bluuebunny/crossref_metadata_2025 huggingface dataset:
Thiols of Intracellular Pathogens
Authors: Spies Hendrik S. C. , Steenkamp Daniel J.
Published: March 2005

Leishmania donovani, the causative agent of visceral leishmaniases, is an intracellular pathogen which proliferates within the host macrophages. Analysis of the thiol composition of L. donovani by means of the thiol‐specific reagent, 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin, indicated that this organism produces substantial amounts of ovothiol A. This observation was further substantiated by HPLC of extracts of L. donovani after derivatization with bromobimane. L. donovani extracts contained a thiol, the bimane derivative of which had identical retention time and fluorescence quenching to a thiol from Crithidia fasciculata, which had previously been identified as ovothiol A. By comparison, the intracellular bacterial pathogen, Mycobacterium bovis, contained only one major low‐molecular‐mass thiol, which was assigned the trivial name mycothiol. The structure of the bimane derivative of mycothiol was solved by a combination of one‐ and two‐dimensional 1H and 13C NMR spectroscopy. Spatial relationships in the molecule were further refined by NOE experiments and allowed identification of mycothiol as 1‐d‐myo ‐inositol‐2‐(N ‐acetyl‐l‐cysteinyl)amino‐2‐deoxy‐α‐d‐glucopyranoside. This assignment was confirmed by positive‐ion fast‐atom‐bombardment mass spectrometry which gave m/z= 677.6 Da and a sodiated species at 699.6 Da. Analysis of the dansylated hydrolysis products of performic‐acid‐oxidized mycothiol indicated the presence of 0.85 mol glucosamine and 1.02 mol cysteic acidmol sulfhydryl groups. Crude extracts of M. bovis contained an enzyme which catalysed the NAD(P)H2‐dependent reduction of mycothiol disulfide to the free thiol. Analysis of perchloric acid extracts of Mycobacterium tuberculosis H37RV indicated the presence of a thiol which comigrated with mycothiol, both as the free thiol and as the 7‐diethylamino‐3‐(4′‐maleimidylphenyl)‐4‐methylcoumarin and bimane derivatives, on reverse‐phase HPLC. The significance of these findings in terms of the evasion of the host defense mechanisms by leishmania parasites and mycobacteria is considered.
ADP‐Induced Changes in Ordering of Spin‐Labelled Myosin Heads in Muscle Fibres
Authors: Belágyi Joseph , Frey István , Pótó László
Published: March 2005

Rotational dynamics and ordering of myosin heads in glycerinated skeletal muscle fibres were studied using an isothiocyanate‐based spin label attached to the fast‐reacting thiol sites of myosin and were compared with data obtained for maleimide and iodoacetamide spin labels attached to the same sites. The ordering of probe molecules on the millisecond time scale in the rigor state, at sarcomere length 2.2–3 ± 0.1 μm static. Isothiocyanate probe molecules showed greater mobility; the segment holding the label rotated in the microsecond time range. In the saturation transfer EPR time domain, MgADP did not produce a significant change in the mobility of spin labels.

The spetcra of isothiocyanate spin‐labelled fibres were analyzed in terms of two narrow distributions with mean angles of 75° and 56°. In the rigor state, the fractions represented approximately 76% and 24% of the total EPR absorbance. In the presence of MgADP, the conventional EPR spectra showed large changes in the ordering of isothiocyanate probe molecules towards a new distribution, the population with a θ value of 56% increased from 24% to 71% at the expense of the 75% population with no change in the mean angles of the distributions. In the case of maleimide and iodoacetamide spin‐labelled fibres, however, the effect of MgADP on the probe angular distribution was small.
Nucleation of Actin Polymerization by Gelsolin
Authors: Ditsch Andrea , Wegner Albrecht
Published: March 2005

The time‐course of assembly of actin with gelsolin was measured by the fluorescence increase of a fluorescent label covalently linked to actin. The actin concentrations ranged from values far below the critical concentration to values above the critical concentration of the pointed ends of actin filaments. If the concentration of actin was in the range of the critical monomer concentration (0.64 μM), the time‐course of the concentration of actin assembled with gelsolin revealed a sigmoidal shape. At higher actin concentrations the time‐course of association of actin with gelsolin approximated an exponential curve. The measured time‐courses of assembly were quantitatively interpreted by kinetic rate equations. A poor fit was obtained if two actin molecules were assumed to bind to gelsolin to form a 1:2 gelsolin‐actin complex and subsequently further actin molecules were assumed to polymerize onto the 1:2 gelsolin‐actin complex toward the pointed end. A considerably better agreement between calculated and measured time‐courses was achieved if additional creation of actin filaments by fast fragmentation of newly formed actin filaments by not yet consumed gelsolin was assumed to occur. This suggests that both polymerization of actin onto gelsolin and fragmentation of actin filaments contribute to formation of new actin filaments by gelsolin. Furthermore it could be demonstrated that below the critical monomer concentration appreciable amounts of actin are incorporated into gelsolin‐actin oligomers.
A Single Amino Acid Mutation Enhances the Thermal Stability of Escherichia coli Malate Dehydrogenase
Authors: Goward Christopher R. , Miller Julie , Nicholls David J. , Irons Laurence I. , Scawen Michael D. , O'brien Ronan , Chowdhry Babur Z.
Published: March 2005

