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1
المؤلفون: Bent O. Petersen, Ilkka M. Helander, Irina Sadovskaya, Evgeny Vinogradov, Jens Ø. Duus, Saïd Jabbouri
المصدر: European Journal of Biochemistry. 270:3036-3046
مصطلحات موضوعية: chemistry.chemical_classification, 0303 health sciences, Strain (chemistry), 010405 organic chemistry, Chemistry, Stereochemistry, Glycosidic bond, Nuclear magnetic resonance spectroscopy, Polysaccharide, 01 natural sciences, Biochemistry, 0104 chemical sciences, Lipid A, 03 medical and health sciences, Hydrolysis, Residue (chemistry), Monosaccharide, lipids (amino acids, peptides, and proteins), 030304 developmental biology
الوصف: The structures of the oligosaccharides obtained after acetic acid hydrolysis and alkaline deacylation of the rough-type lipopolysaccharide (LPS) from Pectinatus frisingensis strain VTT E-82164 were analysed using NMR spectroscopy, MS and chemical methods. The LPS contains two major structural variants, differing by a decasaccharide fragment, and some minor variants lacking the terminal glucose residue. The largest structure of the carbohydrate backbone of the LPS that could be deduced from experimental results consists of 25 monosaccharides (including the previously found Ara4NP residue in lipid A) arranged in a well-defined nonrepetitive structure: We presume that the shorter variant with R1 = H represents the core-lipid A part of the LPS, and the additional fragment is present instead of the O-specific polysaccharide. Structures of this type have not been previously described. Analysis of the deacylation products obtained from the LPS of the smooth strain, VTT E-79100T, showed that it contains a very similar core but with one different glycosidic linkage.
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2
المؤلفون: Rikke Egelund, Kees W. Rodenburg, Peter A. Andreasen, Signe Jensen, Tove Kirkegaard, Susanne L. Schousboe, Helle H. Petersen
المصدر: European Journal of Biochemistry. 263:577-586
مصطلحات موضوعية: Models, Molecular, Time Factors, Protein Conformation, Stereochemistry, Mutant, Temperature, Beta sheet, Substrate (chemistry), Serpin, Protein Engineering, Biochemistry, Recombinant Proteins, Serine, chemistry.chemical_compound, Protein structure, chemistry, Plasminogen activator inhibitor-1, Plasminogen Activator Inhibitor 1, Mutagenesis, Site-Directed, Humans, Amino Acids, Plasminogen activator
الوصف: The serpin (serine proteinase inhibitor) family is of general protein chemical interest because of its ability to undergo large conformational changes, in which the surface-exposed reactive centre loop (RCL) is inserted as strand 4 in the large central beta-sheet A. Loop insertion is an integral part of the inhibitory mechanism and also takes place at conversion of serpins to the latent state, occurring spontaneously only in plasminogen activator inhibitor-1 (PAI-1). We have investigated the importance of beta-strand 5A residues for the activity and latency transition of PAI-1. An approximately fourfold increase in the rate of latency transition resulted from His-substitution of Gln324 (position 334 in the alpha(1)-proteinase inhibitor template numbering), which interacts with the underlying alpha-helix B. The side chains of Gln321 and Lys325 (template residues 331 and 335, respectively) form hydrogen bonds to the peptide backbone of a loop connecting alpha-helix F and beta-strand 3A. While substitution with Ala of Glu321 had only minor effects on the properties of PAI-1, substitution with Ala of Lys325 led to stabilization of the inhibitory activity at incubation conditions leading to conversion of wild-type PAI-1 to a substrate form, and to an anomalous reaction towards a monoclonal antibody, which induced a delay in the latency transition of the mutant, but not wild-type PAI-1. We conclude that the anchoring of beta-strand 5A plays a crucial role in loop insertion. These findings provide new information about the mechanism of an important example of protein conformational changes.
