bluuebunny/crossref_metadata_2025_split huggingface dataset:
Conformational analysis of elcatonin in solution
Authors: OGAWA Kiyoshi , NISHIMURA Shigenori , DOI Masamitsu , KYOGOKU Yoshimasa , HAYASHI Mitsuo , KOBAYASHI Yuji 
Published: March 2005

The conformational characteristics of elcatonin, an analogue of eel calcitonin having a disulfide bond Cys1‐Cys7 replaced by an ethylene linkage between residues 1 and 7, have been analyzed in aqueous trifluoroethanol solutions. Circular dichroic spectra of elcatonin and eel calcitonin itself reveal the presence of α‐helices at trifluoroethanol concentrations above 15%. The spectral changes caused by the trifluoroethanol content of the solutions are interesting. An isosbetic point is detected for eel calcitonin indicating that a conformational transition occurs between two states, namely α‐helical and random coil states. On the other hand, the CD curves of elcatonin at less than 15% trifluoroethanol deviate from the isosbetic point while those at higher concentration are similar to those of eCT. This can probably be attributed to the third element of the ordered structure of elcatonin which is formed in 15% trifluoroethanol.The solution conformation of elcatonin in a mixture of 60% water and 40% trifluoroethanol has been determined by the combined use of 1H‐NMR spectroscopy and distance geometry calculations. The conformation is characterized by an amphiphilic α‐helix between Thr6 and Thr21, which extends into the constrained cyclic portion of the molecule to Thr6. The third structural element of elcatonin found in the CD analysis is detected by some turn structures in the region between residue 1 and Ser5 in the calculated structure.
Cloning and expression of eel natriuretic‐peptide receptor B and comparison with its mammalian counterparts
Authors: KATAFUCHI Takeshi , TAKASHIMA Akira , KASHIWAGI Masahide , HAGIWARA Hiromi , TAKEI Yoshio , HIROSE Shigehisa 
Published: March 2005

A comparative study of the natriuretic‐peptide receptor NPR‐B was performed by cloning and expressing, in COS‐1 cells, the NPR‐B receptor subtype from the eel gill which exhibited a strong C‐type‐natriuretic‐peptide(CNP)‐induced guanylate cyclase activity. Like other mammalian NPR‐B receptors, the eel NPR‐B receptor consisted of a ligand‐binding extracellular domain, a hydrophobic transmembrane domain, a kinase‐like domain and a guanylate cyclase domain. Sequence comparison among the eel and mammalian receptors revealed a relatively low similarity (∼44%) in the extracellular domain compared to a very high similarity (∼ 84 %) in the cytoplasmic regulatory and catalytic domains. This low similarity allowed identification of the amino acid residues or candidate regions important for the ligand‐binding activity. RNase protection analysis of the eel NPR‐B mRNA demonstrated that the message was predominantly expressed in the liver and atrium as well as in the gill with moderate‐to‐small amounts in the brain, ventricle, esophageal sphincter, stomach, posterior intestine and kidney.The high NPR‐B mRNA levels in the liver, atrium and gill were found to decrease markedly when eels were transferred from fresh water to seawater and kept there for 2 weeks. Since similar changes are known to occur in the ligand CNP levels when eels are facing osmotic challenges, the CNP/NPR‐B system appears to play an important role in their successful adaptation to salinity changes.
Purification of a sixth ferredoxin from Rhodobacter capsulatus
Authors: NAUD Isabelle , VINÇON Mathilde , GARIN Jérôme , GAILLARD Jacques , FOREST Eric , JOUANNEAU Yves 
Published: March 2005