The stability of wild‐type Escherichia coli malate dehydrogenase was compared with a mutant form of the enzyme with the amino acid residue at position 102 changed from arginine to glutamine. The mutation occurs on the underside of a mobile loop which closes over the active‐site cleft on formation of the enzyme/cofactor/substrate ternary complex. The mutant enzyme is kinetically compromised while the wild‐type enzyme is highly specific for oxaloacetate. The mutant enzyme was shown to be more resistant to irreversible thermal denaturation by thermal inactivation experiments and high‐sensitivity differential scanning calorimetry than the wild‐type enzyme. In contrast, resistance of both enzymes to reversible unfolding in guanidinium chloride was similar. Circular dichroic spectropolarimetry shows the secondary structures of the enzymes are similar but there is a demonstrable difference in tertiary structure. From the position of the mutation, it is conjectured that the substitution on a mobile surface loop results in partial closure of the loop and greater resistance to thermal inactivation of the mutant enzyme. However, molecular modelling combined with circular dichroic spectropolarimetry indicate that the mutation may have a more widespread effect on the structure than simply partial closure of the mobile surface loop as the environment of distant tyrosine residues is altered. Resistance of the wild‐type enzyme to thermal inactivation can be increased by cofactor addition, which may have the effect of partial closure of the mobile surface loop, but has little effect on the mutant enzyme.
Identification and Partial Purification of Calmodulin‐Binding Microtubule‐Associated Proteins from Neurospora crassa
Authors: Perez Ruben Ortega , Irminger‐Finger Irmgard , Arrighi Jean‐François , Capelli Nicolas , van Tuinen Diederik , Turian Gilbert
Published: March 2005

We have purified microtubule‐associated proteins from Neurospora crassa on the basis of heat stability and affinity to calmodulin. Two proteins of molecular masses 170 kDa and 190 kDa have been partially purified. A third protein of 145 kDa was purified almost to homogeneity, and we present evidence that this protein is a specific substrate for a Ca2+/calmodulin‐dependent protein kinase. The purified 170‐, 190‐, and 145‐kDa proteins induce the assembly of microtubules from purified porcine brain tubulin. We demonstrate that all three proteins are microtubule‐associated proteins on the basis of an in vitro microtubule‐binding assay.
Dissociation Rate of Cognate Peptidyl‐tRNA from the A‐Site of Hyper‐Accurate and Error‐Prone Ribosomes
Authors: Karimi Reza , Ehrenberg Måns
Published: March 2005

The binding stability of the aminoacyl‐tRNA site (A‐site), estimated from the dissociation rate constant kd, of AcPhe‐Phe‐tRNAphe has been studied for wild‐type (wt), for hyperaccurate ribosomes altered in S12 [streptomycin‐dependent (SmD) and streptomycin‐pseudodependent (SmP) phenotypes], for error‐prone ribosomes altered in S4 (Ram phenotype), and for ribosomes in complex with the error‐inducing aminoglycosides streptomycin and neomycin. The AcPhe2‐tRNA stability is slightly and identically reduced for SmD and SmP phenotypes in relation to wt ribosomes. The stability is increased (kd is reduced) for Ram ribosomes to about the same extent as the proofreading accuracy is decreased for this phenotype. kd is also reduced by the action of streptomycin and neomycin, but much less than the reduction in proof‐reading accuracy induced by streptomycin. Similar kd values for SmD and SmP ribosomes indicate that the cause of streptomycin dependence is not excessive drop‐off of peptidyl‐tRNAs from the A‐site.
Three‐Dimensional Structure in Solution of a Wheat Lipid‐Transfer Protein from Multidimensional 1H‐NMR Data
Authors: Gincel Edith , Simorre Jean‐Pierre , Caille Anita , Marion Didier , Ptak Marius , Vovelle Françoise
Published: March 2005

Two‐dimensional and three‐dimensional 1H‐NMR experimental data [Simorre, J. P., Caille, A., Marion, D., Marion, D. & Ptak, M. (1991) Biochemistry 30, 11600–11608] were used to build models of the three‐dimensional structure of a non‐specific wheat lipid‐transfer protein (LTP) by using distance geometry, simulated annealing, energy minimization and molecular dynamics techniques. A first set of 881 distance constraints derived from NOE cross‐peak intensities was used to generate 74 initial structures. One family of topological mirror images of the protein structure was eliminated by considering helical secondary‐structure organization and steric requirements. Back calculations of NOE intensities led us to introduce 535 additional distance constraints. Finally, 21 structures were selected as representative of the structure of the protein. The polypeptide backbone folds into a simple and original right‐handed winding. It is composed of a bundle of four helices linked by flexible loops, which is packed against a C‐terminal fragment forming a non‐standard saxophone‐like shape. The folded protein is stabilized by hydrophobic interactions and the four disulfide bridges combined by pairs on each side of the protein. An hydrophobic cleft, formed by residues located in the second half of the protein could be a potential site for the binding of lipids.
Evidence fromIn vivoManipulations of Lipid Composition in Mutants that the Δ3‐Trans‐Hexadecenoic Acid‐Containing Phosphatidylglycerol is Involved in the Biogenesis of the Light‐Harvesting Chlorophylla/b‐Protein Complex ofChlamydomonas reinhardtii
Authors: Dubertret Guy , Mirshahi Ali , Mirshahi Massoud , Gerard‐Hirne Catherine , Trémolieres Antoine
Published: March 2005

The phosphatidylglycerol containing the unusual Δ3‐transhexadecenoic fatty acid is specifically found in photosynthetic membranes of eukaryotic organisms. Its involvement in the biogenesis and the structure of the light‐harvesting chlorophylla/b‐protein complex has been evidenced byin vivotargeting this lipid to photosynthetic membranes ofChlamydomonas reinhardtiimutants lacking this lipid. In themf1andmf2mutants, this deficiency results in (a) the absence of the oligomeric lightharvesting complex of photosystem 2; (b) an extensive destacking of thylakoid membranes; (c) a very low 77‐K fluorescence emission in the photosystem–2 region.

