bluuebunny/crossref_metadata_2025_split huggingface dataset:
Differential nuclear localization of protein kinase C isoforms in neuroblastoma × glioma hybrid cells
Authors: BECKMANN Roland , LINDSCHAU Carsten , HALLER Hermann , HUCHO Ferdinand , BUCHNER Klaus 
Published: March 2005

The protein kinase C (PKC) α, β and ε isoforms have distinct nuclear localizations in neuroblastoma × glioma hybrid cells NG 108–15. We found by immunoblotting that PKC α, βII, δ and ε are the predominant isoforms in these cells. In contrast to other neuronal cell lines, none of these isoforms is down‐regulated during differentiation.Confocal immunofluorescence microscopy revealed that in undifferentiated cells PKC α is located in the cytoplasm and in the nucleus excluding nucleoli. In differentiated cells PKC α was almost exclusively located in the cytoplasm. Stimulation of the cells with phorbol ester resulted in translocation to the plasma membrane. PKC βII was not detectable in the nuclei. PKC δ was found in the nucleoli and in the cytoplasm, in differentiated cells particularly in the neurites. Phorbol ester failed to induce a translocation to other compartments. PKC δ was localized with the nuclear‐pore complexes at the nuclear envelope. In differentiated cells after stimulation with phorbol ester, partial translocation to the plasma membrane was observed.
Heat‐induced synthesis and tunicamycin‐sensitive secretion of the putative storage glycoprotein conglutin γ from mature lupin seeds
Authors: DURANTI Marcello , SCARAFONI Alessio , GIUS Cristina , NEGRI Armando , FAORO Franco 
Published: March 2005

SDS/PAGE, immune blotting with specific antibodies and amino acid sequence analyses revealed that 90% of the protein released from Lupinus albus seeds incubated in water at 60°C for about 3 h was conglutin γ, a putative storage glycoprotein already present in the protein bodies of mature seeds. Incorporation of [14C]leucine into the protein demonstrated that conglutin γ was newly synthesized during the treatment and the use of protein synthesis inhibitors ruled out the secretion of constitutive conglutin γ. Synthesis and secretion took place only over a narrow temperature range, 57.5–62.5°C, and in a short time interval, 135–180 min, of incubation of the seed. The amount of secreted conglutin γ, i.e. 1 mg/seed, was about three times that present inside the treated or untreated seed.Secreted conglutin γ contained covalently linked carbohydrate as well as the constitutive protein. Inhibition of the glycosylation by tunicamycin did not affect conglutin γ synthesis, but prevented its secretion from the seed, as indicated by quantifying conglutin γ remaining in the seed. An accumulation of the protein outside the protein bodies and at the cotyledonary cell periphery was shown in these samples by immunocytochemistry.Peptide mapping of the fragments obtained by incubation of constitutive and secreted conglutin γ with trypsin and pepsin revealed no difference between the two proteins.Lupin seeds were still viable after the treatment. However no similarities between conglutin γ and heat‐shock proteins were observed either in the amino acid sequence or other molecular features.
Interactions of lipoprotein lipase with the active‐site inhibitor tetrahydrolipstatin (Orlistat)R
Authors: LOOKENE Aivar , SKOTTOVA Nina , OLIVECRONA Gunilla 
Published: March 2005

Lipoprotein lipase (LPL) was rapidly inactivated by low concentrations of the active‐site inhibitor tetrahydrolipstatin (THL). The presence of amphiphils (e.g. long‐chain fatty acids) or of lipid/water interfaces (lipid emulsions) was required for inhibition to occur. Apolipoprotein CII increased the maximal inactivation rate constant by 1.8‐fold in the presence of an emulsion of long‐chain triacylglycerols, but had no effect in the presence of an emulsion of tributyrylglycerol. The fully inhibited enzyme had a ratio of THL/LPL of nearly 2, indicating that both subunits of the LPL homo‐dimer bound THL. The THL‐LPL complex was stable below pH 7.5. At higher pH reactivation occurred indicating that THL was slowly turned over by the enzyme. The apparent reactivation rate constant was increased about threefold by the presence of lipid/water interfaces.Sucrose density gradient centrifugation revealed that THL induces tetramerisation of LPL. This aggregation was reversible on reactivation of the inhibited enzyme. Binding to heparin was not affected by THL. In contrast, binding to lipid droplets and to lipoproteins was increased, indicating exposure of hydrophobic regions in the inhibited LPL. It is suggested that THL induces local conformational changes in LPL, which may involve opening of the putative surface lid structure which covers the active‐site.
Identification of functional sites on bovine leukemia virus envelope glycoproteins using structural and immunological data
Authors: CALLEBAUT Isabelle , PORTETELLE Daniel , BURNY Arsène , MORNON Jean‐Paul 
Published: March 2005