A new ferredoxin has been purified from the photosynthetic bacterium Rhodobacter capsulatus. It is the sixth ferredoxin to be isolated from this bacterium and it was called FdVI.Its primary structure was established based on amino acid sequence analysis of the protein and of peptides derived from it. It is composed of 106 residues including five cysteines. The calculated mass of the polypeptide is 11402.6 Da which matches the experimental value determined by electrospray mass spectrometry. Amino acid sequence comparison revealed that ferredoxin VI (FdVI) is strikingly similar to a ferredoxin from Caulobacter crescentus and to the putidaredoxin from Pseudomonas putida.FdVI exhibited an ultraviolet‐visible absorption spectrum typical for a [2Fe‐2S] ferredoxin. EPR spectroscopy of the reduced protein showed a nearly axial signal similar to that of mitochondrial and P. putida ferredoxins.FdVI is biosynthesized in cells growing anaerobically under either nitrogen‐sufficient or nitrogen‐deficient conditions. Although the function of FdVI is unknown, its structural resemblance to [2Fe‐2S] ferredoxins known to transfer electrons to oxygenases such as P‐450 cytochromes, suggests that FdVI may have a similar role in R. capsulatus.
Primary structure of maize chloroplast adenylate kinase
Authors: SCHILTZ Emile , BURGER Sabine , GRAFMÜLLER Rita , DEPPERT Wolfgang R. , HAEHNEL Wolfgang , WAGNER Edgar 
Published: March 2005

This paper describes the sequence of adenylate kinase (Mg‐ATP + AMP ⇄ Mg‐ADP + ADP) from maize chloroplasts. This light‐inducible enzyme is important for efficient CO2fixation in the C4cycle, by removing and recycling AMP produced in the reversible pyruvate phosphate dikinase reaction.The complete sequence was determined by analyzing peptides from cleavages with trypsin, Asp‐N protease and CNBr and subcleavage of a major CNBr peptide with chymotrypsin. N‐terminal Edman degradation and carboxypeptidase digestion established the terminal residues. Electrospray mass spectrometry confirmed the final sequence of 222 residues (Mr= 24867) including one cysteine and one tryptophan.The sequence shows this enzyme to be a long‐variant‐type adenylate kinase, the nearest relatives being adenylate kinases from Enterobacteriaceae. Alignment of the sequence with the adenylate kinase fromEscherichia colireveals 44% identical residues. Since theE. colistructure has been published recently at 0.19‐nm resolution with the inhibitor adenosine(5′)pentaphospho(5′)adenosine (Ap5A) [Müller, C. W. & Schulz, G. E. (1992)J. Mol. Biol. 224, 159–177], catalytically essential residues could be compared and were found to be mostly conserved. Surprisingly, in the nucleotide‐binding Gly‐rich loop Gly‐Xaa‐Pro‐Gly‐Xaa‐Gly‐Lys the middle Gly is replaced by Ala. This is, however, compensated by an Ile→Val exchange in the nearest spatial neighborhood. A Thr→Ala exchange explains the unusual tolerance of the enzyme for pyrimidine nucleotides in the acceptor site.
Analysis of heparan‐sulphate chains and oligosaccharides from proliferating and quiescent fibroblasts
Authors: SCHMIDTCHEN Artur , FRANSSON Lars‐Åke 
Published: March 2005

Human skin fibroblasts in different growth states were incubated with [3H]glucosamine and/or Na235SO4 and extracted with Triton X‐100 for various periods of time. Free heparan‐sulphate oligosaccharides and protein‐bound heparan‐sulphate chains were separated by chromatography on octyl‐Sepharose and analyzed. A pool of endogenously produced oligosaccharides, present in the cultured cells and isolated after brief extraction, contained fragments of uniform size (approximately 7–10 kDa corresponding to approximately 14–20 disaccharides). Analysis by heparinase I and heparinase III degradations followed by electrophoretic separation (oligosaccharide mapping) showed that the oligosaccharides were rich in glucuronic acid but had a few sulphated iduronic acid residues at the periphery of each molecule. These results indicated that endoheparanase cleavage points were located close to linkages between N‐sulphated glucosamine and sulphated iduronic acid, generating fragments that comprise a major portion of the unmodified segments and a minor portion of the highly modified segments.Prolonged extraction (24–48 h) of cells with Triton X‐100 at 4°C in the presence of proteinase inhibitors resulted in further degradation. There was an increase in the amount of heparan‐sulphate oligosaccharides and a concomitant decrease in the amount of protein‐bound heparan‐sulphate chains present in the same extract. The heparan‐sulphate oligosaccharides obtained after prolonged extraction were more heterogeneous in size comprising, in addition to the major species of approximately 7–10 kDa, intermediate and larger fragments of approximately 17 kDa and 30–40 kDa. This observation suggests that endoheparanase acted at periodically appearing, specific regions in the intact heparan‐sulphate chain. Furthermore, the enzyme and substrate should remain closely associated during cold Triton X‐100 extraction.To determine if the endogenously produced heparan‐sulphate oligosaccharides were derived from a particular heparan‐sulphate species degraded during the growth phase, proteoglycan‐derived heparan‐sulphate chains obtained from proliferating or quiescent fibroblasts were also examined. These chains showed similar oligosaccharide maps, except for a small increase in the amount of glucuronic acid as cell growth was arrested. Hence, an endoheparanase with restricted specificity may generate slightly different oligosaccharides in the various growth states.
Hemoglobin function under extreme life conditions
Authors: CLEMENTI Maria E. , CONDÒ Saverio G. , CASTAGNOLA Massimo , GIARDINA Bruno 
Published: March 2005