We show in this paper that these deficiencies result from modifications in the pigment and polypeptide compositions of the photosystem‐2 light‐harvesting complex; it contains less chlorophyllband some of its constitutive polypeptides are absent or reduced in amount, while immunologically related polypeptides of lower molecular mass accumulate. The direct involvement of the lack oftrans‐C16: 1‐phosphatidylglycerol in these deficiencies is evidenced by the partial restoration of normal characteristics of the light–harvesting complex (pigment and polypeptide composition, oligomerization) after liposome‐mediated,in vivoincorporation of this lipid into the photosynthetic membranes of themflmutant.Trans‐C16: 1‐phosphatidylglycerol, therefore, is involved in the biogenesis of the photosystem‐2 light‐harvesting chlorophylla/b‐protein complex through a mechanism that may prevent degradation processes. Its contribution to the structural conformation of neosynthesized monomers and to their organization into stable oligomeric form is discussed.
The Widespread Expression of Angiogenin in Different Human Cells Suggests a Biological Function not only Related to Angiogenesis
Authors: Moenner Michel , Gusse Michel , Hatzi Elissavet , Badet Josette
Published: March 2005

Angiogenin is a secreted polypeptide that induces neovascularization in vivo. The expression of angiogenin by human cells in culture was investigated by using a specific radioimmunoassay and by cDNA hybridization. Angiogenin immunoreactivity was widely but differentially produced by anchorage‐dependent growing cells including vascular endothelial cells from saphenous and umbilical veins, aortic smooth muscle cells, fibroblasts (from embryos, new‐borns and adults), and tumour cells. Endothelial cells from saphenous veins and the endothelium‐derived EA.hy926 cell line released immunoreactivity whatever the stage of the culture, including release at the lag phase, during exponential growth and at the confluent phase. However, the rate of accumulation of angiogenin varied as a function of EA.hy926 cell density. As compared to anchored cells, normal peripheral blood cells and tumour cells of myelomonocytic and megakaryocytic origin did not noticeably secrete angiogenin except at low levels. A myeloma cell line supernatant contained as much angiogenin cross‐reactivity as did anchored cells, while four tumour T‐cell lines expressed the cross‐reactivity at different levels, i.e. from undetectable levels to a high level. A 0.9‐kb angiogenin messenger RNA was detected by Northern‐blot analyses in a variety of representative cells correlating with the presence of immunoreactivity in the cell‐culture media. The widespread expression pattern of angiogenin suggests a physiological function that is not restricted to the neovascularization process.
Description of the Kinetic Mechanism and the Enantioselectivity of Quinohaemoprotein Ethanol Dehydrogenase from Comamonas testosteroni in the Oxidation of Alcohols and Aldehydes
Authors: Geerlof Aric , Rakels Johannes J. L. , Straathof Adric J. J. , Heijnen Joseph J. , Jongejan Jaap A. , Duine Johannis A.
Published: March 2005

Initial rate studies were performed on the oxidation of (racemic) alcohols as well as aldehydes by quinohaemoprotein ethanol dehydrogenase, type 1, from Comamonas testosteroni with potassium ferricyanide as electron acceptor. The data could be fitted with an equation derived for a mechanism (hexa‐uni ping‐pong) in which alcohols are oxidized to the corresponding carboxylic acids and the intermediate aldehyde becomes released from the enzyme. However, for some substrates it was necessary to assume that they exert uncompetitive inhibition. The same model was used to fit the data of conversion processes. Reversible inactivation of the enzyme takes place during the conversion, the extent being inversely proportional to the concentration of ferricyanide present at the start. From the values of the kinetic parameters obtained for (R)‐ and (S)‐solketal [2,2‐dimethyl‐4‐(hydroxymethyl)‐1,3‐dioxolane] and their corresponding aldehydes, it appeared that the second step in (S)‐solketal conversion is much faster than the first one and that opposite enantiomeric preferences exist for the alcohol and the aldehyde substrates. Since the initial rate measurements as well as the progress curve analysis gave similar kinetic parameter values and product analysis revealed intermediates in the amounts predicted, it is concluded that the kinetic and enantioselective behaviour of the enzyme is adequately described by the model presented here. Finally, the results indicate that both kinetic approaches should be used in conversions with consecutive reactions since they provide complementary information.
Enzymically Produced Cyclic α‐1,3‐Linked and α‐1,6‐Linked Oligosaccharides of d‐Glucose
Authors: Côté Gregory L. , Biely Peter
Published: March 2005

A new type of bacterial enzyme hydrolyzed alternan (Leuconostoc mesenteroides NRRL B‐1355 fraction S dextran, an alternating α‐1,3–α‐1,6‐d‐glucan) to give rise to a series of oligosaccharides. The oligosaccharide formed in the greatest proportion was a cyclic tetrasaccharide of d‐glucosyl residues linked in an alternating α‐1,3–α‐1,6 fashion. Other saccharide products included isomaltose and α‐d‐glucopyranosyl‐1,3–α‐d‐glucopyranosyl‐1,6‐d‐glucose. Oligosaccharides of higher degrees of polymerization were also formed, and included α‐d‐glucosylated derivatives of the cyclic tetrasaccharide. This is the first report of a naturally produced cyclic tetrasaccharide.
Interaction of Plant Profilin with Mammalian Actin
Authors: Giehl Klaudia , Valenta Rudolf , Rothkegel Martin , Ronsiek Melanie , Mannherz Hans‐Georg , Jockusch Brigitte M.
Published: March 2005

The mode of interaction of birch and bovine profilins with actin was compared using a number of techniques. Birch profilin was purified from pollen or as a recombinant protein from Escherichia coli, using poly(l‐proline) affinity chromatography and a monoclonal antibody for the identification of the isolated product. On two‐dimensional gels, the genuine and recombinant proteins were identical in molecular mass and isoelectric point and revealed that birch profilin, in contrast to the basic profilins found in mammals, is an acidic protein, analogous to maize profilins. Bovine profilin was obtained from calf thymus.