Sequence analysis using the sensitive hydrophobic cluster analysis method shows that the bovine leukemia virus envelope glycoproteins conserve the general organization of the influenza hemagglutinin into a ‘stem’, containing the external part of the transmembrane glycoprotein and the N‐terminal and C‐terminal parts of the external glycoprotein, and a ‘head’, containing only external glycoprotein residues. However, our analysis suggests, for the first time, that the bovine leukemia virus envelope head will not adopt the typical ‘jelly‐roll’ fold of the influenza A hemagglutinin head, but most likely folds into another type of ‘Greek‐key’ structure corresponding to the overall topology of constant immunoglobulin domains. We constructed a three‐dimensional model for the bovine leukemia virus envelope head by homology modeling using the crystal structure of the human histocompatibility antigen HLA‐A2 α3 domain. Furthermore, we propose a general model for the oligomeric organization of this head, based on the hemagglutinin trimer. The proposed structural organization of bovine leukemia virus external glycoprotein is further supported by antipeptide and monoclonal antibody reactivities. Our modeling study suggests that the loops of the two neutralizing peptides located in the head are adjacent at the top of the domain and define a potential interaction site of the external glycoprotein with its cellular receptor. This site is topologically similar to the binding site of hemagglutinin with its cellular receptor, sialic acid. The other neutralizing peptides are located within a small domain linking the head to the stem.These data are of interest for defining other oncoviral glycoproteins heads and receptor‐binding sites.
Stability and structural analysis of α‐amylase from the antarctic psychrophile Alteromonas haloplanctis A23
Authors: FELLER Georges , PAYAN Françoise , THEYS Fabienne , QIAN Minxie , HASER Richard , GERDAY Charles 
Published: March 2005

The α‐amylase secreted by the antarctic bacterium Alteromonas haloplanctis displays 66% amino acid sequence similarity with porcine pancreatic α‐amylase. The psychrophilic α‐amylase is however characterized by a sevenfold higher kcat and kcat/Km values at 4°C and a lower conformational stability estimated as 10 kJ · mol−1 with respect to the porcine enzyme. It is proposed that both properties arise from an increase in molecular flexibility required to compensate for the reduction of reaction rates at low temperatures. This is supported by the fast denaturation rates induced by temperature, urea or guanidinium chloride and by the shift towards low temperatures of the apparent optimal temperature of activity.When compared with the known three‐dimensional structure of porcine pancreatic α‐amylase, homology modelling of the psychrophilic α‐amylase reveals several features which may be assumed to be responsible for a more flexible, heat‐labile conformation: the lack of several surface salt bridges in the (β/α)8 domain, the reduction of the number of weakly polar interactions involving an aromatic side chain, a lower hydrophobicity associated with the increased flexibility index of amino acids forming the hydrophobic clusters and by substitutions of proline for alanine residues in loops connecting secondary structures. The weaker affinity of the enzyme for Ca2+ (Kd= 44 nM) and for Cl− (Kd= 1.2 mM at 4°C) can result from single amino acid substitutions in the Ca2+‐binding and Cl−‐binding sites and can also affect the compactness of α‐amylase.
Processing of the precursors of the human thyroid‐stimulating hormone receptor in various eukaryotic cells (human thyrocytes, transfected L cells and baculovirus‐infected insect cells)
Authors: MISRAHI Micheline , GHINEA Nicolae , SAR Sokhavuth , SAUNIER Bertrand , JOLIVET André , LOOSFELT Hugues , CERUTTI Martine , DEVAUCHELLE Gérard , MILGROM Edwin 
Published: March 2005

