bluuebunny/crossref_metadata_2025_split huggingface dataset:
The N‐terminus of A1‐type myosin essential light chains binds actin and modulates myosin motor function
Authors: Timson David J. , Trayer Hylary R. , Trayer Ian P. 
Published: March 2003

There are two isoforms (A1 and A2) of the myosin essential light chain (ELC) and consequently two isoenzymes of myosin subfragment 1 (S1), S1(A1) and S1(A2). The two isoenzymes differ in their kinetic properties with S1(A1) having a lower apparent Km for actin and a slower turnover of MgATP (kcat) than S1(A2). The two forms of the ELC differ only at their N‐termini where A1 has an additional 40‐odd amino acids that are not present in A2.The human atrial ELC (an A1‐type ELC) was overexpressed in Escherichia coli and purified by ammonium sulphate fractionation and ion‐exchange chromatography. The recombinant ELC had actin‐activated MgATPase kinetics similar to those for rabbit skeletal S1(A1) under the same conditions. Deletion of the first 45 amino acid residues resulted in an ELC similar to the rabbit skeletal A2 isoform and, when hybridised into S1, in S1(A2)‐like kinetic properties. Results obtained with an ELC mutant that lacks the first 11 residues were intermediate between these two extremes but tending towards the S1(A2)‐like phenotype.The wild‐type ELC (both hybridised into S1 or free in solution) could be cross‐linked to F‐actin, whereas the deletion mutant lacking the first 45 amino acids could not. The deletion mutant lacking the first 11 amino acids cross‐linked only poorly under the same conditions, consistent with the MgATPase data. We therefore conclude that these N‐terminal eleven amino acids predominantly encode an actin‐binding site which modulates the kinetics of the myosin motor. Furthermore, while free A1‐type ELC cross‐linked to both polymeric F‐actin and the monomeric G‐actin : DNase‐I complex, the same ELC in S1(A1) could only cross‐link to F‐actin. This suggests that the light chain binds to a different actin monomer than the heavy chain.
In Saccharomyces cerevisae, feedback inhibition of homocitrate synthase isoenzymes by lysine modulates the activation of LYS gene expression by Lys14p
Authors: Feller André , Ramos Fernando , Piérard André , Dubois Evelyne 
Published: March 2003

Expression of the structural genes for lysine biosynthesis responds to an induction mechanism mediated by the transcriptional activator Lys14p in the presence of α‐aminoadipate semialdehyde (αAASA), an intermediate of the pathway acting as a coinducer. This activation is reduced by the presence of lysine in the growth medium, leading to apparent repression.  In this report we demonstrate that Saccharomyces cerevisiae possesses two genes, LYS20 and LYS21, encoding two homocitrate synthase isoenzymes which are located in the nucleus. Each isoform is inhibited by lysine with a different sensitivity. Lysine‐overproducing mutants were isolated as resistant to aminoethylcysteine, a toxic lysine analog. Mutations, LYS20fbr and LYS21fbr, are allelic to LYS20 and LYS21, and lead to desensitization of homocitrate synthase activity towards lysine and to a loss of apparent repression by this amino acid. There is a fair correlation between the I0.5 of homocitrate synthase for lysine, the intracellular lysine pool and the levels of Lys enzymes, confirming the importance of the activity control of the first step of the pathway for the expression of LYS genes. The data are consistent with the conclusion that inhibition by lysine of Lys14p activation results from the control of αAASA production through the feedback inhibition of homocitrate synthase activity.
The superoxide dismutase activity of desulfoferrodoxin from Desulfovibrio desulfuricans ATCC 27774
Authors: Romão Célia V. , Liu Ming Y. , Gall Jean Le , Gomes Cláudio M. , Braga Vera , Pacheco Isabel , Xavier António V. , Teixeira Miguel 
Published: March 2003