In viscometric assays, the birch protein was seen to modulate actin filament formation analogous to animal profilin. Birch profilin increased the critical concentration required for muscle and brain actin polymerization in a concentration‐dependent manner, supporting the notion of the formation of a heterologous complex between the plant protein and animal actin. The effect was Mg2+‐sensitive, as had been described for homologous complexes. The dissociation constants obtained for the plant/vertebrate and the vertebrate/vertebrate system were both in the micromolar range. The affinity of birch profilin for muscle actin was slightly lower than that for nonmuscle (brain) actin. A binary complex of birch profilin and skeletal muscle actin could be isolated by gel chromatography.

Cross‐linking experiments with actin, birch profilin, the G‐actin binding peptide thymosin β4 and gelsolin segment 1, the N‐terminal fragment of an actin capping protein, showed that profilin competed with thymosin β4, but had no effect on segment 1 binding to actin.

These data indicate that the actin‐binding domains in plant and animal profilins are functionally highly conserved, although the overall sequence similarity is less than 25%.
Vanadate Inhibition of Hepatocytic Autophagy
Authors: Fosse Margrete , Berg Trond Olav , O'Reilly Denis St. John , Seglen Per Ottar
Published: March 2005

The phosphate analogue vanadate, at 10 mM, strongly (approximately 90%) inhibited the autophagic sequestration of endogenous lactate dehydrogenase in isolated rat hepatocytes. The effect of vanadate was markedly (approximately 80%) antagonized by asparagine (20 mM), and to a lesser extent by glutamine, glycine, and alanine. The antagonism was only observed in the presence of Ca2+ when an isotonic standard incubation medium was used, but by increasing the medium osmolality this Ca2+ requirement could be eliminated. Asparagine induced a cell swelling (17% at 20 mM) that might account for at least part of its vanadate antagonism, since hypotonic cell swelling by itself stimulated autophagy (with a maximal effect at approximately 200 mosM). Conversely, hypertonic media inhibited autophagy and were additive to vanadate. In a strongly hypotonic medium (less than 200 mosM), both asparagine and vanadate were inhibitory. However, since vanadate alone had no effect on cell volume, the vanadate‐asparagine antagonism could not be exerted exclusively at the level of cell volume regulation. An additional mechanism might be a partial deamination of asparagine, generating ammonia, which was found to oppose the vanadate inhibition of autophagy while having no effect on cell volume. Other metabolizable amino acids, like alanine and glycine, were moderately vanadate‐antagonistic while failing to induce cell swelling. These results are compatible with a vanadate‐antagonistic effect of asparagine mediated partly through an unknown mechanism (possibly pH change) by its deamination product, ammonia, partly through cell swelling and a secondary Ca2+ influx that could compensate for a vanadate‐induced depletion of intracellular calcium stores.
The Binding of Divalent Cations to Escherichia coliα‐Haemolysin
Authors: Ostolaza Helena , Soloaga Ana , Goñi Félix M.
Published: March 2005

α‐haemolysin, an extracellular protein toxin of Escherichia coli, is known to disrupt eukaryotic cell membranes. In spite of genetic evidence of Ca2+‐binding motifs in its sequence, conflicting results are found in the literature on the requirement of divalent cations for the membranolytic activity of the toxin. Moreover, Ca2+‐binding sites have not been characterized to date in the native protein. The results in this paper show that when Ca2+ levels are kept sufficiently low during bacterial growth and toxin purification, membrane lysis does not occur in the absence of added divalent cations. Ca2+ and, at higher concentrations, Sr2+ and Ba2+, support the lytic activity, but Mg2+, Mn2+, Zn2+ and Cd2+ appear to be inactive in this respect. Binding of metal ions can be followed by changes in the intrinsic fluorescence of α‐haemolysin; ions supporting lytic activity produce changes in the intrinsic fluorescence that are not caused by the inactive ones. Scatchard analysis of 45Ca2+ binding reveals three equivalent, independent sites, with Kd≈ 0.11 mM. No 45Ca2+ binding is observed when the protein is incubated with Zn2+; conversely, incubation with Ca2+ prevents subsequent binding of 65Zn2+. In the light of three‐dimensional data available for a structurally related protein, alkaline protease of Pseudomonas aeruginosa [Baumann, U., Wu, S., Flaherty, K. M. & McKay, D. B. (1993) EMBO J. 12, 3357–3364] it is suggested that α‐haemolysin may bind a larger number of Ca2+ than the three that are more easily exchangeable and are thus detected in the 45Ca2+‐binding experiments. In addition, structural similarities and conservation of ion‐binding motifs support the hypothesis that His 859 is involved in the mutually exclusive binding of Zn2+ and Ca2+.
The F0 Complex of the Escherichia Coli ATP Synthase
Authors: Birkenhäger Ralf , Hoppert Michael , Deckers‐Hebestreit Gabriele , Mayer Frank , Altendorf Karlheinz
Published: March 2005

Cholate‐solubilized F0 complexes of the ATP synthase (F0F1) from Escherichia coli were studied by application of conventional transmission electron microscopy and electron spectroscopic imaging (ESI) of negatively stained samples. Using the ESI mode, the structural organization of the F0 complex (diameter of 7.5±0.5 nm) could be observed in more detail and defined projections could be distinguished. Projection A appears as a deltoid‐like structure with bilateral symmetry. Projection B has an overall trapezoidal shape with some similarity in shape to the letter W. Applying the ESI mode to the ac complex dissolved in cholate‐containing buffer, an elongated structure consisting of two intensity maxima could be observed. Simulations with models of the F0 and the ac complex revealed that the projections observed can be obtained by tilting and rotating a model in which subunit α and the two copies of subunit b are located outside the subunit c oligomer. This view of structural organization was supported by results obtained with F0 complexes decorated with monoclonal antibodies against subunits a, b or c.
Purification of Turnip Mosaic Potyvirus Viral Protein Genome‐Linked Proteinase Expressed in Escherichia coli and Development of a Quantitative Assay for Proteolytic Activity
Authors: Ménard Robert , Chatel Henrietta , Dupras Robert , Plouffe Céline , LalibertÉ Jean‐François
Published: March 2005

