bluuebunny/crossref_metadata_2025_split huggingface dataset:
Identification and characterization of fragments of major glycoproteins from platelet membrane after chymotrypsin treatment
Authors: McGREGOR John L. , CLEZARDIN Philippe , JAMES Elizabeth , McGREGOR Lilian , DECHAVANNE Marc , CLEMETSON Kenneth J. 
Published: March 2005

Human platelets were surface‐labeled by the periodate/NaB3H4 method or by lactoperoxidase‐catalysed iodination with 125I. The labeled platelets were treated with chymotrypsin under conditions known to give platelets which aggregate with fibrinogen without stimulation with ADP. Platelets and supernatant were then analysed by various gel electrophoretic techniques including isoelectricfocusing/sodium dodecyl sulfate‐polyacrylamide gel electrophoresis under reducing or non‐reducing conditions and two‐dimensional non‐reduced/reduced sodium dodecyl sulfate polyacrylamide gel electrophoresis followed by fluorography or indirect autoradiography.Chymotrypsin‐treatment of surface‐labeled platelets degraded the major glycoproteins Ib, IIb and IIIa but also GP4.9−5.4120, GPlc and GPV. The membrane‐bound fragments of GPIb, IIb and IIIa could be identified and also the supernatant fragments of GPIb and GPV. GPIIIa was also cleaved within a loop structure formed by disulfide bond(s).The fact that remnants of both GPIIb and IIIa are left on chymotrypsin‐treated platelets which aggregate spontaneously with fibrinogen may indicate that a complex formed by these remnants constitutes the fibrinogen‐binding site on platelets.
A high‐affinity site for glutathione in the cytoplasm of Escherichia coli and its possible role in potassium retention
Authors: MEURY Jean , ROBIN Aline 
Published: March 2005

Glutathione‐deficient mutants of Escherichia coli, unlike the wild type, exhibit a fast leak and a fast turnover of K+ at steady state.In a medium with low K+ the growth rate is very slow when unsupplemented with glutathione; when supplemented with glutathione at a concentration as low as 0.1 μM the growth rate is similar to that of the wild type. The ability of glutathione to restore a wild‐type growth rate can be accounted for by an immediate reduction of the K+ leak.Two analogs of glutathione also reduce the K+ leak: γ‐glutamylaminobutyrylglycine (ophthalmic acid) and αγ‐glutamylcystinylbisglycine.Glutathione binds with high affinity (Kd 50 nM) to a cytoplasmic protein; ophthalmic acid and αγ‐glutamylcystinylbisglycine compete with glutathione for the binding site (Ki 0.1 μM and 1 μM), thereby ruling out the possibility that the thiol is involved both in reducing the K+ leak and in binding to the high affinity binding site. For each of the three peptides the Kd for binding is similar to the minimal concentration that achieves the maximal reduction of the K+ leak. The results suggest that the binding of glutathione should play a role in the retention of K+ in E. coli.
Steady‐state kinetic analysis of transcription of cloned tRNASer genes from Drosophila melanogaster
Authors: ST. LOUIS Daniel , SPIEGELMAN George B. 
Published: March 2005

Drosophila melanogaster Schneider II cells contain a factor which inhibits transcription in vitro of cloned tRNA genes in crude extracts made from these cells. The inhibitor could, however, be effectively neutralized by addition of certain non‐template DNAs.In the absence of the transcription inhibitor activity, the steady‐state kinetics of tRNA production from cloned genes followed one‐substrate enzyme kinetics to a high degree of accuracy. Maximal rates of transcription and apparent affinity constants were analyzed for a collection of cloned D. melanogaster tRNASer genes. The stability of the complex formed by the transcription proteins and the template DNA was found to be nearly constant for the genes examined. The transcription rates, however, were greatly influenced by the DNA sequences flanking the tRNA genes.Analysis of transcription competition between DNA templates showed pure competitive behavior. Inhibition constants derived from these experiments indicated that the formation of the transcription complex was affected by sequences flanking the tRNA genes. Furthermore, the rate‐limiting step in complex formation was independent of the stability of the final form of the complex.
Studies of the cellulolytic system of Trichoderma reesei QM 9414
Authors: van TILBEURGH Herman , PETTERSSON Göran , BHIKABHAI Ramagauri , DE BOECK Hilde , CLAEYSSENS Marc 
Published: March 2005

