bluuebunny/crossref_metadata_2025 huggingface dataset:
Involvement of Protein‐Tyrosine Kinase p72syk in Collagen‐Induced Signal Transduction in Platelets
Authors: Fujii Chiyuki , Yanagi Shigeru , Sada Kiyonao , Nagai Katsuya , Taniguchi Takanobu , Yamamura Hirohei
Published: July 2010

Previous studies have demonstrated that activation of platelets by collagen results in a dramatic increase in tyrosine phosphorylation of several cellular proteins, including ppl25FAK, through the interaction of collagen with integrin α2β1 (GP Ia‐IIa). In this study, we report that p72syk is a potential candidate for the protein‐tyrosine phosphorylation event following collagen stimulation in porcine platelets. Washed platelets were stimulated with collagen and the activation of p72syk was assessed in an immunoprecipitation kinase assay. The activity of p72syk increased within 1 min, reached a maximum at 5 min after stimulation by collagen, and the phosphorylation at tyrosine residues of p72syk in platelets also occurred in the same time course as the activation of p72syk. Prior treatment of platelets with cytochalasin D to inhibit actin polymerization, or with aspirin and apyrase to inhibit the secondary reaction, or EGTA and the acetoxymethyl ester of 5,5′‐dimethyl‐bis‐(o‐aminophenoxy)‐ethane‐N,N,N′,N′ ‐tetraacetic acid to chelate both extracellular and intracellular Ca2+, did not affect the activation of p72syk induced by collagen. Furthermore, herbimycin A, a potent proteintyrosine‐kinase inhibitor, was capable of reducing collagen‐evoked p72syk activation, Ca2+ mobilization and platelet aggregation. These results suggest that upon stimulation by collagen p72syk is physically activated by a process that is independent of the effects of Ca2+, ADP, and actin polymerization, and may participate in the regulation of Ca2+ mobilization mediated by collagen in platelets.
The Roles of the N‐linked Carbohydrate Chain of Rice α‐Amylase in Thermostability and Enzyme Kinetics
Authors: Terashima Masaaki , Kubo Atsushi , Suzawa Michimasa , Itoh Yasuaki , Katoh Shigeo
Published: July 2010

The thermostability and kinetics of starch hydrolysis were compared between a rice α‐amylase isozyme Amy1A and its mutant enzyme that lacks an N‐linked carbohydrate chain. Elimination of the N‐glycosylation site in Amy1A reduced the thermostability of the enzyme. The temperature dependence of the kinetic parameters (Vm, and Km) and substrate recognition of the enzymes were also affected by elimination of the N‐glycosylation site. These results suggest that the N‐linked carbohydrate chain of Amy1A has important roles in the thermostability and reaction kinetics of the enzyme.
Overexpression of DNA Methyltransferase in Myoblast Cells Accelerates Myotube Formation
Authors: Takagi Hidekazu , Tajima Shoji , Asano Akira
Published: July 2010

We overexpressed mouse DNA methyltransferase in murine C2C12 myoblast cells and tested the isolated clones for their ability to differentiate. Significant numbers of the clones showed distinct myotubes 24 h after the isolated transformants had been induced to differentiate, whereas the parent C2C12 cells did not form myotubes at this time point. Transfection of the vacant vector or the plasmid containing the reverse‐oriented DNA methyltransferase cDNA did not provide significant numbers of transformants with the accelerated differentiation phenotype, suggesting that the effect is caused by the expression of DNA methyltransferase. The expressions of skeletal muscle myosin and creatine kinase in clones that showed the accelerated differentiation‐phenotype were also induced about 24 h earlier and at higher levels relative to the parent C2C12 or the control cells, indicating that the entire process of myogenesis had been accelerated. All the methyltransferase‐transfected clones, regardless of their phenotypes, demonstrated about threefold higher DNA methyltransferase activity and higher methylation levels than those of the clones transfected with vector alone or the reverse‐oriented plasmid. At the early stage of transfection of the sense‐oriented plasmid, high de novo methylation activities were detected. We consider it likely that this high de novo methylation activity is the reason for the high methylation levels and the accelerated myotube formation of the clones transfected with the sense‐oriented plasmid. In some transformants which showed the accelerated differentiation phenotype, MyoD1 was already fully expressed under the growth conditions while, in control cells, MyoD1 was expressed at low levels. This elevated level of MyoD1 transcription could account for the accelerated myotube formation observed in the transformants. The methylation state of the HpaII sites in exon 1 through exon 2 of the MyoD1 gene and the expression of the MyoD1 transcript are positively correlated.
Recombinant Production and Biological Properties of Rat Cytokine‐Induced Neutrophil Chemoattractants, GRO/CINC‐2α, CINC‐2β and CINC‐3
Authors: Shibata Futoshi , Konishi Kiyoshi , Kato Hideko , Komorita Naruyasu , Al‐Mokdad Maher , Fujioka Motoji , Nakagawa Hideo
Published: July 2010

Recently we found four cytokine‐induced neutrophil chemoattractants, CINC‐1, CINC‐2α, CINC‐2β and CINC‐3/macrophage inflammatory protein 2 (MIP‐2), in conditioned medium of granulation tissue obtained from carrageenin‐induced inflammation in rats [Nakagawa, H., Komorita, N., Shibata, E, Ikesue, A., Konishi, K., Fujioka, M. & Kato, H. (1994) Biochem. J. 301, 545–550]. In the present report, we describe recombinant production of CINC‐2α, CINC‐2β and CINC‐3 in Escherichia coli, and biological properties of these chemokines. Neutrophil chemotactic activities of CINC‐2α and 2βin vitro were the same as the activity of CINC‐1. CINC‐3 had an activity comparable to other CINCs, but showed a decrease at high concentrations. Stimulation of neutrophils with CINCs induced an increase in intracellular [Ca2+] dose‐dependently. CINC‐3 was more potent than the other CINCs and still induced an increase in intracellular [Ca2+] in rat neutrophils stimulated first with other CINCs. CINC‐2α, CINC‐2β and CINC‐3 induced a comparable response to CINC‐1 in the release of cathepsin G from rat neutrophils. Injection of CINC‐2α, 2β and 3 into preformed air‐pouch on the back of rat induced infiltration of neutrophils to an extent similar to that caused by the injection of CINC‐1. These data indicate CINC‐2α, 2β and 3 as well as CINC‐1 are chemoattractants specific for neutrophil in vivo.
Cloning and Expression of Glucosidase I from Human Hippocampus
Authors: Kalz‐Füller Burga , Bieberich Erhard , Bause Ernst
Published: July 2010