The 49‐kDa, nuclear inclusion a‐like, viral protein genome‐linked proteinase (VPg‐Pro) of turnip mosaic potyvirus (TuMV) was expressed in Escherichia coli. The protein was produced in a soluble form at high levels and was active, as demonstrated by intermolecular cleavage of the polymerase capsid protein (Pol‐CP) substrate. The VPg‐Pro was purified by metal‐chelation and ion‐exchange chromato‐graphies. Two forms of VPg‐Pro, which differed in molecular masses, were obtained during isolation; their identities were confirmed by immunoblot analysis and N‐terminal amino acid sequencing. Data indicated that cleavage took place at a site near the C‐terminus of VPg‐Pro and was the result of the proteolytic activity of the viral protein. The purified proteinase retained enzymic activity on its natural substrate (Pol‐CP) and was also capable of hydrolysing the synthetic peptide acyl‐Ala‐Ala‐Val‐Tyr‐His‐Gln‐Ala‐Ala‐NH2, derived from the consensus cleavage site for the TuMV polyprotein. Analysis by mass spectrometry of the two fragments resulting from this reaction indicated that cleavage took place between the Gin and Ala residues, as expected. A fluorogenic derivative of this peptide was hydrolysed by VPg‐Pro, affording a convenient quantitative assay for intermolecular proteolytic activity, and was used to determine the pH‐activity profile. The availability of large quantities of pure proteinase and of a rapid and sensitive assay will permit detailed kinetic and structural studies which are essential to obtain a better understanding of the mode of action of this and related viral proteinases, such as the 3C proteinase of picornaviruses.
Overproduction, Purification and Characterization of the Cellulose‐Binding Domain of the Erwinia Chrysanthemi Secreted Endoglucanase EGZ
Authors: Brun Emmanuel , Gans Pierre , Marion Dominique , Barras Frédéric
Published: March 2005

EGZ is the major endoglucanase secreted by Enwinia chrysanthemi. Functional characterization indicates that it is made of a catalytic N‐terminal domain linked to a C‐terminal cellulose‐binding domain (CBD) by a Ser/Thr‐rich linker. A chimeric plasmid, in which the CBD‐encoding region was fused downstream of the ompA signal sequence, was constructed and introduced into Escherichia coli. This allowed for the production of processed and disulfide‐bonded CBD, mostly recovered from the culture supernatant of E. coli. One‐dimensional NMR analysis of the purified CBD reveals that it folds into a well‐structured domain. Moreover, comparison with the one‐dimensional NMR analysis of full‐length EGZ strongly suggests that the CBD folds autonomously, providing experimental support for the existence of domains of EGZ.
In vivo and in vitro Studies of the Inhibition of the Channel Activity of Colicins by Gadolinium
Authors: Bonhivers Mélanie , Guihard Gilles , Pattus Franc , Letellier Lucienne
Published: March 2005

The primary effects of the ionophoric colicins A, E1 nd B on Escherichia coli cells include triggering an efflux of cytoplasmic potassium, and a decrease of internal ATP as consequences of the opening of ionic channels in the cytoplasmic membrane. We report that micromolar concentrations of gadolinium and other members of the lanthanide family inhibited the efflux of potassium and the ATP decrease and that the cells recovered both ATP and potassium within a few minutes. Gadolinium, in the same concentration range also efficiently inhibited the channel activity of colicins A, E1, B and of the isolated channel‐forming domain of colicin A in planar lipid bilayers. Colicin N was much less sensitive to the trivalent ion in planar lipid bilayers, consistent with the lack of effect of gadolinium on this colicin in vivo. Our data suggest that lanthanide ions act by direct binding to the colicin molecule and that this binding affects both its single‐channel conductance and gating behaviour.
Structure and Function of Mutant Arg44Lys of 4‐Hydroxybenzoate Hydroxylase
Authors: Eppink Michel H. M. , Schreuder Herman A. , Van Berkel Willem J. H.
Published: March 2005

Arg44, located at the si‐face side of the flavin ring in 4–hydroxybenzoate hydroxylase, was changed to lysine by site‐specific mutagenesis. Crystals of [R44K]4‐hydroxybenzoate hydroxylase complexed with 4‐hydroxybenzoate diffract to 0.22‐nm resolution. The structure of [R44K]4‐hydroxybenzoate hydroxylase is identical to the wild‐type enzyme except for local changes in the vicinity of the mutation. The peptide unit between Ile43 and Lys44 is flipped by about 180° in 50% of the molecules. The φ,ψ angles in both the native and flipped conformation are outside the allowed regions and indicate a strained conformation. [R44K]4‐Hydroxybenzoate hydroxylase has a decreased affinity for the flavin prosthetic group. This is ascribed to the lost interactions between the side chain of Arg44 and the diphosphoribose moiety of the FAD. The replacement of Arg44 by Lys does not change the position of the flavin ring which occupies the same interior position as in wild type.