Desulfoferrodoxin (Dfx), a small iron protein containing two mononuclear iron centres (designated centre I and II), was shown to complement superoxide dismutase (SOD) deficient mutants of Escherichia coli[Pianzzola, M.J., Soubes M. & Touati, D. (1996) J. Bacteriol.178, 6736–6742]. Furthermore, neelaredoxin, a protein from Desulfovibrio gigas containing an iron site similar to centre II of Dfx, was recently shown to have a significant SOD activity [Silva, G., Oliveira, S., Gomes, C.M., Pacheco, I., Liu, M.Y., Xavier, A.V., Teixeira, M., Le Gall, J. & Rodrigues‐Pousada, C. (1999) Eur. J. Biochem. 259, 235–243]. Thus, the SOD activity of Dfx isolated from the sulphate‐reducing bacterium Desulfovibrio desulfuricans ATCC 27774 was studied. The protein exhibits a SOD activity of 70 U·mg−1, which increases approximately 2.5‐fold upon incubation with cyanide. Cyanide binds specifically to Dfx centre II, yielding a low‐spin iron species with g‐values at 2.27 (g⊥) and 1.96 (g∥). Upon reaction of fully oxidized Dfx with the superoxide generating system xanthine/xanthine oxidase, Dfx centres I and II become partially reduced, suggesting that Dfx operates by a redox cycling mechanism, similar to those proposed for other SODs. Evidence for another SOD in D. desulfuricans is also presented – this enzyme is inhibited by cyanide, and N‐terminal sequence data strongly indicates that it is an analogue to Cu,Zn‐SODs isolated from other sources. This is the first indication that a Cu‐containing protein may be present in a sulphate‐reducing bacterium.
Contribution of the carbohydrate moiety to conformational stability of the carboxypeptidase Y
Authors: Dumoulin Mireille , Ueno Hiroshi , Hayashi Rikimaru , Balny Claude 
Published: March 2003

The process of pressure‐induced denaturation of carboxypeptidase Y and the role of the carbohydrate moiety in its response to pressure and low temperature were investigated by measuring in situ the catalytic activity and, the intrinsic and 8‐anilino‐1‐naphthalene sulfonic acid binding fluorescences. Pressure‐induced denaturation of carboxypeptidase Y is a process involving at least three transitions. Low pressures (below 150 MPa) induced slight conformational changes characterized by a slight decrease in the center of the spectral mass of intrinsic fluorescence, whereas no changes in 8‐anilino‐1‐naphthalene sulfonic acid binding fluorescence were observed and 80% of the catalytic activity remained. Higher pressure (150–500 MPa) induced further conformational changes, characterized by a large decrease in the center of the spectral mass of intrinsic fluorescence, a large increase in the 8‐anilino‐1‐naphthalene sulfonic acid binding fluorescence and the loss of all catalytic activity. Thus, this intermediate exhibited characteristics of molten globule‐like state. A further increase, in pressure (above 550 MPa) induced transition from this first molten globule‐like state to a second molten globule‐like state. This two‐stage denaturation process can be explained by assuming the existence of two independent structural domains in the carboxypeptidase molecule. A similar three‐transition process was found for unglycosylated carboxypeptidase Y, but, the first two transitions clearly occurred at lower pressures than those for glycosylated carboxypeptidase Y. These findings indicate that the carbohydrate moiety protects carboxypeptidase Y against pressure‐induced denaturation. The origin of the protective effects is discussed based on the known crystallographic structure of CPY.
Structural analysis of 20 oligosaccharide‐alditols released from the jelly coat ofRana palustriseggs by reductive β‐elimination
Authors: Maes Emmanuel , Florea Doina , Coppin Alexandra , Strecker Gérard 
Published: March 2003