Glucosidase I, the first enzyme in the N‐linked oligosaccharide processing pathway, cleaves the distal α1,2‐linked glucose residue from the Glc3‐Man9‐GlcNAc2 oligosaccharide precursor highly specifically. A human hippocampus cDNA library was screened against oligonucleotide probes, generated by PCR using primers derived from the amino acid sequences of tryptic peptides of pig liver glucosidase I. Two independent λ clones were isolated which allowed the construction of a full‐length glucosidase I cDNA of 2881 bp. This cDNA construct encodes, in a single open reading frame, a polypeptide of 834 amino acids corresponding to a molecular mass of 92 kDa. The 92‐kDa protein contains a single N‐glycosylation site of the Asn‐Xaa‐Thr/Ser type at Asn655, as well as a strongly hydrophobic sequence close to its N‐terminus (amino acids 38–58) which, most likely, functions as a transmembrane anchor. The amino acid sequences of all tryptic peptides of the pig liver enzyme were found, with little deviation, within the coding sequence. This demonstrates the authenticity of the cDNA construct and the close evolutionary relationship between the enzymes from human hippocampus and pig liver. In contrast, the nucleotide and amino acid sequence revealed no homology with other processing enzymes cloned so far.

Transfection of COS 1 cells with the glucosidase I cDNA construct resulted in overexpression (about fourfold) of enzymic activity, which was inhibited strongly by 1‐deoxynojirimycin or N,N‐dimethyl‐deoxynojirimycin. The expressed enzyme, with a molecular mass of 95 kDa, is degraded by endoglycosidase H to a 93‐kDa form, indicating that it carries a high‐mannose oligosaccharide chain at Asn655. The presence of this glycan is in line with the localization of glucosidase I in the lumen of the endoplasmic reticulum, shown by immunofluorescence microscopy. The hydrophobicity profile as well as the removal by trypsin of an ≈4‐kDa polypeptide from the membrane‐associated glucosidase I in intact microsomal structures, supports the view that the enzyme is a type‐II transmembrane glycoprotein, which contains a short cytosolic tail of ≈37 amino acids, followed by a single transmembrane domain and a large C‐terminal catalytic domain located on the luminal side of the endoplasmic reticulum membrane.
Characterization of a Ferredoxin from Desulfovibrio Vulgaris (Hildenborough) that Interacts with RNA
Authors: Arendsen Alexander F. , Schalk Jos , Van Dongen Walter M. A. M. , Hagen Wilfred R.
Published: July 2010

The purification and characterization of a ferredoxin from Desulfovibrio vulgaris (Hildenborough) is described. The protein can be isolated in two forms; the major form is strongly complexed to RNA, while a minor form is free from nucleic acid. Bound RNA cannot be removed by digestion with nucleases, or by heating to 70 °C, and it can only be partially removed by rechromatography. The ultraviolet/visible spectrum shows typical absorption maxima at 280 nm and 400 nm for the RNA‐free ferredoxin. The RNA‐bound protein exhibits an additional strong peak at 260 nm. The RNA can be extracted from the protein with phenol. The ferredoxin is a dimer of subunits, each of 7.5 kDa; its pI is 3.9. The protein contains a [4Fe–4S](2+;1+) cluster with an EPR spectrum (g= 1.90, 1.93 and 2.05) in the reduced state. A reduction potential of – 360 mV was determined for the RNA‐free ferredoxin with reversible voltammetry at glassy carbon. From the temperature dependence of the reduction potential, the unusually high standard reaction entropy was calculated as ?S˚=– 230 J · K‐1· mol‐1. No electrochemical response was obtained with the RNA‐bound ferredoxin. Binding of RNA appears to require the presence of an intact cluster, since the absence of absorption at 400 nm runs in parallel with the absence of absorption at 260 nm. The possibility is discussed that the binding to the RNA has a regulatory function and is controlled by the state of the cluster.
Stability, Activity and Structure of Adenylate Kinase Mutants
Authors: Spuergin Peter , Abele Ulrich , Schulz Georg E.
Published: July 2010

Sequence/structure relationships have been explored by site‐directed mutagenesis using a structurally known adenylate kinase. In particular the effects of helix capping and nonpolar core expansion on thermo‐dynamic stability have been analyzed. Six point mutations were produced and characterized by SDS/PAGE, native PAGE, isoelectric focussing, electrophoretic titration, enzyme kinetics, and X‐ray structure analysis. Heat‐denaturation experiments yielded melting temperatures Tm and melting enthalpy changes ?Hm. The heat capacity change ?CP of the wild‐type enzyme was determined by guanidine hydrochloride denaturation in conjunction with Tm and ?Hm. Using the wild‐type ?CP value, Gibbs free energy changes ?G at room temperature were calculated for all mutants. Four mutants were designed for helix capping stabilization, but only one of them showed such an effect. Because of electrostatic interference with the induced‐fit motion, one mutant decreased the catalytic activity strongly. Two mutants expanded nonpolar cores causing destabilization. The mutant with the lower stability could be crystallized and subjected to an X‐ray analysis at 223‐pm resolution which showed the structural changes. The enzyme was stabilized by adding a ‐Pro‐His‐His tail to the C‐terminal α‐helix for nickel‐chelate chromatography. This addition constitutes a helix cap. Taken together, the results demonstrate that stabilization by helix capping is difficult to achieve because the small positive effect is drowned by adverse mutational disruption. Further addition of atoms to nonpolar cores destabilized the protein, although the involved geometry changes were very small, demonstrating the importance of efficient packing.
Transient Acquired Thermotolerance in Drosophila, Correlated with Rapid Degradation of Hsp70 During Recovery
Authors: Danxi LI , Duncan Roger F.
Published: July 2010