[R44K]4‐Hydroxybenzoate hydroxylase fully couples flavin reduction to substrate hydroxylation. Stopped‐flow kinetics showed that the effector role of 4‐hydroxybenzoate is largely conserved in the mutant. Replacement of Arg44 by Lys however affects NADPH binding, resulting in a low yield of the charge‐transfer species between reduced flavin and NADP+. It is inferred from these data that Arg44 is indispensable for optimal catalysis.
Glucocorticoid Regulation of Bone Sialoprotein (BSP) Gene Expression
Authors: Ogata Yorimasa , Yamauchi Masato , Kim Richard H. , Li Jack J. , Freedman Leonard P. , Sodek Jaro
Published: March 2005

Glucocorticoids modulate the development and growth of many organs through interactions with a specific intracellular receptor (glucocorticoid receptor) that regulates gene transcription through a cognate element, the glucocorticoid response element (GRE), in the promoter of target genes. In bone formation glucocorticoids stimulate osteoblast differentiation and the formation of bone matrix. Recent studies have demonstrated that the induction of the bone sialoprotein (BSP) gene is associated with osteoblast differentiation and de novo bone formation. To determine the molecular pathways of glucocorticoid regulation of BSP expression, we have analyzed the effects of the synthetic glucocorticoid, dexamethasone, on the expression of the BSP by bone cells in vitro. At 10 nM, dexamethasone induced BSP expression in association with bone tissue formation by confluent fetal rat calvarial cells and adult rat marrow cells and also stimulated BSP expression up to sixfold in osteoblastic cells (UMR 106–6 and ROS 17/2.8 cells). Most of the stimulation was blocked by cycloheximide, indicating direct and indirect mechanisms of BSP gene regulation. Nuclear ‘run‐on’ transcription analysis revealed an up to twofold increase in transcription corresponding to the increase in mRNA that was unaffected by cycloheximide. Analysis of BSP mRNA in the presence of a transcription inhibitor (5,6–dichloro‐1‐β‐d‐ribofuanosyl benzimidazole) by Northern hybridization revealed that the stability of the BSP mRNA was not significantly altered by dexamethasone, indicating that the major, indirect, stimulation of BSP expression involves a nuclear post‐transcriptional mechanism. To study the direct effects of dexamethasone, nucleotide sequence analysis of the rat BSP promoter was extended upstream to position−2992 and downstream to +2282 in the first intron. Transient transfection analyses, using various rat BSP promoter constructs linked to a luciferase reporter gene, and gel mobility shift assays were used to identify a putative glucocorticoid response unit comprising three GRE half‐sites and a putative AP‐1 site, located within positions –906 to –931 upstream from the translation start site of the BSP gene promoter. BSP transcription was stimulated ≈1.5‐fold by dexamethasone through this GRE, indicating that its direct effects are mediated by glucocorticoid receptor binding to this site. These studies, therefore, have identified both indirect and direct pathways of glucocorticoid regulation of BSP gene expression, the direct effects being mediated by a GRE in the rat BSP promoter through which the effects of glucocorticoids on BSP gene transcription appear to be regulated.
An Infrared Investigation of Palmitoyl‐Coenzyme A and Palmitoylcarnitine Interaction with Perdeuterated‐Chain Phospholipid Bilayers
Authors: Echabe Izaskun , Requero M. Asun , Goñi Félix M. , Arrondo José L. R. , Alonso Alicia
Published: March 2005

Mixtures of di‐(perdeuteropalmitoyl)‐sn‐glycero‐3 choline ([2H62]Pam2GroPCho) with palmitoylcarnitine or palmitoyl‐CoA in aqueous suspension have been examined by Fourier‐transform infrared spectroscopy. The C‐H (or C‐D) region of the spectrum shows that an order‐disorder transition exists in pure aqueous palmitoylcarnitine at 45°C; palmitoylcarnitine mixes with [2H62]Pam2GroPCho without perturbing the gel‐fluid transition of the phospholipid even at [2H62]Pam2GroPCho palmitoylcarnitine 1:2 molar ratios; and palmitoyl‐CoA, however, at similar proportions, smears out the [2H62]Pam2GroPCho transition as detected from C‐D stretching vibrations. Relevant data from the carbonyl region include; the high‐frequency (non‐hydrogen bound) carbonyl subpopulation, but not the low frequency one, detects the gel‐to‐fluid transition of the phospholipid; the carbonyl region detects the thermotropic transition over a wider temperature range than the methylene stretching region, i.e. detects changes starting well below and ending several degrees above the methylene transition temperature, and a significant interaction may occur between some coenzyme A group and the carbonyl groups of the phospholipid. The latter interaction may contribute to explain the coenzyme A/carnitine exchange during mitochondrial fatty acid import.
High‐Affinity Ligand Binding to Subunit HI of the Asialoglycoprotein Receptor in the Absence of Subunit H2
Authors: Bider Marc D. , Cescato Renzo , Jenö Paul , Spiess Martin
Published: March 2005

The hepatic asialoglycoprotein receptor is a hetero‐oligomer composed of two homologous subunits. The specificity and affinity of ligand binding depends on the number and spatial arrangement of several galactose‐binding sites within the receptor complex. Previous studies indicated that both subunits are required for high‐affinity ligand binding, i.e. for the simultaneous interaction with three galactose residues within an N‐linked glycan. However, we found that asialoorosomucoid (ASOR) and asialofetuin (ASF) bind to transfected COS‐7 cells expressing subunit H1 in the absence of the second subunit H2. ASOR binding occurred with a dissociation constant of approximately 40 nM, approximately four‐times higher than the Kd of ASOR binding to the hetero‐oligomeric receptor. Normalized to the amount of H1 expressed, approximately 10–times fewer binding sites were produced by H1 alone. A glycopeptide with a single tri‐antennary N‐linked glycan purified from ASF bound to the hetero‐oligomeric receptor, but did not bind detectably to HI‐expressing COS‐7 cells. H1 is thus unable to simultaneously recognize all three galactose residues in a glycan. From this, we conclude that, at a sufficiently high density of HI on the cell surface, high‐affinity binding of ASOR and ASF is the result of two or more glycans interacting with H1 oligomers with low affinity in a bivalent manner.
tRNA‐Guanine Transglycosylase from Bovine Liver
Authors: Slany Robert K. , Müller Stefan O.
Published: March 2005

