity with other eukaryotic metallothioneins. A comparison on the basis of optimised FASTA scores, shows a closer relationship with horse metallothionein‐1B.
Influence of the Four Leader Sequences of the Human Insulin‐Like‐Growth‐Factor‐2 mRNAs on the Expression of Reporter Genes
Authors: Moor Cornelia H. De , Jansen Maarten , Bonte Edgar J. , Thomas Adri A. M. , Sussenbach John S. , Brande J. Leo Van Den 
Published: March 2005

The human insulin‐like‐growth‐factor‐2 (IGF‐2) gene generates mRNAs with four different leader sequences, but with identical coding and trailing regions. Previous research has revealed that the leader‐2‐containing and leader‐4‐containing mRNAs are completely polysomal, whereas mRNAs possessing leader‐3 are predominantly present in the untranslated free messenger ribonucleoprotein particle (mRNP), both in cell lines and in foetal liver tissue. To investigate the influence of the IGF‐2 leader sequences on expression of the gene, IGF‐2 leader–luciferase and leader‐chloramphenicol acetyltransferase fusion constructs were transfected transiently into different cell lines. In these experiments, the levels of expression obtained by constructs with leader‐1, leader‐2 and leader‐4 were very similar, both at the level of mRNA and protein. Leader‐3, however, strongly repressed the expression of the fusion mRNA via an unknown mechanism. This repression appeared to be confined to nucleotides at positions 328–906 of the leader sequence. The remaining 5′ part of the leader sequence was efficient both in RNA expression and in translation, but the 3′ part of the leader (nucleotides 906–1180) again moderately repressed luciferase expression, possibly due to endonucleolytic cleavage in this region of the RNA.To evaluate the effect of the IGF‐2 leaders on in vitro translation, leader–chloramphenicol acetyltransferase fusion mRNAs were synthesized and translated in reticulocyte lysates. Compared to a chloramphenicol acetyltransferase control RNA, leader‐1‐chloramphenicol acetyltransferase mRNA translated over 20‐fold less efficiently, whereas leader‐2 repressed translation of its chloramphenicol acetyltransferase mRNA moderately (3–5 fold). Despite a general improvement of the translation efficiency upon translation in HeLa lysate, these discrepancies with the transfection data persisted. Translation of leader‐3‐containing dNAs in reticulocyte lysates was barely detectable. The whole 5′ region of leader‐3, up to nucleotide 614, could be shown to be repressive. Only leader‐4 directed translation of the chloramphenicol acetyltransferase open reading frame efficiently. As with leader‐1 and leader‐2, this L4‐chloramphenicol acetyltransferase mRNA translated in a cap‐dependent manner under the conditions of our experiments; translation of this mRNA was relatively resistant to addition of cap analogue.We conclude that all four IGF‐2 leader sequences differ in their translational properties. This makes it likely that changes in the translational machinery will affect the expression of the various IGF‐2 mRNAs differentially.
Luminescence spectroscopy of pyridoxic acid and pyridoxic acid bound to proteins
Authors: KWON Oh‐Shin , BLAZQUEZ Manuel , CHURCHICH Jorge E. 
Published: March 2005

Luminescence techniques, i.e. fluorescence and phosphorescence, have been employed to study pyridoxic acid bound to proteins through a stable amide linkage. Proteins tagged with 4‐pyridoxic acid display the following fluorescence properties: (a) emission and excitation spectra centered at around 430 and 320 nm, respectively; (b) fluorescence quantum yields of 0.3–0.4 and (c) average decay times covering the range 8–9.6 ns. The fluorescence properties of the probe have been used to study the dynamics of the protein in the nanosecond time scale.In the absence of molecular oxygen, free and bound 4‐pyridoxic acid exhibit long‐lived emission at room temperature. The long‐lived emission is red‐shifted when compared to fluorescence and decays with average life times ranging over 2.2–0.6 ms depending on the nature of the protein.The fluorophore pyridoxic acid covalently linked to proteins is suitable to study the dynamics of proteins, i.e. fast and slow motions of the macromolecule in the nanosecond and millisecond time scales, respectively.
Kinetic resolution of the reaction catalysed by proton‐translocating transhydrogenase from Escherichia coli as revealed by experiments with analogues of the nucleotide substrates
Authors: HUTTON Mike , DAY Joanna M. , BIZOUARN Tania , JACKSON J. Baz 
Published: March 2005

The mechanism, by which transhydrogenase couples transfer of H− equivalents between NAD(H) and NADP(H) to the translocation of protons across a membrane, has been investigated in the solubilised, purified enzyme from Escherichia coli using analogues of the nucleotide substrates. The key observation was that, at low pH and ionic strength, solubilised transhydrogenase catalysed the very rapid reduction of acetylpyridine adenine dinucleotide (an analogue of NAD+) by NADH, but only in the presence of either NADP+ or NADPH. This indicates that the rates of release of NADP+ and NADPH from their binary complexes with the enzyme are slow. The dependences on pH and salt concentration suggest that (a) release of both NADP+ and NADPH are accompanied by the release of H+ from the enzyme and (b) increased ionic strength decreases the value of the pKa of the group responsible for H+ release. Modification of the enzyme with N,N1‐dicyclohexylcarbodiimide led to inhibition of the rate of release of NADP+ and NADPH from the enzyme, but had a much smaller effect on the binding and release of NAD+, NADH and their analogues and on the interconversion of the ternary complexes of the enzyme with its substrates.It is considered that the binding and release of H+, which accompany the binding and release of NADP+/NADPH, might be central to the mechanism of proton translocation by the enzyme in its membrane‐bound state.
Purification, characterization, primary structure, crystallization and preliminary crystallographic study of a serine proteinase from Streptomyces fradiae ATCC 14544
Authors: KITADOKORO Kengo , TSUZUKI Hiroshige , NAKAMURA Etsuo , SATO Tomohiro , TERAOKA Hiroshi 
Published: March 2005

A proteinase having wide substrate specificity was isolated from Streptomyces fradiae ATCC 14544. This proteinase, which we propose to call SFase‐2, was purified from the culture filtrate by S‐Sepharose chromatography. The purified enzyme showed an apparent molecular mass of 19 kDa on SDS/PAGE. When synthetic peptides were used as substrates, SFase‐2 showed broad substrate specificity. It also hydrolyzed keratin, elastin and collagen as proteinaceous substrates. It was completely inhibited by diisopropylfluorophosphate and chymostatin, but not by tosylphenyl‐alaninechloromethane, tosyllysinechloromethane or EDTA, indicating that it can be classified as a serine proteinase.The matured protein sequence of SFase‐2 was determined by a combination of amino acid sequencing and the DNA sequencing of the gene. SFase‐2, consisting of 191 amino acids, is a novel proteinase. It showed 76% similarity in the amino acid sequence with Streptomyces griseus proteinase A [Johnson P. and Smillie L. B. (1974) FEBS Lett. 47, 1–6].For insight into the three‐dimensional structure of SFase‐2, we obtained single crystals by the vapor diffusion method using sodium phosphate as a precipitant. These crystals belonged to the orthorhombic, space group P212121 with cell dimensions a= 6.92 nm, b= 7.28 nm, c= 2.99 nm; one molecule was present in the asymmetric unit.