The complementary DNA for human thyroid‐stimulating hormone (TSH) receptor encodes a single protein with a deduced molecular mass of 84.5 kDa. This protein is cleaved during its maturation in the human thyroid since the receptor protein has been shown to be composed of two subunits (α subunit of ∼ 53 kDa and β subunit of ∼ 38 kDa) held together by disulfide bridges [Loosfelt, H., Pichon, C., Jolivet, A., Misrahi, M., Caillou, B., Jamous, M., Vannier, B. & Milgrom, E. (1992) Proc. Natl Acad. Sci. USA 89, 3765–3769]. A similar processing occurs in an L cell line permanently expressing the human TSH receptor. The processing is however incomplete, resulting in a permanent accumulation of a 95‐kDa high‐mannose precursor which is present only in trace amounts in the thyroid. Pulse‐chase experiments show the successive appearance in the L cells of two precursors: initially the ∼ 95‐kDa high‐mannose glycoprotein followed by a ∼ 120‐kDa species containing mature oligosaccharides. This latter precursor is then processed into the α and β subunits. In primary cultures of human thyrocytes precursors of similar size are detected. Spodoptera frugiperda insect cells (Sf9 and Sf21) infected with a recombinant baculovirus encoding the human TSH receptor synthesize a monomeric protein of about 90 kDa soluble only in denaturing conditions. Comparison with the product of in vitro transcription‐translation experiments (∼ 80 kDa), suggests that it may be incompletely or improperly glycosylated. The TSH receptor expressed in these cells is unable to bind the hormone.Immunoelectron microscopy studies show that in human thyrocytes most of the receptor is present on the cell surface; in L cells the receptor is detected on the cell surface, as well as in the endoplasmic reticulum and in the Golgi apparatus (this intracellular pool of receptor molecules probably corresponding to the high‐mannose precursor); in insect cells nearly all the receptor molecules are trapped in the endoplasmic reticulum. These differences in receptor distribution are concordant with the differences observed for receptor processing.
Catabolite inactivation of heterologous fructose‐1,6‐bisphosphatases and fructose‐1,6‐bisphosphatase‐β‐galactosidase fusion proteins inSaccharomyces cerevisiae
Authors: GAMO Francisco‐Javier , NAVAS M. Angeles , BLAZQUEZ Miguel A. , GANCEDO Carlos , GANCEDO Juana M. 
Published: March 2005

Fructose‐1,6‐bisphosphatase (FruP2ase) fromSaccharomyces cerevisiaeis rapidly inactivated upon addition of glucose to a culture growing on non‐sugar carbon sources. Under the same conditions the FruP2ases fromSchizosaccharomyces pombeorEscherichia coliexpressed inS. cerevisiaewere not affected. A chimaeric protein confaining the first 178 amino acids from the N‐terminal half ofS. cerevisiaeFruP2ase fused toE. coliβ‐galactosidase was susceptible to catabolite inactivation. Elimination of a putative destruction box, RAELVNLVG…KK….K., beginning at amino acid 60 did not prevent catabolite inactivation. Similarly a change of the vacuole‐targeting sequence QKKLD, amino acids 80–84, to QKNSD did not affect significantly the course of inactivation of β‐galactosidase. A fusion protein carrying only the first 138 amino acids from FruP2ase was inactivated at a higher rate than the one carrying the first 178, suggesting the existence of a protective region between amino acids 138 and 178. A fusion protein carrying the first 81 amino acids from FruP2ase was inactivated by glucose at a similar rate to the one carrying the 178 amino acids, but one with only the first 18 amino acids was resistant to catabolite inactivation.Inactivation of FruP2ase in mutantsubr1that lack a protein required for ubiquitin‐dependent proteolysis, orpralthat lack vacuolar protease A, proceeded as in a wild type. Our results suggest that at least two domains of FruP2ase may mark β‐galactosidase for catabolite inactivation and that FruP2ase can be inactivated by a mechanism independent of transfer to the vacuole.
NMR studies of recombinant Coprinus peroxidase and three site‐directed mutants
Authors: VEITCH Nigel C. , TAMS Jeppe W. , VIND Jesper , DALBØGE Henrik , WELINDER Karen G. 
Published: March 2005