The eggs of amphibians are surrounded by three to eight layers of jelly coats. This extracellular matrix is mainly composed of hydrated mucin‐type glycoproteins. These highly glycosylated molecules are synthesized by oviduct and play an important role in the fertilization process. Recent structural analyses have shown the strict species‐specificity of the O‐linked oligosaccharides which constitute 60–70% of these oviducal mucins. Consequently, these carbohydrate chains represent new phenotypic markers, and from a biological point of view, can influence parasite tropism or can be involved in species‐specific interaction of gametes. The primary structure of 20 oligosaccharide‐alditols, released by alkali/borohydride treatment from the mucin ofRana palustrisegg jelly coats, was established by1H and13C‐NMR analysis. Thirteen of these components possess new structures and the polymerization of the sequence Gal(β1–3)GalNAc(α1–4) characterizes the species‐specificity ofR. palustris.
Polypeptide neurotoxins from spider venoms
Authors: Grishin Eugene 
Published: March 2003

Spider venoms contain a variety of toxic components. The polypeptide toxins are divided into low and high molecular mass types. Small polypeptide toxins interacting with cation channels display spatial structure homology. They can affect the functioning of calcium, sodium, or potassium channels. A family of high molecular mass toxic proteins was found in the venom of the spider genus Latrodectus. These neurotoxins, latrotoxins, cause a massive transmitter release from a diversity of nerve endings. The latrotoxins are proteins of about 1000 amino acid residues and share a high level of structure identity. The structural and functional properties of spider polypeptide toxins are reviewed in this paper.
Structural studies on lipopolysaccharides of serologically non‐typable strains of Helicobacter pylori, AF1 and 007, expressing Lewis antigenic determinants
Authors: Knirel Yuriy A. , Kocharova Nina A. , Hynes Sean O. , Widmalm Göran , Andersen Leif P. , Jansson Per‐Erik , Moran Anthony P. 
Published: March 2003

In contrast to other Helicobacter pylori strains, which have serologically detectable Lewisx (Lex) and Lewisy (Ley) antigenic determinants in the O‐specific polysaccharide chains of the lipopolysaccharides, H. pylori AF1 and 007 were non‐typable with anti‐Lex and anti‐Ley antibodies. The carbohydrate portions of the lipopolysaccharides were liberated by mild acid hydrolysis and subsequently studied by sugar and methylation analyses, 1H‐NMR spectroscopy and electrospray ionization‐mass spectrometry. Compared with each other, and with lipopolysaccharides of strains studied previously, the lipopolysaccharides of both AF1 and 007 showed similarities, but also differences, in the structures of the core region and O‐specific polysaccharide chains. The O‐specific polysaccharide chains of both strains consisted of a short or long polyfucosylated poly‐N‐acetyl‐β‐lactosamine chains, which were distinguished from those of other strains by a high degree of fucosylation producing a polymeric Lex chain terminating with Lex or Ley units:where n = 0 or 1 in strain AF1 and 0 in strain 007, m = 0–2, 6–7 in strain AF1 and m = 0–2, 6–7 or ≈ 40 in strain 007, the medium‐size species being predominant. Therefore, compared with other strains, the lack of reactivity of lipopolysaccharide of H. pylori AF1 and 007 with anti‐Lex and anti‐Ley may reflect the presence of a polymeric Lex chain and has important implications for serological and pathogenesis studies. As the substitution pattern of a dglycerodmanno‐heptose residue in the outer core varied in the two strains, and an extended dd‐heptan chain was present in some lipopolysaccharide species but not in others, this region was less conservative than the inner core region. The inner core lglycerodmanno‐heptose region of both strains carried a 2‐aminoethyl phosphate group, rather than a phosphate group, as reported previously for other H. pylori strains.
Structure of a 2‐aminoethyl phosphate‐containing O‐specific polysaccharide of Proteus penneri 63 from a new serogroup O68
Authors: Shashkov Aleksander S. , Kondakova Anna N. , Senchenkova Sof’ya N. , Zych Krystyna , Toukach Filip V. , Knirel Yuriy A. , Sidorczyk Zygmunt 
Published: March 2003

