bluuebunny/crossref_metadata_2025_split huggingface dataset:
Associations Between Erythrocyte Band 3 Protein and Aldolase in Detergent Solution
Authors: Huber Elfriede , Bäumert Hans Georg , Spatz‐kümbel Gerhard , Schubert Dieter 
Published: July 2004

The cytoplasmic domain of band 3, the predominant polypeptide of the erythrocyte membrane, represents a binding site for certain glycolytic enzymes. We have studied the association between human band 3 protein and aldolase, in order to clarify the role of the different band 3 oligomers as ligand binding sites. The experiments were performed on mixtures of solubilized band 3 and aldolase in solutions of a nonionic detergent, nonaethyleneglycol lauryl ether. The main technique applied was sedimentation equilibrium analysis in an analytical ultracentrifuge. In addition, nonequilibrium centrifugation techniques were used. To facilitate the evaluations, the aldolase was labelled with a dye. The following results were obtained. (1) With unmodified band 3, aldolase is bound exclusively or at least predominantly to the band 3 tetramer (but not to monomers or dimers). (2) The band 3 tetramer can bind up to four aldolase tetramers. (3) The band 3 tetramer/aldolase complex is unstable on the time scale of the techniques used. (4) Stable band 3 dimers (stabilized either covalently or noncovalently) can also associate with aldolase and can bind up to two aldolase tetramers.The results described, together with those reported previously, point at a prominent role of the band 3 tetramer in ligand binding.
The Structure of the Keratan Sulphate Chains Attached to Fibromodulin Isolated from Articular Cartilage
Authors: Lauder Robert M. , Huckerby Thomas N. , Nieduszynski Ian A. 
Published: July 2004

Fibromodulin has been isolated from bovine and equine articular cartilage and the attached keratan sulphate chains subjected to digestion by keratanase II. The oligosaccharides generated have been reduced and subsequently isolated by strong anion‐exchange chromatography. Their structures have been determined by high‐field 1H‐NMR spectroscopy and high‐pH anion‐exchange chromatography.Both α(2‐6)‐ and α(2‐3)‐linked N‐acetylneuraminic acid have been found in the capping oligosaccharides, and, fucose which is α(1‐3)‐linked to N‐acetylglucosamine has been found as a branch in both repeat region and capping oligosaccharides. These data demonstrate that there are fundamental differences between the structures present in the N‐linked keratan sulphate chains attached to fibromodulin from articular cartilage and those from tracheal cartilage, which lack both α(2‐6)‐linked N‐acetylneuraminic acid and α(1‐3)‐linked fucose.It has been confirmed that the keratan sulphate chains are short, being only eight or nine disaccharides in length. Very significant differences in the levels of galactose sulphation have been identified at the non‐reducing end of the chain. The galactose residue adjacent to the non‐reducing cap is sulphated in only 1–3% of chains, compared with a sulphation level of over 40% closer to the reducing end. This highlights the difference between the chain termini and the repeat region in terms of structure and points to the potential for functional importance.The repeat region and capping fragments of the N‐linked keratan sulphates from bovine and equine articular cartilage fibromodulin have been found to have the following general structure: NeuAc‐(α2–3/6)Gal[6SO3−](β1–4)GlcNAc6SO−3(β1–3)Gal[6SO3−](β1‐4)‐[Fuc(α1‐3)]0–1GlcNAc6SO−3(β1‐3)Gal[6SO3−](β1–4))6–7GlcNAc6SO−3.
4‐Hydroxybenzoate Hydroxylase from Pseudomonas Sp. CBS3
Authors: Seibold Birgit , Matthes Martina , Eppink Michel H. M. , Lingens Franz , Van Berkel Willem J. H. , Müller Rudolf 
Published: July 2004