Proton nuclear magnetic resonance spectroscopy has been used to characterise and compare wild‐type fungal and recombinant Coprinus cinereus peroxidase (CIP) and three mutants in which Gly156 and/or Asn157 was replaced by Phe. Analysis of one‐ and two‐dimensional NMR spectra of recombinant CIP was undertaken for comparison with the fungal enzyme and in order to establish a meaningful basis for solution studies of CIP mutants. Proton resonance assignments of haem and haem‐linked residues obtained for the cyanide‐ligated form of recombinant CIP revealed a high degree of spectral similarity with those of lignin and manganese‐dependent peroxidases and extend previously reported NMR data for fungal CIP.The three mutants examined by NMR spectroscopy comprised site‐specific substitutions made to a region of the structure believed to form part of the peroxidase haem group access channel for substrate and ligand molecules. Proton resonances of the aromatic side‐chains of Phe156 and Phe157 were found to have similar spectral characteristics to those of two phenylalanine residues known to be involved in the binding of aromatic donor molecules to the plant peroxidase, horseradish peroxidase isoenzyme C. The results are discussed in the context of complementary reactivity studies on the mutants in order to develop a more detailed understanding of aromatic donor molecule binding to fungal and plant peroxidases.
Structural changes of high‐density‐lipoprotein apolipoproteins following incubation with human polymorphonuclear cells
Authors: COGNY Anne , PAUL J. Louis , ATGER Véronique , SONI Théophile , MOATTI Nicole 
Published: March 2005

Based on the analogy in mechanisms and events between the pathogenesis of atherosclerosis and the inflammatory reaction, we investigated the impact of human polymorphonuclear leukocyte (PMN) degranulation and oxidative process on high‐density‐lipoprotein (HDL) structure.HDL were incubated (37°C) with PMN at a physiological ratio (370 nmol cholesterol‐HDL/ml with 2X106 PMN/ml) for 15, 30 and 60 min with or without stimulating agent. PMN activation was assessed by measurement of superoxide anion generation and elastase production, which both reached peak concentration at 15 min.HDL apolipoproteins (apo) analysed by immunoblotting after SDS/PAGE and electrofocusing evidenced the following modifications: (a) a slow hydrolysis of apo AII and apo Cs; (b) a rapid hydrolysis of apo E; (c) a change in apo AI isoform distribution with an increase in the most acidic isoform (AI‐2) at the expense of a less acidic form (AI‐1); (d) a shift of the major apo AII isoform into two more basic forms.In contrast, no quantifiable lipid modification nor lipid oxidation, assessed by thiobarbituric‐acid‐reactive substances (TBARS) were noted. Despite a lack of variation of TBARS, a decrease in HDL vitamin E content by 80% was observed. Since this decrease was prevented by addition of superoxide dismutase in the medium, we concluded the occurence of an oxidative process affecting HDL.Experiments with proteolytic inhibitors showed that elastase caused the proteolytic cleavage of apolipoprotein E, AII and Cs. In contrast, apo AI modification might involve both oxidative and proteolytic processes.
Metabolism of malate in bovine adrenocortical mitochondria studied by 13C‐NMR spectroscopy
Authors: PERRIN Anne , GOUT Elisabeth , CHAMBAZ Edmond M. , DEFAYE Geneviève 
Published: March 2005

13C‐NMR spectroscopy was used to study the metabolism of [13C]malate in bovine coupled adrenocortical mitochondria. The most apparent difference between the mitochondria from steroidogenic tissues and mitochondria from other tissues is the presence, in addition to the normal respiratory chain, of a second electron‐transport system responsible for steroid hydroxylation.[13C]malate was synthesized from [13C]succinate by isolated adrenocortical mitochondria. The basic functional suspension consisted of oxygenated mitochondria to which were added ADP, inorganic phosphate (Pi) and [13C]malate, both in the absence or presence of the steroid substrate, deoxycorticosterone. These mitochondria synthesized [13C]citrate and [13C]pyruvate from [13C]malate. The 13C labeling of these two metabolites demonstrated an important role of the malic enzyme and the kinetics depended on the presence of the steroid substrate; the citric acid cycle was stopped during the hydroxylation pathway. The addition of cyanide, a strong inhibitor of the respiratory chain, confirmed an increased malic enzyme activity when hydroxylation occurred, since pyruvate was trapped by formation of a cyanohydrin.The relative enzymic activities of malic enzyme and isocitrate dehydrogenase were compared, both in the absence or presence of the steroid substrate, by supplementing the basic suspension with unlabeled exogenous metabolites, such as pyruvate or oxaloacetate.
NMR spectroscopy of exchangeable protons of glucoamylase and of complexes with inhibitors in the 9–15‐ppm range
Authors: FIRSOV Leonid M. , NEUSTROEV Kirill N. , ALESHIN Alexander E. , METZLER Carol M. , METZLER David E. , SCOTT Robert D. , STOFFER Bjarne , CHRISTENSEN Trine , SVENSSON Birte 
Published: March 2005