Acquired thermotolerance, measured either as increased cell viability following a lethal heat shock or by translational thermotolerance, appears rapidly following a ‘priming’ heat treatment, but also decays rapidly. 4 hours after priming heating thermotolerance is reduced by >50% and by 9 hours it is virtually undetectable. Heat‐shock protein 70 (Hsp70) turns over with a half‐life of approximately 2 hours, and the decline in its intracellular abundance parallels the loss of acquired thermotolerance. Continuous heat shock extends the half‐life of Hsp70 to ≈7 hours. When Hsp70 is expressed at normal temperature using a metallothionein promoter, only partial acquired translational thermotolerance results. The results suggest that acquired thermotolerance is tightly regulated in Drosophila and partly, but not wholly, determined by post‐translational regulation of Hsp70 levels.
Pattern Formation on Cardiac Troponin I by Consecutive Phosphorylation and Dephosphorylation
Authors: Jaquet Kornelia , Thieleczek Rolf , Heilmeyer Ludwig M. G.
Published: July 2010

Two serine residues located adjacently in the heart‐specific N‐terminus of cardiac troponin I can be phosphorylated in vivo. Both residues are sequentially phosphorylated and dephosphorylated by cAMP‐dependent protein kinase (PKA) and protein phosphatase 2A (PP2A). The concentration changes of the different troponin I species have been determined separately for the phosphorylation and dephosphorylation reaction and approximated by time courses predicted by a reaction model. Dependent on the concentration ratio of active protein kinase/protein phosphatase, four different troponin I species can be generated; one nonphosphorylated, two monophosphorylated and one bisphosphorylated. This pattern generation will be observed in proteins phosphorylated and dephosphorylated by a single protein kinase and phosphatase on more than one site and is a new principle inherent in signal cascades.
Human Topoisomerase IIα is Phosphorylated in a Cell‐Cycle Phase‐Dependent Manner by a Proline‐Directed Kinase
Authors: Wells Nicholas J. , Hickson Ian D.
Published: July 2010

Topoisomerase II is essential for chromosome condensation and segregation at mitosis in eukaryotic cells, but the mechanism of its regulation is not clearly understood. We have investigated whether or not the a isozyme of human topoisomerase II is phosphorylated in a cell‐cycle phase‐dependent manner. Two‐dimensional tryptic phosphopeptide mapping revealed that several sites on HeLa topoisomerase IIα protein were phosphorylated predominantly or exclusively during the G2 and M phases. To identify the protein kinases involved in this cell‐cycle phase‐specific phosphorylation, oligohistidine‐tagged recombinant domains of the topoisomerase IIα protein were expressed inEscherichia coli, purified by affinity chromatography and phosphorylatedin vitroby different protein kinases. Phosphorylation of the C‐terminal domain of the topoisomerase IIα protein by the universal mitotic controller, p34cdc2, generated multiple tryptic phosphopeptides, many of which corresponded to the G2/M‐phase‐specific phosphorylation sites observedin vivo.The same phosphopeptides were obtained following phosphorylation of the C‐terminal domainin vitroby the mitogen‐activated protein kinase. Site‐directed mutagenesis studies identified five of these sites of phosphorylation, each of which comprised a serine‐proline motif. Our data implicate one or more proline‐directed kinases in the cell‐cycle‐dependent regulation of topoisomerase IIα enzyme activity in human cells.
Site‐Directed Mutagenesis of Thermus thermophilus Elongation Factor Tu
Authors: Zeidler Waltraud , Egle Christian , Ribeiro Sofia , Wagner Annett , Katunin Vladimir , Kreutzer Roland , Rodnina Marina , Wintermeyer Wolfgang , Sprinzl Mathias
Published: July 2010

His85 in Thermus thermophilus elongation factor Tu (EF‐Tu) was replaced by glutamine, leucine and glycine residues, leading to [H85Q]EF‐Tu, [H85L] EF‐Tu and [H85G]EF‐Tu, respectively. Asp81 was replaced by alanine leading to [D81A]EF‐Tu, and replacement of Arg300 provided [R300I]EF‐Tu. Glycine in position 85 of domain I induces a protease‐sensitive site in domain II and causes complete protein degradation in vivo. A similar effect was observed when Asp81 was replaced by alanine or Arg300 by isoleucine. Degradation is probably due to disturbed interactions between the domains of EF‐Tu · GTP, inducing a protease‐sensitive cleavage site in domain II. [H85Q]EF‐TU, which can be effectively overproduced in Escherichia coli, is slower in poly(U)‐dependent poly(Phe) synthesis, has lower affinity to aminoacyl‐tRNA but shows only a slightly reduced rate of intrinsic GTP hydrolysis compared to the native protein. The GTPase of this protein variant is not efficiently stimulated by aminoacyl‐tRNA and ribosomes. The slow GTPase of [H85Q]EF‐Tu increases the fidelity of translation as measured by leucine incorporation into poly(Phe) in in vitro poly(U)‐dependent ribosomal translation. Replacement of His85 in T. thermophilus EF‐Tu by leucine completely deactivates the GTPase activity but does not substantially influence the aminoacyl‐tRNA binding. [H85L]EF‐Tu is inactive in poly(U)‐dependent poly(Phe)‐synthesis. The rate of nucleotide dissociation is highest for [H85L]EF‐Tu, followed by [H85Q]EF‐Tu and native T. thermophilus EF‐Tu. Mutation of His85, a residue which is not directly involved in the nucleotide binding, thus influences the interaction of EF‐Tu domains, nucleotide binding and the efficiency and rate of GTPase activity.
Structural and Functional Characterization of Proteolytic Fragments Derived from the C‐Terminal Regions of Bovine Fibrinogen
Authors: Litvinovich Sergei V. , Henschen Agnes H. , Krieglstein Kerstin G. , Ingham Kenneth C. , Medved Leonid V.
Published: July 2010