4‐Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was purified by five consecutive steps to apparent homogeneity. The enrichment was 50‐fold with a yield of about 20%. The enzyme is a homodimeric flavoprotein monooxygenase with each 44‐kDa polypeptide chain containing one FAD molecule as a rather weakly bound prosthetic group. In contrast to other 4‐hydroxybenzoate hydroxylases of known primary structure, the enzyme preferred NADH over NADPH as electron donor. The pH optimum for catalysis was pH 8.0 with a maximum turnover rate around 45°C. Chloride ions were inhibitory, and competitive with respect to NADH.4‐Hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 has a narrow substrate specificity. In addition to the transformation of 4‐hydroxybenzoate to 3,4‐dihydroxybenzoate, the enzyme converted 2‐fluoro‐4‐hydroxybenzoate, 2‐chloro‐4‐hydroxybenzoate, and 2,4‐dihydroxybenzoate. With all aromatic substrates, no uncoupling of hydroxylation was observed.The gene encoding 4‐hydroxybenzoate hydroxylase from Pseudomonas sp. CBS3 was cloned in Escherichia coli. Nucleotide sequence analysis revealed an open reading frame of 1182 bp that corresponded to a protein of 394 amino acid residues. Upstream of the pobA gene, a sequence resembling an E. coli promotor was identified, which led to constitutive expression of the cloned gene in E. coli TG1. The deduced amino acid sequence of Pseudomonas sp. CBS3 4‐hydroxybenzoate hydroxylase revealed 53% identity with that of the pobA enzyme from Pseudomonas fluorescens for which a three‐dimensional structure is known. The active‐site residues and the fingerprint sequences associated with FAD binding are strictly conserved. This and the conservation of secondary structures implies that the enzymes share a similar three‐dimensional fold. Based on an isolated region of sequence divergence and site‐directed mutagenesis data of 4‐hydroxybenzoate hydroxylase from P. fluorescens, it is proposed that helix H2 is involved in determining the coenzyme specificity.
In vivo Modulation of Annexins I, II and V Expression by Thyroxine and Methylthiouracil
Authors: El Btaouri Hassan , Claisse Didier , Bellon George , Antonicelli Frank , Haye Bernard 
Published: July 2004

Regulation of annexin concentration and localization were investigated in thyroid tissues of hypothyroid [methylthiouracil (MeSur) treatment], euthyroid (control) and hyperthyroid [thyroxine (T4) treatment] rats.A low level of circulating thyroid hormones induces a decrease of total thyroid calcium‐binding protein concentration when compared with the concentration in unstimulated animals. Conversely, concentrations of annexins I, II and V increase. The accumulation of these proteins in two subcellular compartments (cytosolic and particulate fractions) can be reversed by addition of thyroid hormones. The finding of a specific increase in annexins concentration in thyroid‐hormone‐deficient rats, with a general decrease of the total calcium‐binding protein content points to a very important role of these proteins in the cells. Furthermore, hyperthyroidism gives opposite results. To investigate the transduction pathway of annexins I‐, II‐ and V‐induced biosynthesis by thyroid hormones in thyroid glands, we used cultured pig thyroid cells as in vitro model system. In previous work [16], we have shown that annexin concentrations and localization are under TSH control via the adenylate cyclase pathway. In the presence of MeSur (in the culture medium), the protein‐binding iodine remains low, indicative of weak thyroid hormone synthesis (data not shown) and that the annexins content is unchanged.These results suggest that, in thyroid tissue, an indirect mechanism links thyroid hormones to annexin expressions via the TSH feed‐back loop, and excludes autocrine regulation.
The Emerging Three‐Dimensional Structure of a Receptor
Authors: Hucho Ferdinand , Tsetlin Victor I. , Machold Jan 
Published: July 2004

The nicotinic acetylcholine receptor is the neurotransmitter receptor with the most‐characterized protein structure. The amino acid sequences of its five subunits have been elucidated by cDNA cloning and sequencing. Its shape and dimensions (approximately 12.5 nm×8 nm) were deduced from electron‐microscopy studies. Its subunits are arranged around a five‐fold axis of pseudosymmetry in the order (clockwise) αHγαLδβ. Its two agonist/competitive‐antagonist‐binding sites have been localized by photola‐belling studies to a deep gorge between the subunits near the membrane surface. Its ion channel is formed by five membrane‐spanning (M2) helices that are contributed by the five subunits. This finding has been generalized as the Helix M2 model for the superfamily of ligand‐gated ion channels. The binding site for regulatory non‐competitive antagonists has been localized by photolabelling and site‐directed‐mutagenesis studies within this ion channel.Therefore a three‐dimensional image of the nicotinic acetylcholine receptor is emerging, the most prominent feature of which is an active site that combines the agonist/competitive‐antagonist‐binding sites, the regulatory site and the ion channel within a relatively narrow space close to and within the bilayer membrane.
Conformational Study of [Met5]Enkephalin‐Arg‐Phe in the Presence of Phosphatidylserine Vesicles
Authors: D'alagni Maria , Delfini Maurizio , Di Nola Alfredo , Eisenberg Moises , Paci Maurizio , Roda L. Giorgio , Veglia Gianluigi 
Published: July 2004