The eucaryotic tRNA‐modifying enzyme tRNA‐guanine transglycosylase (Tgt) exchanges a guanine residue in the anticodon of tRNAs specific for aspartic acid, asparagine, histidine and tyrosine with the nutritionally derived deazaguanine base queuine (q), and with queuine precursors and guanine. In higher eucaryotes, the amount of the resulting queuosine nucleoside (Q) is dependent on the developmental state of the respective cells. Neoplastically transformed and fast‐proliferating cells usually are almost Q‐deficient. The Tgt enzyme from bovine liver was purified 14000‐fold by DEAE cellulose chromatography, ammonium sulfate precipitation, and two subsequent affinity chromatography steps on heparin and tRNA agarose. The purest preparations contained two major proteins of 66 kDa and 32 kDa as revealed by SDS/PAGE and silver staining. The Km of the Tgt enzyme for guanine was 1.4 μM and the value for a purified Q‐specific tRNATyr was 0.08 μM. The enzyme was active over a broad pH range; the activity was independent of metal ions and was strongly inhibited by salt concentrations higher than 50 mM. The determination and comparison of the N‐terminal amino acid sequences from endoproteinase Lys‐C cleavage products of the two subunits revealed no significant similarity to any known proteins.
Purification of Paracoccus Denitrificans Cytochrome c552 and Sequence Analysis of the Gene
Authors: Turba Andreas , Jetzek Martin , Ludwig Bernd
Published: March 2005

Unlike mitochondria, many bacteria use a large repertoire of c ‐type cytochromes in different branches of their electron transport system. Among the many cytochromes c present in the soil bacterium Paracoccus denitrificans, a membrane‐bound cytochrome (c552) has been suggested to mediate the electron transport between the cytochrome bc1, complex and cytochrome‐c oxidase [Berry, E. A. & Trumpower, B. L. (1985) J. Biol. Chem. 260, 2458–2467]. We have purified this cytochrome from cytoplasmic membranes, and cloned and sequenced its gene, cyc M. Sequence analysis reveals that, while its C‐terminal portion is highly similar to type‐I cytochromes c, its N‐terminal part contains a hydrophobic segment providing membrane attachment. In addition, we present immunological evidence for its functional role in respiration.
Hepatocyte‐Derived Interleukin‐6 and Tumor‐Necrosis Factor α Mediate the Lipopolysaccharide‐Induced Acute‐Phase Response and Nitric Oxide Release by Cultured Rat Hepatocytes
Authors: Saad Bashar , Frei Karl , Scholl Florence A. , Fontana Adriano , Maier Peter
Published: March 2005

The regulation of acute‐phase protein production and nitric oxide (NO) release in lipopolysaccharide‐induced liver injury is thought to occur in response to monocytes/macrophages and Kupfrer‐cell‐derived cytokines. In this study, we used primary cultured rat hepatocytes maintained as a differentiated phenotype to investigate the direct effects of endotoxin (lipopolysaccharide) on the production of the acute‐phase proteins and on NO release.

Lipopolysaccharide (10 (μg/ml) increased the production of α2‐macroglobulin 2.5‐fold compared to untreated cultures and decreased the production of albumin by 50%. The effect of lipopolysaccharide was mimicked by adding interleukin‐6 (IL‐6) and tumor‐necrosis factor α(TNF‐α), cytokines being induced by treatment of hepatocytes with lipopolysaccharide. Maximal TNF‐α (600 pg/ml) and IL‐6 (1800pg/ml) concentrations were observed 4 h and 6 h after lipopolysaccharide stimulation, respectively. The lipopolysaccharide‐induced acute‐phase protein response was blocked by anti‐(IL‐6) but not by anti‐(TNF‐α) IgG. The latter reduced the lipopolysaccharide‐induced IL‐6 production by 60%. Besides its effects on the acute‐phase proteins, endotoxin caused a significant increase in NO production in cultured rat hepatocytes. Unlike anti‐(IL‐6) IgG, anti‐(TNF‐α) IgG reduced the lipopolysaccharide‐induced NO production by 50% indicating that endotoxin‐induced NO production is partially mediated by TNF‐α but not by IL‐6. Precul‐ture with gadolinium chloride (GdCl3), an inhibitor of Kupffer cells, did not change the response of hepatocytes to lipopolysaccharide indicating that the observed findings are direct endotoxin effects on hepatocytes. The data demonstrate that by their production of TNF‐α and IL‐6 rat hepatocytes respond to lipopolysaccharide treatment with an IL‐6 mediated acute‐phase protein and a TNF‐a‐mediated NO production. These features have previously been attributed to monocytes/macrophages and Kupffer cells.
Purification and Characterization of the Arylsulfatase Synthesized by Pseudomonas aeruginosa PAO During Growth in Sulfate‐Free Medium and Cloning of the Arylsulfatase Gene (atsA)
Authors: Beil Stefan , Kehrli Hans , James Peter , Staudenmann Werner , Cook Alasdair M. , Leisinger Thomas , Kertesz Michael A.
Published: March 2005

An arylsulfatase (EC 3.1.6.1) was extracted from Pseudomonas aeruginosa PAO1 and purified 2700‐fold to homogeneity. Synthesis of this enzyme was repressed when sulfate, cysteine or thiocyanate was supplied as the sole sulfur source for growth, but derepressed with all other sulfur sources tested. The apparent molecular mass was determined by SDS/PAGE to be 57 kDa, and the enzyme was presumed to be a monomer after gel filtration chromatography. The arylsulfatase showed maximal activity at 57°C and pH 8.9, and a Km of 105 μM for 4‐nitrocatecholsulfate. Despite previous reports that both inducible and derepressible forms of arylsulfatase exist in P. aeruginosa, we found only one enzyme under a variety of growth conditions: a sulfate‐repressed enzyme with a native isoelectric point of 4.76. The gene encoding this enzyme (atsA) was isolated by complementation of a Tn5–751 mutant of P. aeruginosa PAO1. Sequencing revealed a 1602‐bp reading frame encoding a 534‐amino‐acid protein with sequence similarity to known bacterial and eukaryotic arylsulfatases (30–40% and 25–30% identity, respectively), but lacking the signal peptide which is present in all known sequences. The lack of this signal peptide suggests that the P. aeruginosa arylsulfatase is neither periplasmic nor membrane‐associated, unlike other known arylsulfatases. The atsA gene was located at 15–17′ on the P. aeruginosa genome by Southern hybridization. Only a single copy was observed under moderate stringency conditions.
Use of Mathematical Models for Predicting the Metabolic Effect of Large‐Scale Enzyme Activity Alterations
Authors: Schuster Ronny , Holzhütter Hermann‐Georg
Published: March 2005