A number of new as well as previously described fragments derived from the D region of bovine fibrinogen by limited proteolysis have been characterized by sequence analysis, differential scanning calorimetry and circular dichroism. Determination of the extremities of the polypeptide chains forming individual fragments allowed the scheme of proteolysis and the borders between domains in the D region of fibrinogen to be established. It was also found that the most thermostable region of the D fragment (TSD) can be substantially reduced in size without loss of its compact structure. The α‐helical content of the newly prepared 21‐kDa TSD2 and 16‐kDa TSD3 fragments were 82% and 75%, respectively, strongly supporting a coiled‐coil structure for this region of the fibrinogen molecule. The DX and DZ fragments, prepared from a chymotryptic digest of the DLA fragment, were found to be similar to the DL and DY fragments, respectively, except for an internal cleavage at K393‐T394 in their β chains. This cleavage leads to destabilization of all thermolabile domains, indicating interaction between them. The DL and DY fragments, containing only one polymerization site in their β chains, were able to inhibit fibrin polymerization at high concentration. However, these same fragments failed to bind to fibrin‐Sepharose under conditions where their structural analogues, DX and DZ, were tightly bound, indicating that cleavage after K393 substantially increases the affinity of this site.
The Use of Methyl‐Substituted Benzhydroxamic Acids as Structural Probes of Peroxidase Substrate Binding
Authors: Veitch Nigel C. , Williams Robert J. P.
Published: July 2010

The interactions between cyanide‐ligated horseradish peroxidase (isoenzyme C) and a series of mono‐substituted methylbenzhydroxamic acids have been studied using proton NMR techniques, and compared with analogous data for benzhydroxamic acid itself. Use of the methyl‐substituted compounds in two‐dimensional NOE experiments provides additional data on the structure of the aromatic donor molecule binding site through identification of new NOE connectivities with amino acid side‐chains in the heme binding pocket. In this context the methyl group of the substituted benzhydroxamic acids acts as an effective probe of their local environment in the aromatic donor molecule binding site. While all the benzhydroxamic acids interact strongly with the heme edge at heme methyl C18H3, the methyl groups of 3‐methylbenzhydroxamic and 4‐methylbenzhydroxamic acids principally interact with the distal His42 Cɛ1H and Phe A side‐chains respectively. This confirms that the binding site is situated towards the distal side of the heme plane and that the Phe A side‐chain known to participate in aromatic donor binding is located towards the entrance of the site. Dynamic properties of these peroxidase complexes are addressed in terms of ligand exchange phenomena and a comparison made with data available for benzhydroxamic acid. The existence of alternative binding modes in the case of the methylbenzhydroxamic acids is also discussed.
Characterization and Evolutionary Relationships of Magnolia Legumin‐Encoding cDNAs Representing two Divergent Gene Subfamilies
Authors: Fischer Hilde , Haake Volker , Horstmann Christian , Jensen Uwe
Published: July 2010

We have cloned and sequenced three different cDNAs encoding legumins of Magnolia salicifolia. Analysis of the nucleotide and derived amino acid sequences shows that the cDNAs designated A2, A11, and B14 represent two divergent subfamilies with nucleotide similarities of only about 55%. The B14 cDNA codes for a relatively methionine‐rich legumin precursor, and the β‐chain of this protein is shown to be glycosylated; neither feature is common in legumins. In an evolutionary analysis, the B14 legumin cDNA is relatively similar to gymnospermous legumin sequences and paralogous to all angiosperm legumins hitherto known. The A legumin sequence clusters with those of monocot legumins in a low angiosperm branch. We conclude that the evolution of legumin genes in angiosperms involved an early gene duplication which resulted in the progenitor of the B14 legumin, on the one hand, and the progenitor of A2, A11 and modem angiosperm legumins, on the other hand.
Photoaffinity Labeling of Protoporphyrinogen Oxidase, the Molecular Target of Diphenylether‐Type Herbicides
Authors: Camadro Jean‐Michel , Matringe Michel , Thome Françloise , Brouillet Nicole , Mornet René , Labbe Pierre
Published: July 2010

Diphenylether‐type herbicides are extremely potent inhibitors of protoporphyrinogen oxidase, a membrane‐bound enzyme involved in the heme and chlorophyll biosynthesis pathways. Tritiated acifluorfen and a diazoketone derivative of tritiated acifluorfen were specifically bound to a single class of high‐affinity binding sites on yeast mitochondrial membranes with apparent dissociation constants of 7 nM and 12.5 nM, respectively. The maximum density of specific binding sites, determined by Scatchard analysis, was 3 pmol · mg−1 protein. Protoporphyrinogen oxidase specific activity was estimated to be 2500 nmol protoporphyrinogen oxidized · h−1· mol−1 enzyme. The diazoketone derivative of tritiated acifluorfen was used to specifically photolabel yeast protoporphyrinogen oxidase. The specifically labeled polypeptide in wild‐type mitochondrial membranes had an apparent molecular mass of 55 kDa, identical to the molecular mass of the purified enzyme. This photolabeled polypeptide was not detected in a protoporphyrinogen‐oxidase‐deficient yeast strain, but the membranes contained an equivalent amount of inactive immunoreactive protoporphyrinogen oxidase protein.
Cloning, Properties, Site‐Directed Mutagenesis Analysis of the Subunit Structure, Tissue Distribution and Regulation of Expression of the Type‐CEel Natriuretic Peptide Receptor
Authors: Takashima Akira , Katafuchi Takeshi , Shibasaki Manabu , Kashiwagi Masahide , Hagiwara Hiromi , Takei Yoshio , Hirose Shigehisa
Published: July 2010