The interaction of [Met5]enkephalin‐Arg‐Phe with phosphatidylserine (PtdSer) was studied by circular dichroism (CD), two‐dimensional nuclear magnetic resonance spectroscopy, hybrid distance geometry simulated annealing (DG–SA) and molecular dynamics (MD) calculations.The very low solubility of [Met5]enkephalin‐Arg‐Phe and the instability of the solution containing PtdSer vesicles at low pH values did not allow us to observe the amide proton resonances in the usual two‐dimensional NMR work. NOESY cross‐peaks of protons of side chains from two‐dimensional NMR were converted into distances which were used as restraints for modelling with DG‐SA and MD. Our results indicate that, in aqueous solutions at pH 7.68 [Met5]enkephalin‐Arg‐Phe exists in the absence of PtdSer as a random distribution of conformers, whereas in the presence of PtdSer it adopts conformations containing a common orientation of the bonds of Cα2, Cα3, Cα4 and Cα5, although different orientations of the peptide planes are consistent with the results.Two of the reported conformers from MD simulations are characterized by the presence of a 2←4 γ and inverse γ turns centered on Gly3. A gradual decline of order was observed when moving from the central moiety of the peptide to both the N‐terminus and C‐terminus. Finally, the DG‐SA and MD calculations resulted in a structure such that the orientation of the Phe4 and Met5 side chains favours hydrophobic interactions with the apolar portion of the PtdSer vesicle to form a hydrophobic cluster. These data support the hypothesis of a role of lipids to modify the conformation of [Met5]enkephalin‐Arg‐Phe to permit the interactions with the receptor site.
Structural and Serological Characterisation of Two O‐Specific Polysaccharides of Acinetobacter
Authors: Vinogradov Evgeny V. , Pantophlet Ralph , Dijkshoorn Lenie , Brade Lore , Holst Otto , Brade Helmut 
Published: July 2004

Extraction of dry bacteria of Acinetobacter strain 34 (DNA group 2) or Acinetobucter strain 108 (DNA group 13) by phenol/water yielded a polymer that was identified by means of serological studies and fatty acid analysis as S‐form lipopolysaccharide. Degradation of the lipopolysaccharides of strains 34 and 108 in 1% acetic acid and 5% acetic acid, respectively, and gel‐permeation chromatography gave the respective O‐antigenic polysaccharides, the structures of which were determined, by compositional analysis and NMR spectroscopy of the polysaccharide, as
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for strain 34, and
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for strain 108, where d‐FUCp 3NBUOH represents 3‐[(R)‐3‐hydroxybutyramido]‐3,6‐dideoxy‐d‐galactose and d‐Galp ANAc represents 2‐acetamido‐2‐deoxy‐D ‐galacturonic acid.Both structures were specifically recognised in Western blots by polyclonal rabbit antisera and there was no cross‐reaction between these two structures.
Structural Characterisation of Human Recombinant Glycohormones Follitropin, Lutropin and Choriogonadotropin Expressed in Chinese Hamster Ovary Cells
Authors: Amoresano Angela , Siciliano Rosa , Orrù Stefania , Napoleoni Roberta , Altarocca Valter , De Luca Eva , Sirna Antonino , Pucci Piero 
Published: July 2004

The α and β chains from human recombinant gonadotropins follitropin, lutropin and choriogonadotropin expressed in CHO cells have been structurally characterised both at the protein and at the carbohydrate level by using advanced mass spectrometric procedures. The three α chains share the same amino acid sequence while they display different glycosylation patterns. The oligosaccharide structures detected belong to the complex‐type glycans with different degree of sialylation. Partial proteolytic processing occurred at the N‐terminus of the follitropin β chain and at the C‐terminus of the lutropin β chain. The N‐linked glycans from the three β chains were found to contain fucosylated and sialylated diantennary and triantennary complex‐type structures. The follitropin β chain showed the presence of N‐acetyllactos‐amine repeats on the antennae.The overall structure of the recombinant glycohormones corresponds to their natural counterparts with the exception that sulphated terminal glycosylation is missing.
Molecular Cloning and Characterization of a Putative Glutathione Reductase Gene, the PfGR2 Gene, from Plasmodium falciparum
Authors: Färber Petra M. , Becker Katja , Müller Sylke , Schirmer R. Heiner , Franklin Richard M. 
Published: July 2004