There are numerous examples showing that the metabolism of cells can be severely impaired if the activity of only one of the participating enzymes undergoes large‐scale alterations, resulting, for example, from spontaneous mutations (inherited or aquired enzymopathies), the administration of toxic drugs or self‐inactivation of enzymes during cell aging. However, a quantitative relationship between the degree of enzyme deficiency and the extent of metabolic dysfunction is very difficult to establish by experimental means. An alternative is to tackle this problem by mathematical modelling. Our approach is based on a comprehensive mathematical model of the energy and redox metabolism for human erythrocytes. We calculate stationary states of the cell metabolism, varying the activity of each of the participating enzymes by several orders of magnitude. The metabolic states are then evaluated in terms of a performance function which relates the metabolic variables to the overall functional fitness of the cell. The performance function for the erythrocyte takes into account the homeostasis of three essential metabolic variables: the energetic state (ATP), the reductive capacity (reduced glutathione), and the osmotic state. Based on the behaviour of the performance function at varying enzyme activities, we estimate those ranges of enzyme activities, in which the metabolic alterations should be either tolerable, associated with non‐chronic or chronic diseases, or letal. For most enzymopathies, the experimental and clinical observations can be satisfactorily rationalized by the computational results. Moreover, a surprisingly high correlation is found between the range of the activity range where disease is predicted by the model and the observed number of diseased probands.

Another objective of our study was to contribute to the theory of metabolic control. The well‐elaborated concept of the metabolic control theory is restricted to (infinitely) small activity alterations. In order to quantify the metabolic effect of finite (large‐scale) changes in the activity of an enzyme, we propose, as a control measure, the effective activity Ea, defined as the relative activity of an enzyme (with respect to the activity in a reference state) required to bring about a change in the stationary value of a metabolic variable by the (finite) factor α. We demonstrate that none of the existing extrapolation methods using the conventional control coefficient is capable to provide reliable predictions of the effective activities for all enzymes of erythrocyte metabolism.
Macrophage Scavenger Receptor Mediates the Endocytic Uptake and Degradation of Advanced Glycation End Products of the Maillard Reaction
Authors: Araki Norie , Higashi Takayuki , Mori Takashi , Shibayama Rie , Kawabe Yoshiki , Kodama Tatsuhiko , Takahashi Kiyoshi , Shichiri Motoaki , Horiuchi Seikoh
Published: March 2005

Modification of proteins by long‐term incubation with glucose leads to the formation of advanced glycation end products (AGE). Recent immunological demonstration of the presence of AGE proteins in several human tissues suggests that they may be involved in aging, diabetic complications and atherosclerosis. AGE proteins are taken up by macrophages via the AGE receptor, which is similar to the macro‐phage scavenger receptor (MSR). In the present study, we examined whether MSR could mediate the endocytic uptake of AGE proteins by using Chinese hamster ovary (CHO) cells overexpressing bovine type II MSR (CHO‐SRII). 125I‐labelled AGE bovine serum albumin (125I‐AGE‐BSA) as well as 125I‐acetylated low‐density lipoprotein (125I‐acetyl‐LDL) underwent endocytic degradation by CHO‐SRII cells, but not by control CHO cells. Endocytic degradation of 125I‐acetyl‐LDL and 125I‐AGE‐BSA by CHO‐SRII cells was significantly inhibited by unlabeled AGE‐BSA, as well as by acetyl‐LDL. Immunoelectron microscopic studies using both AGE‐BSA conjugated with gold particles and anti‐(bovine MSR) antibody (D2) revealed co‐localization of gold particles and the reactive sites for the antibody at coated pits of plasma membranes as well as in endosomes. These results clearly show that MSR mediates the endocytic uptake and degradation of AGE proteins, suggesting a new role of MSR in biological recognition of AGE in vivo.
Alternative Splicing of Calretinin mRNA Leads to Different Forms of Calretinin
Authors: Schwaller Beat , Celio Marco R. , Hunziker Willi
Published: March 2005

cDNA clones for calretinin, a member of the troponin‐C family of calcium‐binding proteins, were isolated from a cDNA library of the human colon carcinoma cell line WiDr. Sequence analysis revealed two forms of alternatively spliced calretinin mRNAs encoding C‐terminally truncated proteins. Exon 7 was either spliced to exon 9 (8) or to exon 10 (8,9); both resulted in a frame shift and a translational stop at the second codon of exon 9 (8), or at codon 15 of exon 10 (8,9), respectively. The presence of 8 and 8,9 calretinin mRNA in WiDr cells was confirmed using reverse‐transcriptase PCR and sequence analysis of the amplicon, as well as by a ribonuclease protection assay. Co115/3 and three other human colon carcinoma cell lines were found, by reverse‐transcriptase PCR to also contain 8,9 calretinin mRNA. The truncated proteins were able to bind calcium, as evidenced by a calcium blot of the 8 form (calretinin‐20k) and 8,9 form (calretinin‐22k) expressed in Escherichia coli. Immunohistochemical staining using an antiserum specific for the novel C‐terminus of calretinin‐22k confirmed its presence in WiDr, Co115/3 and three additional colon carcinoma cell lines. The fact that alternative splicing of calretinin was found in five different cell lines suggests that alternatively spliced calretinins fulfill a physiological function.