Eel natriuretic peptide receptor C (NPR‐C) was cloned, characterized and found to have a unique interchain disulfide linkage when compared to that of mammalian NPR‐C. The NPR‐C cDNA was obtained from an eel gill cDNA library; the open reading frame codes for a polypeptide of 502 amino acids exhibiting the known features of NPR‐C, including a weak ligand specificity and a disulfide‐linked homo‐dimeric structure. The deduced amino acid sequence shares approximately 60% similarity with the mammalian NPR‐C but it lacks the Gly‐rich prosequence present in the mammalian counterparts. Site‐directed mutagenesis revealed that eel and mammalian NPR‐C are quite different in their interchain disulfide‐bonding pattern; eel uses the second Cys residue and mammals the fifth Cys residue for the covalent dimerization. The ligand‐binding activity of the extracellular domain is not independent of the short cytoplasmic tail. RNase protection analysis revealed that the eel receptor is highly expressed in the gill and heart and, to a much lesser extent, in other tissues including the brain and intestine. The NPR‐C mRNA levels were found to be down‐regulated in most tissues when eels were transferred from fresh water to seawater; however, in the anterior intestine, the levels were up‐regulated, suggesting that NPR‐C plays a role in the adaptation to salinity changes in the euryhaline eel.
High‐Level Expression of Recombinant Pea Chloroplast Fructose‐1,6‐Bisphosphatase and Mutagenesis of Its Regulatory Site
Authors: Jacquot Jean‐Pierre , Lopez‐Jaramillo Javier , Chueca Ana , Cherfils Jacqueline , Lemaire Stéphane , Chedozeau Bruno , Miginiac‐Maslow Myroslawa , Decottignies Paulette , Wolosiuk Ricardo , Lopez‐Gorge Julio
Published: July 2010

The cDNA fragment coding for mature chloroplast pea fructose‐1,6‐bisphosphatase [Fru(1,6)P2ase] was introduced by PCR into the expression vector pET‐3d resulting in the construction pET‐FBP. After transformation of BL21(DE3) Escherichia coli cells by the pET‐FBP plasmid and induction with isopropyl thio‐β‐d‐galactoside, high‐level expression of the recombinant enzyme was achieved. The protein could be purified in three days by a simple procedure which includes heat treatment, ammonium sulfate fractionation, DEAE Sephacel and ACA 44 chromatographies with a yield of 20 mg/l culture. In every respect, the recombinant enzyme was similar to plant chloroplast Fru(1,6)P2ase and, in particular, its reactivity with Mg2+ and redox regulatory properties were conserved. In a second series of experiments based on three‐dimensional modeling of the chloroplast protein and sequence alignments, two cysteine residues of the recombinant enzyme (Cys173 and Cys178) were mutated into serine residues. An active enzyme, which did not respond to thiol reagents and to light activation, was obtained, confirming the putative regulatory role of the insertional sequence characteristic of the chloroplast enzyme.
Binding Properties and Protease Stability of Recombinant Human Nidogen
Authors: Mayer Ulrike , Zimmermann Katrin , Mann Karlheinz , Reinhardt Dieter , Timpl Rupert , Nischt Roswitha
Published: July 2010

Recombinant human nidogen was obtained from transfected kidney cell clones as a 150‐kDa protein with a three‐globule structure. It was modified by sulfation and O‐glycosylation and a lower level of N‐glycosylation than mouse nidogen. Recombinant nidogens of both species were, however, indistinguishable in their affinities for laminin‐1 and a recombinant laminin γ1 chain fragment and showed a similar binding to collagen IV and the heparan sulfate proteoglycan perlecan. The two nidogens were also equivalent in the promotion of ternary complex formation between these ligands, indicating that this function has been conserved during mammalian evolution. Fewer zinc‐binding sites could be identified in human nidogen and correlated with a lower capacity of zinc to prevent binding to laminin and collagen IV. Most remarkable was the greater sensitivity of human nidogen to endogenous proteolysis in cell culture, yielding fragments of 90–145 kDa. Studies with several exogenous proteases, including thrombin and leucocyte elastase, showed lack of stability of the N‐terminal globular domain G1 in contrast to what was found for mouse nidogen. Since such degradation could be important for basement membrane remodelling, this difference between human and mouse may be biologically significant.
Role of Amino Acid Sequences Flanking Dibasic Cleavage Sites in Precursor Proteolytic Processing
Authors: Rholam Mohamed , Brakch Noureddine , Germain Doris , Thomas David Y. , Fahy Christine , Boussetta Hamadi , Boileau Guy , Cohen Paul
Published: July 2010

The amino acid sequences flanking 352 dibasic moieties contained in 83 prohormones and pro‐proteins listed in a database were examined. Frequency calculations on the occurrence of given residues at positions P6 to P′4 allowed us to delineate a number of features which might be in part responsible for the in vivo discrimination between cleaved and uncleaved dibasic sites. These include the following: amino acids at these positions were characterized by a large variability in composition and properties; no major contribution of a given precursor subsite to endoprotease specificity was observed; some amino acid residues appeared to occupy preferentially certain precursor subsites (for instance, Met in P6 and P3, Asp and Ala in P′1, Pro in P6, Gly in P3 and P′2 etc.) whereas some others appeared to be excluded.