1H‐NMR spectra have been recorded for glucoamylases I and II from Aspergillus awamori var. X100 and from A. niger in the 9–15‐ppm reion. At least 17 distinct peaks, many of them arising from single protons, are observed. These are designated A–Q, A being the furthest downfield. At least 9 of these are lost rapidly by exchange when the enzyme is placed in D2O. Peaks A, B, E and H undergo distinct shifts with pH change in the pH region 3–7. Several others undergo smaller shifts. Small differences are also seen between the enzymes from the two different sources. Binding of the pseudotetrasaccharide inhibitor acarbose leads to a 0.50‐ppm downfield shift of peak B, other smaller changes, and retention of two additional protons in D2O. δ‐d‐Gluconolactone induces shifts in peaks E, H, and L. The slow substrate maltitol causes peak A to broaden and shift, peaks J and K to shift and a new or greatly shifted resonance to appear at 15.4 ppm. It disappears as the maltitol is hydrolyzed.Treatment with iodoacetamide or diethyl pyrocarbonate leads to disappearance of peak D at 12.3 ppm. When this peak was irradiated strong nuclear Overhauser effects (NOE) were observed at 8.01 ppm and 7.22 ppm, positions expected for the Cε1 and Cδ2 protons of an uncharged imidazole ring. We identify D as arising from the Nε2 proton of His254 which is uncharged except at the lowest pH values. Other NOE and two‐dimensional NOE spectra have provided additional information.Three mutant forms of the A. niger enzyme, in which tryptophan residues have been replaced by phenylalanine, have been examined. Because of shifts induced by changes in ring current and other environmental effects it is hard to make a direct identification of the resonances from the replaced indole NH protons. However, on the basis of a distinct NOE between peaks E and H we have identified these resonances as arising from the indole NH protons of Trp52 and Trp120. Other possible assignments are considered.The NMR spectra of the glucoamylases I, which have a starch binding domain of about 104 residues at the carboxyl terminus, show four sharp resonances in the 9.7–10.6‐ppm range that are not present in the glucoamylases II, which lack this domain. These resonances no doubt represent the four indole NH ring protons from Trp543, Trp562, Trp590 and Trp615. Three of these are very sharp suggesting a high mobility of this domain.
The thermostability of natural variants of bacterial plasminogen‐activator staphylokinase
Authors: GASE Ariane , BIRCH‐HIRSCHFELD Eckhard , GÜHRS Karl‐Heinz , HARTMANN Manfred , VETTERMAN Stefan , DAMASCHUN Gregor , DAMASCHUN Hilde , GAST Klaus , MISSELWITZ Rolf , ZIRWER Dietrich , COLLEN Désiré , SCHLOTT Bernhard 
Published: March 2005

Three natural variants (wild‐type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase) of the bacterial plasminogen‐activator staphylokinase, a 136‐amino‐acid protein secreted by certain Staphylococcus aureus strains, have been characterized. These variants differ at amino acid positions 34, 36 and 43 only, and have a very similar plasminogen‐activating capacity and conformation in solution, as revealed by fluorescence spectroscopy, dynamic light scattering and circular dichroism. However, the thermostability of these variants is significantly different.At 70°C and 0.5 mg protein/ml, irreversible inactivation occurred with apparent half‐life (t1/2) values 0.54 ± 0.13, 0.81 ± 0.20 and 3.7 ± 0.7 h (mean ± SEM) for wild‐type staphylokinase, [R36G, R43H]staphylokinase, and [G34S, R36G, R43H]staphylokinase, respectively, with corresponding values at 0.08 mg/ml of 5.3 ± 1.4 h and 11 ± 2.0 h for wild‐type staphylokinase and [R36G, R43H]staphylokinase, respectively. Dynamic light‐scattering measurements indicated that inactivation was associated with protein aggregation, which precluded accurate determination of transition temperatures and enthalpies of unfolding. 0.08–0.34 mg/ml [G34S, R36G, R43H]staphylokinase, however, did not aggregate at 70°C but underwent unfolding as revealed by a 20% increase in the Stokes’ radius and a 30% decrease in circular dichroism. The unfolding was reversible upon cooling and was associated with full recovery of functional activity.Thus, these natural variants of staphylokinase have a different sensitivity to thermal inactivation, that is mediated by reversible unfolding of the protein and concentration‐dependent irreversible aggregation. [G34S, R36G, R43H]staphylokinase, the most resistant natural variant, has a stability approaching the minimal requirements for pasteurization, which would facilitate its development for clinical use.