1. The 1,4‐β‐glucan cellobiohydrolase II (CBH II) from Trichoderma reesei QM 9414 catalyses the hydrolysis of the 4‐methylumbelliferyl β‐d‐glycosides derived from cellotriose, cellotetraose and cellopentaose [MeUmb(Glc)n; n= 3–5]. The reaction has been followed by quantitative high‐performance liquid chromatography. Specific activity for cellobiose removal at apparent substrate saturation were determined as (0.8 ± 0.2) min−1 for MeUmb(Glc)3 and (9 ± 2) min−1 for MeUmb(Glc)4. The enzyme showed a deviant specificity with MeUmb(Glc)5 as substrate. Two chromophoric products were formed simultaneously [MeUmb(Glc)3 and MeUmb(Glc)2] with turn‐over numbers (17 ± 4) min−1 and (21 ± 6) min−1, respectively.2. Methylumbelliferyl β‐glucoside (MeUmbGlc) and the corresponding cellobioside [MeUmb(Glc)2] were used in equilibrium binding experiments. Both ligands yielded one binding site per molecule of Mr= 54000 upon forced flow dialysis (diafiltration). The association constants found were in fair agreement with those determined from MeUmb fluorescence quenching titrations. Quenching was total at all temperatures investigated for MeUmb(Glc)2, whereas for MeUmbGlc it increased from 80% to 100% between 2°C and 20°C. The association constants fitted linear van't Hoff plots in both cases.3. MeUmb(Glc)2 and MeUmbGlc were also used as indicator ligands to determine the association constants and thermodynamic parameters of several non‐chromophoric ligands of CBH II. The binding of glucose increased the affinity for MeUmb(Glc)2 whereas it displaced MeUmbGlc from its complex.4. A putative binding site of the CBH II containing four subsites can be proposed. The thermodynamic data for methyl β‐d‐glucopyranoside and cellobiose as ligands also point at an extended binding site.
The protein phosphatases involved in cellular regulation
Authors: STRÅLFORS Peter , HIRAGA Akira , COHEN Philip 
Published: March 2005

A type‐1 protein phosphatase (protein phosphatase‐1G) was purified to homogeneity from the glycogenprotein particle of rabbit skeletal muscle. Approximately 3 mg of enzyme were isolated within 4 days from 5000 g of muscle. Protein phosphatase‐1G had a molecular mass of 137 kDa and was composed of two subunits G (103 kDa) and C (37 kDa) in a 1:1 molar ratio. The subunits could be dissociated by incubation in the presence of 2 M NaCl, separated by gel‐filtration on Sephadex G‐100, and recombined at low ionic strength. The C component was the catalytic subunit, and was identical to the 37‐kDa type‐1 protein phosphatase catalytic subunit (protein phosphatase‐1C) isolated from ethanol‐treated muscle extracts, as judged by peptide mapping. The G component was the glycogen‐binding subunit. It was very asymmetric, extremely sensitive to proteolytic degradation, and failed to silver stain on SDS/polyacrylamide gels.Protein phosphatase‐1G was inhibited by inhibitor‐1 and inhibitor‐2, but unlike protein phosphatase‐1c, the rate of inactivation was critically dependent on the ionic strength, temperature and time of preincubation with the inhibitor protein. At near physiological temperature and ionic strength, protein phosphatase‐1G was inactivated very rapidly by inhibitor‐1. Protein phosphatase‐1G interacted with inhibitor‐2 (1–2) to form an inactive species, with the structure GCI‐2. This form could be activated by preincubation with Mg‐ATP and glycogen synthase kinase‐3.The G subunit could be phosphorylated on a serine residue(s) by cyclic‐AMP‐dependent protein kinase, but not by phosphorylase kinase or glycogen synthase kinase‐3. Phosphorylation was rapid and stoichiometric, and increased the rate of inactivation of protein phosphatase‐1G by inhibitor‐1. The relationship of the G subunit to the ‘deinhibitor protein’ is discussed.
Transcription in methanogens
Authors: THOMM Michael , STETTER Karl O. 
Published: March 2005

The purification of the DNA‐dependent RNA polymerase of Methanococcus thermolithotrophicus is described. As the first step of purification the endogenous template was removed from the enzyme by hydrophobic interaction chromatography. The purified enzyme consists of seven components with different molecular masses.Transcription studies on T7 DNA and the recombinant plasmid pMV15, containing rRNA genes of Methanococcus vannielii, revealed that only the methanogen DNA is transcribed specifically, indicating a principal structural difference between archaebacterial and eubacterial promoters. This could be shown both by analysis of ternary transcription complexes and Southern hybridization. The site of initiation was found within a restriction fragment harbouring the first 390 nucleotides of the sequence coding for mature 16S rRNA and 1100 base pairs of upstream sequences. The specific initiation on this fragment strongly suggests that the enzyme can start in vitro transcription from the promoter(s) of rRNA synthesis.
Nucleotide sequence and high‐level expression of the major Escherichia coli phosphofructokinase
Authors: HELLINGA Homme W. , EVANS Philip R. 
Published: March 2005