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3
المؤلفون: Egon Persson, Lars Christian Petersen, Per-Ola Freskgård
المصدر: European journal of biochemistry. 261(1)
مصطلحات موضوعية: Protein Denaturation, Alkylation, Stereochemistry, Macromolecular Substances, Protein Conformation, medicine.medical_treatment, Factor VIIa, In Vitro Techniques, Biochemistry, Thromboplastin, Tissue factor, chemistry.chemical_compound, Zymogen, otorhinolaryngologic diseases, medicine, Humans, Denaturation (biochemistry), Binding site, Protease, Binding Sites, biology, Factor VII, Active site, Recombinant Proteins, Cross-Linking Reagents, chemistry, Chromogenic Compounds, biology.protein, Thermodynamics, Salt bridge, Protein Binding
الوصف: Activation of the zymogen factor VII yields an enzyme form, factor VIIa, with only modest activity. The thermal effect on this low activity of factor VIIa and its enhancement by the cofactor tissue factor was investigated. Factor VIIa activity measured with a chromogenic peptide substrate is characterized by an unusual temperature dependency which indicates that the activated protease exists in an equilibrium between a latent (enzymatically inactive) and an active conformation. As shown by calorimetry and activity measurements the thermal effects on factor VIIa are fully reversible below the denaturation temperature of 58.1 degrees C. A model for factor VIIa has been proposed [Higashi, S., Nishimura, H., Aita, K. & Iwanaga, S. (1994) J. Biol. Chem. 269, 18891-18898] in which the protease is supposed to exist primarily as a latent enzyme form because of the poor incorporation into the protease structure of the N-terminal Ile153 released by proteolytic cleavage during activation of factor VII. Binding of tissue factor to factor VIIa is assumed to shift the equilibrium towards an active conformation in which the N-terminal Ile153 forms a salt bridge with Asp343. We corroborate the validity of this model by: (a) chemical modification of factor VIIa; this suggests that the thermal effect on the equilibrium between the active and inactive conformation is reflected in the relative accessibility of the active site and the N-terminal Ile153; (b) measurements of factor VIIa binding to tissue factor indicating that complex formation is favoured by stabilization of the active conformation; and (c) activity measurements of a cross-linked factor VIIa-tissue factor complex; this showed that cross-linking stabilized the active conformation of factor VIIa and essentially prevented its thermally-induced transformation into the inactive state.
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4
المصدر: European journal of biochemistry. 251(1-2)
مصطلحات موضوعية: Stereochemistry, Streptokinase, Biochemistry, Fibrin, Serine, Enzyme activator, Cricetinae, medicine, Animals, Cysteine, Disulfides, Fibrinolysin, biology, Chemistry, Lysine, Serine Endopeptidases, Plasminogen, Kallikrein, Trypsin, Urokinase-Type Plasminogen Activator, Recombinant Proteins, Enzyme Activation, Tissue Plasminogen Activator, biology.protein, Electrophoresis, Polyacrylamide Gel, Plasminogen activator, medicine.drug
الوصف: Plasminogen contains a unique disulphide bond, Cys558-Cys566, responsible for the cyclic nature of the peptide sequence surrounding the activation site at Arg561-Val562. A recombinant [Ser558, Ser566]-Lys78-plasminogen variant was produced in which the two cysteine residues were replaced by serine residues. The variant was used to study the functional implications of removing the structural restrains imposed to the activation loop by this disulphide bond. Elimination of the Cys558-Cys566 bond attenuated activation by urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA), but resulted in an increased susceptibility to cleavage by trypsin and plasma kallikrein. Two opposite effects on the interaction of plasminogen with streptokinase were produced by modification of this bond; (a) attenuation of the rate at which the active complex with streptokinase was formed and (b) a 7.5-fold increase in plasminogen activation catalysed by this complex. Activation by tPA in the presence of fibrin, in contrast to activation in its absence, was not attenuated by elimination of this disulphide bond. However, the activation rate as a function of plasminogen concentration followed a different saturation curve, and the fibrin degradation pattern was changed. The results suggest that the Cys558-Cys566 disulphide bond is of importance for the specificity of plasminogen. This applies to its activation and also to its role in subsequent fibrin clot degradation.
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5
المؤلفون: Helmut Brade, Bent O. Petersen, Klaus Bock, Otto Holst, Evgeny Vinogradov
المصدر: European journal of biochemistry. 243(1-2)
مصطلحات موضوعية: chemistry.chemical_classification, Lipopolysaccharides, Magnetic Resonance Spectroscopy, biology, Lipopolysaccharide, Acinetobacter, Stereochemistry, Molecular Sequence Data, Oligosaccharides, Nuclear magnetic resonance spectroscopy, Carbohydrate, Oligosaccharide, biology.organism_classification, Mass spectrometry, Biochemistry, Mass Spectrometry, chemistry.chemical_compound, Pyranose, chemistry, Carbohydrate Sequence, Monosaccharide
الوصف: The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter strain ATCC 17905 was studied. After deacylation of the lipopolysaccharide, a mixture of two compounds (ratio approximately 2:1) was isolated by high-performance anion-exchange chromatography, the structures of which were determined by NMR spectroscopy and electrospray-mass spectrometry as [STRUCUTRE IN TEXT] [Sug, 3-deoxy-D-manno-2-octulopyranosonic acid (Kdo) in oligosaccharide 1 (major portion) and D-glycero-D-talo-2-octulopyranosonic acid (Ko) in oligosaccharide 2 (minor portion)]. All monosaccharide residues also possess the D-configuration and are present in the pyranose form.