Lipopolysaccharide of Proteus penneri strain 63 was degraded by mild acid to give a high molecular mass O‐specific polysaccharide that was isolated by gel‐permeation chromatography. Sugar and methylation analyses and NMR spectroscopic studies, including two‐dimensional 1H,1H COSY, TOCSY rotating‐frame NOE spectroscopy, H‐detected 1H,13C and 1H,31P heteronuclear multiple‐quantum coherence (HMQC), and 1H,13C HMQC‐TOCSY experiments, demonstrated the following structure of the polysaccharide:where FucNAc is 2‐acetamido‐2,6‐dideoxygalactose and PEtn is 2‐aminoethyl phosphate. The polysaccharide studied shares some structural features, such as the presence of d‐GlcNAc6PEtn and an α‐L‐FucNAc‐(1→3)‐d‐GlcNAc disaccharide, with other Proteus O‐specific polysaccharides. A marked cross‐reactivity of P. penneri 63 O‐antiserum with P. vulgaris O12 was observed and substantiated by a structural similarity of the O‐specific polysaccharides of the two strains. In spite of this, the polysaccharide of P. penneri 63 has the unique structure among Proteus O‐antigens, and therefore a new, separate serogroup, O68, is proposed for this strain.
A small‐angle X‐ray solution scattering study of bovine α‐crystallin
Authors: Vanhoudt Jos , Abgar Saı¨d , Aerts Tony , Clauwaert Julius 
Published: March 2003

The native high molecular mass form of α‐crystallin, the most important soluble protein in the eye lens, and its low molecular mass form obtained at 37 °C in dilute solutions were investigated by synchrotron radiation small‐angle X‐ray scattering. The α‐crystallin solutions are polydisperse and good fits to the experimental data can be obtained using distributions of spheres with radii varying between about 5 and 10 nm.In spite of the polydispersity, two different ab initio methods were used to retrieve low resolution shapes from the scattering data. These shapes correspond to the z‐average structure of the oligomers. In the absence of any symmetry constraints, the scattering curves of the two forms of α‐crystallin yield bean‐like shapes. The shape corresponding to the low molecular mass form has about 20% less mass at the periphery. Imposing tetrahedral symmetry on the average structures worsens the fit to the experimental data.We emphasized the apparent contradiction between hydrodynamic and molecular properties of α‐crystallin. An explanation was put forward based on the presence of solvent‐exposed flexible C‐terminal extensions. We present two bead models (‘hollow globule with tentacles’ and ‘bean with tentacles’) based on NMR and cryo‐electron microscopy studies and discuss how well they correspond with our data from X‐ray scattering, light scattering and analytical ultracentrifugation.
Can yeast glycolysis be understood in terms of in vitro kinetics of the constituent enzymes? Testing biochemistry
Authors: Teusink Bas , Passarge Jutta , Reijenga Corinne A. , Esgalhado Eugenia , van der Weijden Coen C. , Schepper Mike , Walsh Michael C. , Bakker Barbara M. , van Dam Karel , Westerhoff Hans V. , Snoep Jacky L. 
Published: March 2003

This paper examines whether the in vivo behavior of yeast glycolysis can be understood in terms of the in vitro kinetic properties of the constituent enzymes. In nongrowing, anaerobic, compressed Saccharomyces cerevisiae the values of the kinetic parameters of most glycolytic enzymes were determined. For the other enzymes appropriate literature values were collected. By inserting these values into a kinetic model for glycolysis, fluxes and metabolites were calculated. Under the same conditions fluxes and metabolite levels were measured.In our first model, branch reactions were ignored. This model failed to reach the stable steady state that was observed in the experimental flux measurements. Introduction of branches towards trehalose, glycogen, glycerol and succinate did allow such a steady state. The predictions of this branched model were compared with the empirical behavior. Half of the enzymes matched their predicted flux in vivo within a factor of 2. For the other enzymes it was calculated what deviation between in vivo and in vitro kinetic characteristics could explain the discrepancy between in vitro rate and in vivo flux.
Cloning, expression and chromosomal localization of a novel human dipeptidyl peptidase (DPP) IV homolog, DPP8
Authors: Abbott Catherine A. , Yu Denise M. T. , Woollatt Erica , Sutherland Grant R. , McCaughan Geoffrey W. , Gorrell Mark D. 
Published: March 2003