The gene for the major phosphofructokinase enzyme in Escherichia coli, pfk A, has been sequenced. Comparison of the amino acid sequence with other phosphofructokinases showed that this enzyme is related to the Bacillus stearothermophilus and rabbit muscle enzymes, but is different from the second, minor phosphofructokinase found in E. coli.The region which has been sequenced comprises the complete pfkA – tpi interval on the E. coli genetic map. Two other genes have been identified from the nucleotide sequence: a gene for a periplasmic sulphate‐binding protein, sbp, and for a membrane‐bound enzyme, CDP‐diglyceride hydrolase, cdh. This establishes the complete gene arrangement in this region as pfkA‐sbp‐cdh‐tpi.The pfkA gene has been subcloned into a high‐copy‐number plasmid under the control of a strong, chimaeric promoter which arose as an artefact in the construction of the plasmid gene bank from which the original pfkA recombinant was isolated. A specialised recombinant has been constructed which carries a 1.4 × 103‐nucleotide insert containing just the pfkA gene flanked by two HindIII recognition sites providing a simple system for the recloning of this gene into different vectors. This recombinant expresses the enzyme at high levels (40–50% of total cell protein is active, soluble phosphofructokinase). This expression system is now being used to study the enzyme using ‘reverse genetics’.
l‐Phenylalanine ammonia‐lyase from Phaseolus vulgaris
Authors: BOLWELL G. Paul , BELL John N. , CRAMER Carole L. , SCHUCH Wolfgang , LAMB Chris J. , DIXON Richard A. 
Published: March 2005

l‐Phenylalanine ammonia‐lyase (EC 4.3.1.5) has been purified over 200‐fold from cell cultures of bean (Phaseolus vulgaris L.) exposed to elicitor heat‐released from the cell walls of the phytopathogenic fungus Colletotrichum lindemuthianum. Four forms of the enzyme, with identical Mr but differing apparent pI values of 5.4, 5.2, 5.05 and 4.85, were observed following the final chromatofocussing stage of the purification.
A preparation (purified 43‐fold by ammonium sulphate precipitation, gel‐filtration and ion‐exchange chromatography) containing all four forms exhibited apparent negative rate cooperativity with respect to substrates. However, the individual forms displayed normal Michaelis‐Menten kinetics, with Km values of 0.077 mM, 0.122 mM, 0.256 mM and 0.302 mM in order of decreasing apparent pI value.
A preparation purified 200‐fold and containing all four forms was used to immunise rabbits for the production of anti‐(phenylalanine ammonia‐lyase) serum. The antiserum was characterised by: (a) immunotitration experiments; (b) solid phase enzyme‐linked immunosorbent assays; (c) comparison of immunoprecipitates of 35S‐labelled phenylalanine ammonia‐lyase subunits (synthesized both in vivo and in vitro) on both one‐dimensional and two‐dimensional polyacrylamide gels after immunoprecipitation with the bean antiserum or antisera raised against pea and parsley phenylalanine ammonia‐lyase preparations and (d) immune blotting.
Experiments involving (a) immunoprecipitation followed by analysis on SDS/polyacrylamide gels and (b) SDS/polyacrylamide gel electrophoresis followed by immune blotting, indicated that the Mr of newly synthesized (in vivo and in vitro) bean phenylalanine ammonia‐lyase subunits is 77000; a 70000‐Mr form is readily generated as a partial degradation product during purification.
Immunoprecipitates of bean phenylalanine ammonia‐lyase synthesized both in vivo and in vitro showed the presence of multiple subunit types of identical Mr but differing in pI. Furthermore, treatment of bean cultures with Colletotrichum elicitor resulted in a 10‐fold increase in phenylalanine ammonia‐lyase extractable activity within 8 h, and chromatofocussing analysis indicated that this was associated with differential increased appearance of the high‐pI, low‐Km forms as compared to the two higher Km forms. This differential induction was further confirmed by immune blotting of crude extracts subjected to isoelectric focussing.
The mitochondrial location of protoporphyrinogen oxidase
Authors: DEYBACH Jean‐Charles , DA SILVA Vasco , GRANDCHAMP Bernard , NORDMANN Yves 
Published: March 2005