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6
المؤلفون: Peter Højrup, Torben E. Petersen, Lone K. Rasmussen
المصدر: European journal of biochemistry. 203(3)
مصطلحات موضوعية: Iodoacetic acid, Formates, Stereochemistry, Dimer, Molecular Sequence Data, Peptide, Antiparallel (biochemistry), Biochemistry, Mass Spectrometry, chemistry.chemical_compound, Protein structure, Animals, Chymotrypsin, Trypsin, Amino Acid Sequence, Disulfides, Chromatography, High Pressure Liquid, chemistry.chemical_classification, Performic acid, Protein primary structure, Caseins, chemistry, Cattle, Peptides, Oxidation-Reduction, Cysteine
الوصف: Carboxymethylation of bovine skimmed milk with 14C-labelled iodoacetic acid followed by purification of the alpha s2-casein dimer showed that all four cysteine residues in the protein are engaged in disulfide linkages. Mass spectrometry and sequence analysis of cystine-containing tryptic peptides revealed the presence of two interchain disulfide bridges in the protein. Sequence analysis of disulfide-linked peptides resulting from an enzymatic cleavage between the bridges demonstrated that the individual chains in the dimers are either aligned in an antiparallel or a parallel orientation. The identity of some of the disulfide-linked peptides was further verified by performic acid oxidation followed by sequence analysis of the resulting peptides.
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7
المؤلفون: Hans Chr. Thøgersen, Torben E. Petersen, Karna Skorstengaard
المصدر: European Journal of Biochemistry. 140:235-243
مصطلحات موضوعية: Bridged-Ring Compounds, Chemical Phenomena, Stereochemistry, Biology, Biochemistry, Homology (biology), Species Specificity, Animals, Humans, Amino Acid Sequence, Disulfides, Threonine, Peptide sequence, chemistry.chemical_classification, Binding Sites, Chymotrypsin, Protein primary structure, Peptide Fragments, Fibronectins, Amino acid, Fibronectin, Chemistry, chemistry, biology.protein, Cattle, Collagen, Protein Binding, Binding domain
الوصف: The complete amino acid sequence of the collagen-binding domain of bovine plasma fibronectin has been determined. The fragment, generated by digestion of fibronectin with plasmin and chymotrypsin, contains 340 residues (260-599 of fibronectin) with threonine and tryptophan as the amino-terminal and carboxyl-terminal amino acids, respectively. 24 half-cystines and no cysteines are present in the sequence. Three glucosamine-based oligosaccharide groups are attached to Asn-399, Asn-497 and to Asn-511, respectively. Two of the three types (I and II) [Petersen et al. (1983) Proc. Natl Acad. Sci. USA 80, 137-141] of internal homology occur in the fragment, namely four of the at least twelve stretches of type I sequence homology, 'fingers', and two stretches of type II homology. The type I homology is present in two other plasmic fragments from fibronectin, while the type II homology has been found in the collagen-binding domain only.
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8
المؤلفون: Lars Chr. Petersen
المصدر: European journal of biochemistry. 85(2)
مصطلحات موضوعية: Carbon Monoxide, biology, Cytochrome, Cytochrome b6f complex, Stereochemistry, Chemistry, Cytochrome c peroxidase, Cytochrome c, Myocardium, Cytochrome c Group, Ascorbic Acid, Biochemistry, Aerobiosis, Electron Transport Complex IV, Kinetics, Cytochrome C1, Coenzyme Q – cytochrome c reductase, biology.protein, Cytochrome c oxidase, Animals, Cytochromes, Cattle, Anaerobiosis, Cytochrome aa3, Mathematics
الوصف: 1 The anaerobic-aerobic transition of the CO-inhibited ascorbate cytochrome c cytochrome aa3 system is investigated using purified oxidase. 2 In this system the presence of CO in the medium facilitates the measurements of the oxygen kinetics and at the same time allows a spectral resolution of cytochromes a and a3. Measurements of the oxygen consumption, and the steady-state reduction of the cytochromes c, a, and a3 as a function of the oxygen concentration are reported. 3 A double reciprocal plot of oxygen consumption rate versus oxygen concentration is nonlinear at low concentration of cytochrome c but becomes linear as the cytochrome concentration is increased. 4 The plot of oxygen consumption rate against [O2] × [a2+3· CO], where a2+3· CO is the CO complex of the oxidase measured at 590 nm, is a straight line through the origin. This suggests that the binding of CO to the oxidase does not depend on the redox state of cytochrome a but is described by a single dissociation constant and also that the reaction between reduced cytochrome a3 and O2 is a second-order reaction, k+1= 1.4 · 108 M−1 s−1. 5 The results indicate that transfer of electrons from cytochrome a to cytochrome a3 during turnover is an irreversible second-order reaction. But a similar simple relation seems not to be valid for the transfer of electrons from cytochrome c to a or from cytochrome c to a3. 6 The assumption of a quasi-equilibrium between cytochrome c and cytochrome a leads to the conclusion that the midpoint potential of cytochrome a changes with the redox state of cytochrome a3, probably as a consequence of allosteric interaction between the hemes.
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