Most amino acid residues occupying the P′1 position in these precursor cleavage sites were tolerated. But the β‐carbon branched side chain residues (Thr, Val, Leu, Ile) and Pro, Cys, Met and Trp were either totally excluded or poorly represented, suggesting that they might be unfavourable to cleavage. The biological relevance of these observations to the efficacy of dibasic cleavage by model propeptide convertases was in vitro tested using both pro‐ocytocin convertase and Kex2 protease action on a series of pro‐ocytocin related synthetic substrates reproducing the Pro7→Leu15 sequence of the precursor in which the Ala13 residue (P′1 in the LysArg‐Ala motif) was replaced by various amino acid residues. A good correlation was obtained on this model system indicating that P′1 residue of precursor dibasic processing sites is an important feature and may play the role of anchoring motif to S′1 convertase subsite.

We tentatively propose that the present database, and the corresponding model, may be used for further investigation of dibasic endoproteolytic processing of propeptides and pro‐proteins.
Introduction of Specific Carbohydrates into Eucalyptus gunnii Cells Increases their Freezing Tolerance
Authors: Leborgne Nathalie , Teulieres Chantal , Travert Sébastien , Rols Marie‐Pierre , Teissie Justin , Boudet Alain Michel
Published: July 2010

The comparison of soluble sugar content in various cell lines of Eucalyptus gunnii exhibiting different freezing resistances revealed that the most resistant cell line contained the highest soluble sugar content. It was possible to increase the freezing resistance of the sensitive cell line by progressive exposure to low temperatures (acclimation). During the early stage of cold acclimation, an increase of soluble sugar concentration was observed in the cells confirming the correlation between freezing resistance and soluble carbohydrate content in this species. In addition, feeding experiments on the sensitive cell line were performed to introduce specific sugars into the cells. Both electropulsation and long‐term incubation in the presence of fructose and raffinose led to an increase in the tolerance of the cells during a freezing programme. Using radioactive fructose, the uptake of the sugar into cells and protoplasts was checked. In the light of these results, hypotheses are presented concerning the possible role of intracellular sugars in cryoprotection.
Protein Phosphatase 2B of Saccharomyces Cerevisiae is Required for Tolerance to Manganese, in Blocking the Entry of ions into the Cells
Authors: Farcasanu Ileana C. , Hirata Dai , Tsuchiya Eiko , Nishiyama Fumitaka , Miyakawa Tokichi
Published: July 2010

The role of protein phosphatase 2B (PP2B/calcineurin) of Saccharomyces cerevisiae in the tolerance to divalent cations was investigated. PP2B‐deficient mutants were found to be sensitive to MnCl2, but not to ZnCl2, CuCl2, NiCl2, and CoCl2. By measuring both manganese uptake and its efflux, it was found that the sensitivity of the mutant cells was due to an increase in manganese uptake and that the wild‐type cells were able to prevent manganese entry into the cells, rather than export it in a more efficient manner. In the presence of the immunosuppressant FK506, the behavior of wild‐type cells became similar to that of PP2B mutants. Out of various divalent cations tested, externally added magnesium ions were able to block manganese uptake in both wild‐type and PP2B mutant strains.
Construction of Stable BHK‐21 Cells Coexpressing Human Secretory Glycoproteins and Human Gal(β1‐4)GlcNAc‐R α2,6‐Sialyltransferase
Authors: Grabenhorst Eckart , Hoffman Andrea , Nimtz Manfred , Zettlmeissl Gerd , Conradt Harald S.
Published: July 2010

The human β‐trace protein has been cloned and has been expressed for the first time in a mammalian host cell line. Stable BHK‐21 cell lines exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT‐βTP or pAB3‐1 which contain the cDNAs encoding the human secretory glycoproteins β‐trace protein or antithrombin III and pABSial containing the human Golgi enzyme CMP‐NeuAc:Gal(β1‐4)GlcNAc‐R α2,6‐sialyltransferase (ST6N) gene. The β‐trace protein was purified by immunoaffinity chromatography and N‐linked oligosaccharides were subjected to carbohydrate structural analysis. The enzymically liberated oligosaccharides were found to consist of 90% of diantennary chains as is the case for natural β‐trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fractions in a ratio of 1:5. The monosialylated oligosaccharides of β‐trace protein coexpressed with human ST6N were found to contain NeuAc in α2,6‐or α2,3‐linkage in the same ratio. From 1H‐NMR analysis as well as calculations of peak areas obtained by HPLC, 60% of the molecules of the disialo fraction were found to contain NeuAc in both α2,3‐and α2,6‐linkage to Galβ(1‐4)GlcNAc‐R, whereas 40% of the molecules of this fraction contained NeuAc in only α2,3‐linkage to Gal(β1‐4)GlcNAc‐R. The α2,6‐linked NeuAc was shown to be attached preferentially to the Gal(β1‐4)GlcNAc(β1‐2)Man(α1‐3) branch of the diantennary structure. Therefore the in vivo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specificity of the bovine colostrum ST6N activity. Furthermore, these studies demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human‐type glycosylated secretory recombinant polypeptides.
Nature of the Masking of Nucleotide‐Binding Sites in Brown Adipose Tissue Mitochondria
Authors: Huang Shu‐Gui , Klingenberg Martin
Published: July 2010