Using the digitonin method and subsequent fractionation of rat liver mitochondria, protoporphyrinogen oxidase (penultimate enzyme in the heme biosynthesis pathway) was found to be closely associated with the mitochondrial inner membrane fraction.Chemical treatment with non‐specific probes (trypsin and diazobenzene sulfonate) of either intact or inverted mitoplasts, indicated that protoporphyrinogen oxidase was anchored within the lipid bilayer of the inner membrane. Protoporphyrinogen had an equal access to the active site of the enzyme from both sides of the inner membrane and its transformation to protoporphyrin did not appear to be energy‐dependent.Studies of protoporphyrinogen synthesis from exogenously added coproporphyrinogen in either intact or hypoosmotically treated mitochondria underlined the importance of the peculiar submitochondrial location of coproporphyrinogen oxidase and protoporphyrinogen oxidase for the transfer of substrates to the inner membrane.
Amino acid biosynthesis and sodium‐dependent transport in Methanococcus voltae, as revealed by 13C NMR
Authors: EKIEL Irena , JARRELL Ken F. , SPROTT G. Dennis 
Published: March 2005

Of several methanogenic bacteria examined only Methanococcus voltae readily incorporated exogenous amino acids into cell protein. This was easily shown, since growth in the presence of exogenous amino acids resulted in a loss of signal intensities from those carbon atoms normally labelled by [13C]acetate during biosynthesis. From 80% to 95% of the Ser, Lys, Pro or Val incorporated into protein could be supplied directly from the growth medium. In contrast, Asp and Glu, if supplied to the medium, accounted for only a small percentage of the total acidic amino acid used in protein synthesis. Constitutive transport systems took up a wide range of amino acids at rates of 0.1–4.1 nmol min−1 mg−1. The transport systems required Na+, with the possible exception of the basic amino acid lysine, and were inhibited by N‐ethylmaleimide or 3,3′,4′,5‐tetrachlorosalicylanilide. No interconversion of Ile to other amino acids was detected when cells were given [13C]Ile during growth, whereas the expected labelling of the Asp and Glu families of amino acids resulted when [13C]Asp was provided to the culture.
Mc. voltae synthesized its amino acids from acetate via routes fully consistent with those found in Methanospirillum hungatei [Ekiel, I., Smith, I. C. P. & Sprott, G. D. (1983) J. Bacteriol. 156, 316–326]. Propionate could substitute for an auxotrophic requirement for Ile, resulting in the synthesis of Ile with the β‐carbon originating from the carboxyl of acetate and the α‐carbon from the carboxyl of propionate. No labelling of Ile from [13C]acetate could occur without the fatty acid. These results provide strong evidence for the carboxylation of propionate to form 2‐oxobutyrate as intermediate in Ile biosynthesis, and show that the metabolic defect in Ile biosynthesis occurs prior to 2‐oxobutyrate synthesis. The presence of constitutive amino acid transport systems and multiple routes for ile biosynthesis make Methanococcus voltae an attractive methanogen for genetic studies.
Modification of the N‐linked oligosaccharides in cell surface glycoproteins during chick embryo development
Authors: CODOGNO Patrice , BOTTI Joëlle , FONT Jacqueline , AUBERY Michèle 
Published: March 2005