Dipeptidyl peptidase (DPP) IV has roles in T‐cell costimulation, chemokine biology, type‐II diabetes and tumor biology. Fibroblast activation protein (FAP) has been implicated in tumor growth and cirrhosis. Here we describe DPP8, a novel human postproline dipeptidyl aminopeptidase that is homologous to DPPIV and FAP. Northern‐blot hybridization showed that the tissue expression of DPP8 mRNA is ubiquitous, similar to that of DPPIV. The DPP8 gene was localized to chromosome 15q22, distinct from a closely related gene at 19p13.3 which we named DPP9. The full‐length DPP8 cDNA codes for an 882‐amino‐acid protein that has about 27% identity and 51% similarity to DPPIV and FAP, but no transmembrane domain and no N‐linked or O‐linked glycosylation. Western blots and confocal microscopy of transfected COS‐7 cells showed DPP8 to be a 100‐kDa monomeric protein expressed in the cytoplasm. Purified recombinant DPP8 hydrolyzed the DPPIV substrates Ala‐Pro, Arg‐Pro and Gly‐Pro. Thus recombinant DPP8 shares a postproline dipeptidyl aminopeptidase activity with DPPIV and FAP. DPP8 enzyme activity had a neutral pH optimum consistent with it being nonlysosomal. The similarities between DPP8 and DPPIV in tissue expression pattern and substrates suggests a potential role for DPP8 in T‐cell activation and immune function.
Contribution of tryptophan residues to the CD spectrum of the extracellular domain of human tissue factor
Authors: Andersson Dick , Carlsson Uno , Freskgård Per‐Ola 
Published: March 2003

The contribution to the circular dichroism (CD) spectrum made by each of the four Trp residues in the extracellular domain of human tissue factor, sTF (s designates soluble), was determined from difference CD spectra. The individual Trp CD spectra showed that all four residues contributed to the CD spectrum in almost the entire wavelength region investigated (180–305 nm). The sum of the individual spectra of each Trp residue in the near‐UV region was qualitatively identical to the wild‐type spectrum, clearly demonstrating that the Trp residues are the major contributors to the spectrum in this wavelength region. Trp CD bands interfere with the peptide bands in the far‐UV region, leading to uncertainty in the predictions of the amounts of various types of secondary structure. Accordingly, the best prediction of secondary sTF structure content was achieved using a hypothetical Trp‐free CD spectrum obtained after subtraction of all individual Trp spectra from the wild‐type spectrum. The mutated Trp residues were also exploited as intrinsic probes to monitor the formation of local native‐like tertiary structure by kinetic near‐UV CD measurements. The global folding reaction was followed in parallel with a novel functional assay that registered the recovery of cofactor activity, i.e. stimulation of the amidolytic activity of Factor VIIa. From these measurements, it was found that sTF appears to regain FVIIa cofactor activity before the final side‐chain packing of the Trp residues. The combined kinetic refolding results suggest that the compact asymmetric environments of the individual Trp residues in sTF are formed simultaneously, leading to the conclusion that the native tertiary structure of the whole protein is formed in a cooperative manner.
Recombinant expression of N‐terminal truncated mutants of the membrane bound mouse, rat and human flavoenzyme dihydroorotate dehydrogenase
Authors: Ullrich Alexandra , Knecht Wolfgang , Fries Markus , Löffler Monika 
Published: March 2003