Binding of the fluorescent nucleotide derivative 2′‐O ‐dansyl GTP and purine nucleotides to brown adipose tissue mitochondria from hamster was studied. 2′‐O ‐Dansyl GTP binds with enhanced fluorescence to the uncoupling protein (UCP) in the mitochondria, similar to the isolated protein. The fluorescence signal showed biphasic fast and slow increases. Treatment of the mitochondria with an anion exchanger (Dowex) increased the total fluorescence but decreased the slower phase. The biphasic fluorescence response was restored by incubation with only 1 μM ATP, indicating that residual bound ATP may be responsible for the observed slow phase. The binding of [14C]GTP and GDP also increased after Dowex treatment. The dissociation of bound [14C]ATP but not of bound [14C]ADP was slow and apparently limited the binding assays. Short incubation (5 min) resulted in a curvature of the Scatchard plot, where the ‘high‐affinity sites’ correspond to the free UCP sites; GDP had apparently higher affinity than GTP. Dowex treatment and incubation for 60 min produced a more linear Scatchard plot. Under such conditions, one measures the maximal UCP‐binding sites (1.2 μmol/g protein); GTP exhibited higher affinity (Kd= 0.64 μM) than GDP (Kd= 3.1 μM). Acute cold adaptation (40 min at 4°C) of hamsters caused an increase by over 40% of [14C]GTP binding, as compared to the control warm‐(28°C)‐adapted animals. Dowex treatment completely abolishes this unmasking/masking effect, where both mitochondria had identical binding capacity and affinity for GTP. The inhibition by purine nucleotides of H+ transport as measured by potassium‐acetate‐induced mitochondrial swelling was dependent on the incubation time. Diphosphates inhibited faster and triphosphates required longer incubation (10 min) but inhibited more strongly. A linear correlation between the mitochondrial swelling rate and GDP binding was observed for mitochondria with depleted endogenous ATP or with added ATP. These data indicate that residual bound ATP from the tissue is responsible for the masking phenomenon.
Degradation of the D1 Protein of Photosystem‐II Reaction Centre by Ultraviolet‐B Radiation Requires the Presence of Functional Manganese on the Donor Side
Authors: Barbato Roberto , Frizzo Annalisa , Friso Giulia , Rigoni Fernanda , Giacometti Giorgio M.
Published: July 2010

The in vivo effects of ultraviolet‐B radiation (280–320 nm) on photosystem‐II activity and degradation of the D1 protein are investigated and compared with the in vitro results on isolated thylakoids and other detergent‐extracted photosystem‐II preparations. A cleavage site in the second transmembrane segment of the D1 protein, giving rise to a 20‐kDa C‐terminal and a 13‐kDa N‐terminal fragment pair, is detected after irradiation of entire leaves as well as in all photosystem‐II preparations, irrespective of their actual ability to evolve oxygen but depending on the presence of Mn ions associated with the water‐splitting system. Damage to the plastoquinone moiety, observed by other authors, is confirmed and is proposed to be responsible for the impairment of electron‐transport activity, but not for the observed cleavage of the D1 protein.
Characterization and Solubilization of Novel Aldosterone‐Binding Proteins in Porcine Liver Microsomes
Authors: Meyer Christiane , Christ Michael , Wehling Martin
Published: July 2010

Using the radioligand [1,2,6,7‐3H]aldosterone ([3H]aldosterone), specific binding sites for aldosterone were identified and characterized in microsomal preparations from porcine liver. The maximum binding capacity is approximately 700 fmol · mg‐1 microsomal protein. The reversible binding of [3H]aldosterone was saturable and Scatchard analysis revealed two apparent dissociation constants (Kd), Kd1≤ 11 nM and Kd2= 118 nM. Binding was optimal at pH 7.2, thermolabile, and was reduced by more than 70% when membrane vesicles were pretreated with trypsin. Binding was selective for aldosterone with cortisol being a weak agonist at 1000‐fold higher concentrations only. Among those detergents tested to optimize conditions for solubilization, n ‐octylglucoside (75 mM) was most favorable and solubilized 25% of the radio‐ligand‐binding protein complex in the undissociated form. These binding sites have unique pharmacological properties, which are similar to those found for aldosterone membrane binding in human lymphocytes and pig kidney, and for rapid aldosterone effects on sodium‐proton exchange.
The Long‐Term Regulation of Skeletal Muscle Pyruvate Dehydrogenase Kinase by Dietary Lipid is Dependent on Fatty Acid Composition
Authors: Fryer Lee G. D. , Orfali Karen A. , Holness Mark J. , Saggerson E. David , Sugden Mary C.
Published: July 2010

The provision of a diet high in saturated and monounsaturated fat for 28 days evoked a significant (1.9‐fold) increase in pyruvate‐dehydrogenase kinase activity measured in isolated mitochondria from representative slow‐twitch (oxidative) skeletal muscles (pooled soleus and adductor longus muscles) from adult rats. The increase observed in response to 28 days of high‐fat feeding in slow‐twitch skeletal muscle mitochondria was similar in magnitude to that observed in heart mitochondria. Pyruvate‐dehydrogenase kinase activity was not increased in response to the provision of the high‐fat diet in mitochondria prepared from a representative fast‐twitch muscle (tibialis anterior), while the increases evoked by 28 days of high‐fat feeding in cardiac and slow‐twitch skeletal muscle were prevented by the replacement of 7% of the dietary fatty acids with long‐chain ω–3 fatty acids from marine oil. Cardiac myocytes prepared from the high‐fat‐fed rats showed impaired responses of this enzyme to n ‐octanoate (1 mM) and N6,2‐O ‐dibutyryl‐adenosine 3′,5′‐monophosphate (50 μM) individually in cultured cardiac myocytes and of glucose uptake to insulin at low concentrations in freshly prepared cardiac myocytes, compared with control rats maintained on standard low‐fat/high‐carbohydrate diet. These impairments in responses to agonists were substantially improved by the inclusion of long‐chain ω–3 fatty acids in the high‐fat diet. The results indicate that pyruvate‐dehydrogenase kinase activity in oxidative skeletal muscle is a target for longer‐term regulation by high‐fat feeding and that the fatty acid composition of the diet, rather than the fat content, is a key influence.
An Essential Lysyl Residue (Lys208) in the Substrate‐Binding Site of Porcine FAD‐Containing Monooxygenase
Authors: Wu Ru‐Feng , Ichikawa Yoshiyuki
Published: July 2010

The substrate (amine)‐binding site of porcine FAD‐containing monooxygenase (FMO) (EC 1.14.13.8) was examined using pyridoxal 5′‐phosphate (pyridoxal‐P) to modify lysyl residues. The enzymic activity of the FMO was inhibited competitively by pyridoxal‐P. Upon reduction of pyridoxal‐P‐treated FMO with NaBH4, a new characteristic absorption peak of substituted pyridoxal‐Pappeared at 325 nm. The amino acid residue compositions of the native and pyridoxal‐P‐treated FMOs indicated that the lysyl residues were modified by pyridoxal‐P. The about 74% inactivation of the enzymic activity on covalent pyridoxal‐Ptreatment of the FMO was nearly completely prevented in the presence of the substrate,N,N‐dimethylaniline. The FMO covalently modified with pyridoxal‐Pin the presence or absence ofN,N‐dimethylaniline was digested with trypsin treated with tosylphenylalanylchloromethane and the resultant peptide fragments were separated with a reverse‐phase high‐performance liquid chromatography system; only one peptide was specifically labeled with pyridoxal‐Pand was detected at 325 nm in the absence ofN,N‐dimethylaniline. The modified peptide was analyzed and identified as that comprising the amino acid residues 186–208.