Recently, glutathione reductase (GR) has emerged as a promising target for antiparasitic drugs. The central role of GR in cellular antioxidant defence, the particular susceptibility of intracellular parasites like Plasmodium falciparum to oxidative stress, and successful inhibitor studies substantiate this approach. However, more information is required on the structural and functional characteristics of GR from malarial parasites and differences from the enzyme of host erythrocytes.We have identified a putative P. falciparum GR gene coding for a polypeptide (PfGR2) of 500 ami no acids that exhibits 40–45% sequence identity with GR enzymes from other species. 18 out of 19 residues contributing to glutathione binding are identical in the putative PfGR2 and human GR.According to Southern blot analysis, the PfGR2 gene is present as a single‐copy gene. It is expressed during the intraerythrocytic life cycle. Stage‐specific Northern blot analysis demonstrates that the PfGR2 gene is only weakly transcribed in ring, early trophozoite, and segmenter stages; major transcription occurs in the late trophozoite/early schizont stage. This is consistent with the high glutathione reductase activity found in early schizonts. Other data also suggest that PfGR2 corresponds to the enzyme isolated from parasitized erythrocytes. These criteria include the subunit molecular mass (56.2 kDa), the N‐terminal sequence (VYDLIVIGGGSGGMA), the presence of specific sequence motifs at ligand‐binding sites, and, as demonstrated by Western blotting, the occurrence of a unique chain segment in the core of the central domain.In view of these data, the function(s) of PfGR2 as well as PfGR1, the product of another GR‐like gene of P falciparum (Müller et al., 1995) should be carefully assessed.
Mutational Studies of the Amino Acid Residues in the Combining Site of Erythrina corallodendron Lectin
Authors: Adar Rivka , Sharon Nathan 
Published: July 2004

High‐resolution X‐ray crystallography of the complex of the Gal/GalNAc‐specific Erythrina corallodendron lectin with lactose identified the amino acid side chains that form contacts with the galactose moiety of the disaccharide. The contribution of these amino acids to the binding of different monosaccha‐rides and oligosaccharides by the lectin was examined by site‐directed mutagenesis.Replacement of Phe131, on which the galactose is stacked, by tyrosine, gave a mutant with the same hemagglutinating activity and carbohydrate specificity as the parent lectin, but replacement by alanine or valine resulted in loss of activity. Mutations of Ala88, Asp89, and Asn133 produced mutants that were also inactive whereas those of the other combining site residues, Tyr106, Ala218, and Gln219, were biologically active. None of the active mutants interacted with mannose or glucose. Thus, contiary to an earlier assumption, Ala218 is not responsible for the inability of E. corallodendron lectin to bind these sugars. Our findings also demonstrate that Gln219 is not involved in galactose binding in solution, even though this is implicated by the crystal data. Instead, our data suggest that Gln219 assists in the ligation of N ‐acetyllactosamine to the lectin, by interacting with the acetamide group of the disaccharide.Comparison with other legume lectins specific for mannose/glucose, galactose, N‐acetylgalactosam‐ine, l‐fucose or N ‐acetylglucosamine, shows that only three of the combining site residues of E. corallodendron lectin occupy invariant positions both in their primary and tertiary structures. These residues are an aspartic acid and an asparagine corresponding to positions 89 and 133, respectively, in E. corallodendron lectin, and an aromatic residue, either phenylalanine (as Phe131 in this lectin), tyrosine or tryptophan. We therefore postulate that these three residues are essential for ligand binding by all such lectins, irrespective of their specificity.
α‐Oxidation of 3‐Methyl‐Substituted Fatty Acids in Rat Liver
Authors: Croes Kathleen , Casteels Minne , De Hoffmann Edmond , Mannaerts Guy P. , Van Veldhoven Paul P. 
Published: July 2004

α‐Oxidation of 3‐methyl‐substituted fatty acids in rat liver was studied in intact and permeabilized rat hepatocytes, and in homogenates and subcellular fractions. The experiments revealed that the primary end product of α‐oxidation is formic acid, which is then converted to CO2, Rates of α‐oxidation identical to those observed in intact hepatocytes were obtained in the permeabilized hepatocytes and liver homogenates when ATP, Mg2+ and CoA, and Fe2+, 2‐oxoglutarate and ascorbate were added, suggesting that α‐oxidation involves a fatty acid activation reaction and a dioxygenase reaction. Subcellular fractionation by differential and density gradient centrifugation demonstrated that α‐oxidation is confined to peroxisomes, which produce formic acid that is converted to CO2, mainly in the cytosol. α‐Oxidation in broken cell systems went hand in hand with the formation of a 2‐hydroxy‐3‐methylacyl‐CoA ester. Formation of the metabolite was strictly dependent on the presence of the above‐mentioned cofactors, was confined to peroxisomes and was inhibited by fenoprofen and propyl gallate, inhibitors of α‐oxidation in intact cells, indicating that the 2‐hydroxyacyl‐CoA ester is a bona fide intermediate of α‐oxidation. Selective omission of cofactors from the reaction mixture and analysis of the incubation mixtures for 3‐methyl fatty acids, 3‐methyl fatty acyl‐CoAs and their respective 2‐hydroxy derivatives revealed that the activation reaction precedes the dioxygenase (hydroxylase) reaction.Our experiments demonstrate that α‐oxidation is a peroxisomal process that consists of at least three reactions: fatty acid activation, hydroxylation and the reaction(s) involved in the release of formic acid.
Structures of the Antigenic O‐Polysaccharides of Lipopolysaccharides Produced by Actinobacillus actinomycetemcomitans Serotypes a, c, d and e
Authors: Perry Malcolm B. , Maclean Leann M. , Brisson Jean‐Robert , Wilson Mark E. 
Published: July 2004