Important differences in asparagine‐linked glycopeptides were observed in in vitro cultured fibroblasts derived from chick embryo at different stages of development. Cells from 8‐day and 16‐day embryos were labeled metabolically with [3H]mannose. Cell surface glycopeptides obtained after mild trypsin treatment were extensively digested with pronase and then chromatographed on concanavalin‐A‐Sepharose and other immobilized lectins. The most important changes concerned the complex type chains. The ratio between triantennary plus tetraantennary and biantennary chains increased about 2.5‐fold from the 8th to the 16th day of development. In the same way, complex chains with bisecting N‐acetylglucosamine increased from 8‐day to 16‐day cells as shown by Phaseolus‐vulgaris‐erythroagglutinin – agarose chromatography. In 16‐day cells, the majority of triantennary chains (60%) with α‐linked mannose substituted at C2 and C6 positions and biantennary chains (50%) wer shown to contain fucosyl (α1 → 6)N‐acetylglucosaminyl structure in the core region by their ability to bind to a lentil lectin affinity column. Similarly, in 8‐day cells, triantennary chains (50%) were more fucosylated than biantennary chains (35%). Thus, complex structures exhibited an increased fucosylation of their invariable core from the 8th to the 16th day of development, except for fucosylated triantennary chains which were retained on Phaseolus vulgaris Leucoagglutin and on lentil lectin. These latter structures were present at the surface of 8‐day cells and absent at the surface of 16‐day cells.After chromatography on Bio‐Gel P6 and treatment with endo‐β‐N‐acetylglucosaminidase H, the [3H]mannose‐labeled glycopeptides were separated by high resolution chromatography into glycopeptides with complex chains and glycopeptides with high‐mannose chains. Analysis of the high‐mannose oligosaccharides released after endo‐β‐N‐acetylglucosaminidase H treatment by chromatography on Bio‐Gel P4 indicated that the same type of high‐mannose chains were present at the surface of 8‐day and 16‐day cells.Quantification of mannose, galactose and sialic acid residues using gas liquid chromatography was consistent with a decrease of the relative amount of oligomannose chains and an increase of the relative amount of complex type chains in 16‐day cells compared to 8‐day cells. Thus N‐linked oligosaccharides derived from cell surface glycoproteins undergo changes during embryo development resulting in greater complexity of carbohydrate chains.
Pulse‐chase studies of the synthesis of apolipoprotein B in a human hepatoma cell line, Hep G2
Authors: WETTESTEN Margit , BOSTRÖM Kristina , BONDJERS Göran , JARFELDT Marianne , NORFELDT Per‐Ingvar , CARRELLA Massimo , WIKLUND Olov , BORÉN Jan , OLOFSSON Sven‐Olof 
Published: March 2005

We have used pulse‐chase methodology to study the synthesis of apolipoprotein B in a human hepatoma‐derived cell line, the Hep G2 cells. A 2‐min pulse with [35S]methionine was followed by a chase period varying from 5–90 min. A protein of large molecular mass (estimated molecular mass: 312 ± 41 kDa, mean ± SD, n= 8) could be immunoprecipitated from the cells at all chase periods between 5 min and 60 min with both monoclonal antibodies to a narrow density cut of the low density lipoprotein LDL‐2 (density: 1.030–1.055 g/ml) and polyclonal antibodies to the apolipoprotein B apoB 100 or to a narrow density cut of LDL‐2 (density: 1.030–1.055 g/ml). In addition to this large molecular mass protein, nascent polypeptides could be precipitated after 5, 10 and 15 min of chase.The apolipoprotein B molecules that had been labelled during the pulse disappeared from the cells after 60–90 min of chase, while they started to appear in the medium after 30–35 min of chase.The results obtained indicate (a) that apolipoprotein B is synthesized as one polypeptide with a large molecular mass, (b) that newly synthesized apolipoprotein B molecules are secreted after a delay of 30–35 min, (c) that no intracellular accumulation of apolipoprotein B occurs, and (d) that apolipoprotein B is recovered in the density fraction < 1.21 g/ml of the medium suggesting that it is secreted in lipoprotein form.
Regulation of gene expression in Tetrahymena pyriformis under heat‐shock and during recovery
Authors: GALEGO Lisete , RODRIGUES‐POUSADA Claudina 
Published: March 2005

Proteins extracted from Tetrahymena pyriformis cells, which were shifted from 28°C to 34°C (heat‐shocked) and then labelled with [35S]‐methionine, were analysed by two‐dimensional gel electrophoresis according to the method of O'Farrell. In the fluorogram obtained after 2.5 min heat‐shock new polypeptide spots already appear in the region of 70–75 kDa and 25–29 kDa, indicating that the induction response is very rapidly triggered. Other newly induced proteins become more intense after 15 min and 30 min of heat‐shock. After a 60‐min treatment, the electrophoretic analysis shows the normal pattern of proteins resembling that of exponentially growing cells. Our experiments in vivo and in vitro also show that the messengers coding for stress proteins have a short lifetime, suggesting that modulation of the translation efficiency of heat‐shock protein mRNAs is accompanied by an alteration in their biological stability.Studies using the inhibitor actinomycin D suggest that control of this response is exercise mainly at the transcriptional level for the 70–75‐kDa and at the translational level for the 25–29‐kDa heat‐shock proteins.When T. pyriformis cells are allowed to recover at 28°C the heat‐shock proteins disappear completely and normal protein synthesis is completely re‐established. Although the level of normal protein synthesis attained is lower, cells are able to recover completely from heat‐shock when new mRNA synthesis is blocked by actinomycin D, indicating a conservation of normal pre‐existing mRNAs during heat‐shock.