These results suggest that Lys208 plays an important role in the substrate (amine)‐binding site of FMO.
Protein Disulphide Isomerase and a Lumenal Cyclophilin‐Type Peptidyl Prolyl Cis‐Trans Isomerase are in Transient Contact with Secretory Proteins During Late Stages of Translocation
Authors: Klappa Peter , Freedman Robert B. , Zimmermann Richard
Published: July 2010

The transport of a presecretory protein into the mammalian endoplasmic reticulum can be divided into early translocation events which include specific targeting of the presecretory protein to and insertion into the endoplasmic reticulum membrane and late translocation events, comprising signal sequence cleavage, completion of translocation and folding of the secretory protein into a functional conformation. The microsomal membrane proteins Sec61αp and translocating‐chain‐associating membrane protein were previously identified as being in close contact with a nascent presecretory protein at an early step of translocation. Here, we investigated whether additional microsomal proteins are in contact with translocating chains during or immediately after transit. This was addressed by crosslinking after release of the nascent chain from Sec61αp. We observed two additional membrane proteins interacting with the nascent precursor in the early stages of translocation and three lumenal proteins interacting with the processed polypeptide chain in the late stages of translocation. One of the lumenal proteins was identified as protein disulphide isomerase by immunoprecipitation. Another of the lumenal proteins was suggested to be a lumenal cyclophilin‐type peptidyl prolyl cis‐trans isomerase by the effect of cyclosporin A. We propose that molecular chaperones, such as protein disulphide isomerase and cyclophilin may represent two of the lumenal proteins which are involved in completion of translocation.
Identification of Factor‐XIIIa‐Reactive Glutaminyl Residues in the Propolypeptide of Bovine von Willebrand Factor
Authors: Takagi Junichi , Aoyama Toshihiro , Ueki Shoko , Ohba Hirotsugu , Saito Yuji , Lorand Laszlo
Published: July 2010

von Willebrand factor is a large multimeric plasma protein which plays important roles in platelet aggregation, blood coagulation and probably also in the adhesion of endothelial cells. A 100‐kDa propeptide, called the propolypeptide of von Willebrand factor (pp‐vWF), is generated during biosynthesis. We found that pp‐vWF served as a substrate for transglutaminases including human factor XIIIa and guinea pig liver transglutaminase [Usui, T., Takagi, J. & Saito, Y. (1993) J. Biol. Chem. 268, 12311–123161. As such, it could form cross‐linked copolymers with the extracellular matrix protein, laminin, making it all the more likely that pp‐vWF plays a role in cell adhesion phenomena [Takagi, J., Sudo, Y., Saito, T. & Saito, Y. (1994) Eur. J. Biochem. 222, 861–867].

In this work, we identified the Gln residues in pp‐vWF specifically reacting with blood coagulation factor XIIIa as amine acceptors. The fluorescent amine, dansylcadaverine, was employed for labeling the enzyme‐reactive sites of the protein. Following partial proteolysis, fragments containing the labeled Gln residues were isolated by passage through an anti‐dansyl affinity chromatographic column. Amino acid sequence analyses of the fragments revealed that, out of about 40 Gln residues in pp‐vWF, only four could be modified in the factor‐XIIIa‐catalyzed reaction.
Identification by UV Cross‐Linking of Oligo(U)‐Binding Proteins in Mitochondria of the Insect Trypanosomatid Crithidia fasciculata
Authors: Leegwater Peter , Speijer Dave , Benne Rob
Published: July 2010

RNA editing in trypanosomes is the process of insertion and deletion of U residues at specific sites of mitochondrial transcripts mediated by short guide RNAs (gRNAs) that have a 3′ oligo(U) extension. Here ‘we describe the identification by UV cross‐linking of proteins present in mitochondrial extracts from Crithidia fasciculata with a high affinity for gRNAs, and the characterization of the binding specificity. A 65‐kDa protein binds to gRNAs provided they are equipped with a U tail, to post‐transcriptionally labelled mitoribosomal 9S and 12S RNAS that also possess a 3′ terminal stretch of U residues, and to free oligo(U) sequences with a minimal length of 23–29 nucleotides. It does not bind to a number of control RNAs, one of which has an internal U stretch of 13 residues. Poly(U), but not poly(C) or total yeast RNA, efficiently competes for binding to gRNA. Proteins of 88 kDa and 30 kDa also bind to gRNAs with a U tail, to miochondrial ribosomal RNAs and to oligo(U). These proteins, however, require longer oligo(U) for binding (>39 nucleotides) and they also have an affinity for other U‐rich RNAs and poly(C). For comparison, part of the analysis was also carried out with a mitochondrial extract from Trypanosoma brucei. In this organism, gRNA‐binding proteins of 83 kDa and 64 kDa were found with the same preference for 3′‐terminal oligomeric U stretches as the C. fasciculata 65‐kDa protein, whereas the binding specificity of a 26‐kDa protein resembled that of the C. fasciculata 88‐kDa and 30‐kDa proteins. The possible involvement of the proteins in the editing process is discussed.