Actinobacillus actinomycetemcomitans is a gram‐negative capnophilic coccobacillus that has been implicated in the etiology of certain forms of early‐onset periodontitis as well as non‐oral infections, mostly bacterial endocarditis. Five distinct serotypes of A. actinomycetemcomitans have been described. Although the O‐polysaccharide (O‐PS) of lipopolysaccharide (LPS) has been shown to define the serologic specificity for this species. only the structure of the O‐PS of serotype b has been characterized. The focus of the current study was to define the structures of the O‐PS of A. actinomycetemcomitans serotypes a, c, d and e. Structure determination was accomplished through the use of methylation, periodate oxidation, and one‐dimensional and two‐dimensional NMR methods.The O‐PS of A. actinomycetemcomitans (OMZ‐542) serotype d had [α]D+15° and was composed of D‐glucose, D‐mannose and L‐rhamnose (L‐Rha) in a molar ratio of 1:2:1. Methylation, periodate oxidation, and two‐dimensional 1H‐NMR and 13C‐NMR studies showed that the antigenic O‐PS was a high‐molecular‐mass polymer composed of repeating tetrasaccharide units with the structure:
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The O‐PS of the LPS produced by A. actinomycetemcomitans (ATCC 29523) serotype a had [α]D+ 150° and was found to contain 6‐deoxy‐D‐talose (6dTalp) and O ‐acetyl (2:1) and was a high‐molecular‐mass polymer composed of O ‐acetyl–substituted repeating disaccharide units with the structure:
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The O‐PS of the LPS of A. actinomycetemcomitans (SUNY 67) serotype c had [α]D ‐170° and was composed of 6‐deoxy‐L‐talose and O ‐acetyl (2:1). Structural analysis showed that the O‐PS was a high‐molecular‐mass polymer of repeating disaccharide units with the structure:
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The O‐PS of the LPS of A. actinomycetemcomitans (OMZ 534) serotype e had [4aL]D+57° and was composed of 2‐acetamido‐2‐deoxy‐D‐glucose and L‐rhamnose (1:1) and by chemical and NMR analysis was found to be a polymer of repeating disaccharide units with the structure:
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Cytochrome c′ of Methylococcus Capsulatus Bath
Authors: Zahn James A. , Arciero David M. , Hooper Alan B. , Dispirito Alan A. 
Published: July 2004

Cytochrome c′ was isolated from the obligate methylotroph Methylococcus capsulatus Bath. The native and subunit molecular masses of the cytochrome were 34.9 kDa and 16.2 kDa, respectively, with an isoelectric pH of 7.0. The amino acid composition and N‐terminal amino acid sequence were consistent with identification of the protein as a cytochrome c′. The electron paramagnetic resonance spectrum of the monoheme cytochrome indicated the presence of a high spin, S= 5/2, heme center that is diagnostic of cytochromes c′. The optical absorption spectra of ferric or ferrous cytochrome c′ were also characteristic of cytochromes c′. The ferrocytochrome bound carbon monoxide and nitric oxide, but not isocyanide, cyanide, or azide. Changes in physical properties due to binding of CO or NO to some other c′‐type cytochromes have been interpreted as an indication of dimer dissociation. In the case of cytochrome c′ from M. capsulatus Bath, analytical ultracentrifugation of the ferricytochrome, the ferrocytochrome, and the ferrocytochrome–CO complex indicate that the changes induced by binding of CO are conformational and are not consistent with dimer dissociation.EPR spectra show that cytochrome c′ was reduced in the presence of hydroxylamine only when in a complex with cytochrome P‐460. The value of the midpoint potential, Em 7.0 was –205 mV for cytochrome c′ from M. capsulatus Bath, which is well below the range of values reported for other cytochromes c′. The values of midpoint potentials for cytochrome P‐460 (Em 7.0=–300 mV to –380 mV) and cytochrome c555 (Em 7.0=+175 mV to +195 mV) are less than and greater than, respectively, the value for cytochrome c′ and suggest the possibility that the latter may function as an electron shuttle between cytochrome P‐460 and cytochrome c555.