Mammalian dihydroorotate dehydrogenase, the fourth enzyme of pyrimidine de novo synthesis is an integral protein of the inner mitochondrial membrane that faces the intermembrane space and is functionally connected to the respiratory chain via ubiquinone. Here, we describe the first cloning and analyzing of the complete cDNA of mouse dihydroorotate dehydrogenase. Based on our recent functional expression of the full‐length rat and human dihydroorotate dehydrogenase, here we expressed N‐terminal‐truncated C‐terminal‐histidine‐tagged constructs of the mouse, rat and human enzymes in Escherichia coli. These proteins were devoid of the N‐terminal bipartite sequence consisting of the mitochondrial targeting sequence and adjacent hydrophobic domain necessary for import and proper location and fixation of the enzyme in the inner mitochondrial membrane. By employing metal‐chelate affinity chromatography under native conditions, the enzymes were purified without detergents to a specific activity of more than 100 µmol·min−1·mg−1 at pH optimum of 8.0–8.1. Flavin analyses by UV‐visible spectrometry of the native enzymes gave fairly stoichiometric ratios of 0.6–1.2 mol flavin per mol protein. The kinetic constants of the truncated rat enzyme (Km = 11 µm dihydroorotate; Km = 7 µm ubiquinone) and human enzyme (Km = 10 µm dihydroorotate; Km = 14 µm ubiquinone) were very close to those recently reported for the full‐size enzymes. The constants for the mouse enzyme, Km = 26 µm dihydroorotate and Km = 62 µm ubiquinone, were slightly elevated in comparison to those of the other species. The three truncated enzymes were tested for their efficacy with five inhibitors of topical clinical relevance against autoimmune disorders and tumors. Whereas the presence of the N‐terminus of dihydroorotate dehydrogenase was essentially irrelevant for the efficacy of the malononitrilamides A77‐1726, MNA715 and MNA279 with the rat and human enzyme, the N‐termini were found to be important for the efficacy of the dianisidine derivative redoxal. Moreover, the complete N‐terminal part of the human enzyme seemed to be of crucial importance for the ‘slow‐binding’ features of the cinchoninic acid derivative brequinar, which was suggested to be one of the reasons for the narrow therapeutic window reported from clinical trials on its anti‐proliferative and immunosuppressive action.
Protein engineering of Bacillus megaterium CYP102
Authors: Carmichael Angus B. , Wong Luet‐Lok 
Published: March 2003

Cytochrome P450 (CYP) enzymes are involved in activating the carcinogenicity of polycyclic aromatic hydrocarbons (PAHs) in mammals, but they are also utilized by microorganisms for the degradation of these hazardous environmental contaminants. Wild‐type CYP102 (P450BM‐3) from Bacillus megaterium has low activity for the oxidation of the PAHs phenanthrene, fluoranthene and pyrene. The double hydrophobic substitution R47L/Y51F at the entrance of the substrate access channel increased the PAH oxidation activity by up to 40‐fold. Combining these mutations with the active site mutations F87A and A264G lead to order of magnitude increases in activity. Both these mutations increased the NADPH turnover rate, but the A264G mutation increased the coupling efficiency while the F87A mutation had dominant effects in product selectivity. Fast NADPH oxidation rates were observed (2250 min−1 for the R47L/Y51F/F87A mutant with phenanthrene) but the coupling efficiencies were relatively low (< 13%), resulting in a highest substrate oxidation rate of 110 min−1 for fluoranthene oxidation by the R47L/Y51F/A264G mutant. Mutation of M354 and L437 inside the substrate access channel reduced PAH oxidation activity. The PAHs were oxidized to a mixture of phenols and quinones. Notably mutants containing the A264G mutation showed some similarity to mammalian CYP enzymes in that some 9,10‐phenanthrenequinone, the K‐region oxidation product from phenanthrene, was formed. The results suggest that CYP102 mutants could be useful models for PAH oxidation by mammalian CYP enzymes, and also potentially for the preparation of novel PAH bioremediation systems.