Considering the variety of species that depend on hemoglobin for oxygen transport, these molecules must execute their primary function under extreme environmental conditions. Hence, a thermodynamic analysis of oxygen binding with hemoglobins from different species reveals a series of adaptive mechanisms which are based on the thermodynamic connection between the binding of heterotropic effectors and the reaction with oxygen.The examples reported, from fishes to human fetus, illustrate how evolution can alter the structural basis of the heterotropic interactions to optimize the oxygenation‐deoxygenation cycle in dependence of the physiological needs of the particular organisms. Moreover they show that a thermodynamic analysis of the reaction with oxygen overcomes the meaning of a detailed structural and functional characterization going deeper into the physiology of the specific organism.
Lipid modulation of glycolipid receptor function
Authors: BOYD Beth , MAGNUSSON Göran , ZHIUYAN Zhang , LINGWOOD Clifford A. 
Published: March 2005

Verotoxins bind to glycosphingolipids containing terminal Gal(α1‐4)Gal residues. Globotriaosylceramide is the most effective receptor for verotoxin‐1 in vitro and is the functional plasma‐membrane receptor which mediates cytopathology for most sensitive cells. Binding of verotoxin‐1 to a series of galabiose‐containing or globotriaose‐containing synthetic glycolipids with monoalkylsulfides and bisalkylsulfides or sulfones as the lipid moiety, have been studied for toxin binding by TLC overlay and in solid phase in the presence of auxiliary lipids. The results demonstrate that for an identical carbohydrate, binding is dramatically altered according to the nature of the lipid moiety. The close proximity of the galabiose sequence and the hydrophobic species also compromised recognition.The lipid environment is also a major determinant of receptor function, since species that were effective, even preferred toxin receptors as monitored by TLC overlay, were not necessarily recognized in the presence of auxiliary lipids. Certain glycolipids, which were not recognized by TLC overlay, were nevertheless found to be effective receptors in an auxiliary lipid matrix.These results demonstrate the crucial role of the lipid moiety in verotoxin/glycolipid recognition and are discussed in relation to toxin pathogenesis and glycolipid receptor function.
High affinity of ergopeptides for cytochromes P450 3A
Authors: PEYRONNEAU Marie‐Anne , DELAFORGE Marcel , RIVIERE René , RENAUD Jean‐Paul , MANSUY Daniel 
Published: March 2005

The interaction between rat and human liver cytochromes P450 with a series of lysergic acid derivatives and ergopeptide alkaloids was studied by difference visible spectroscopy. Ergopeptides, like bromocriptine, ergocryptine and dihydroergotamine, strongly interacted with rat liver microsomes with the appearance of a difference spectrum which is characteristic of their binding to a protein site close to the heme. The intensity of this spectrum was clearly dependent on the amounts of P450s 3A in the microsomes and was at its maximum in dexamethasone‐treated rat microsomes. All the ergopeptides studied exhibited a high affinity for rat P450s 3A (Ks around 1 μM), although lysergic acid derivatives not bearing the tripeptide moiety failed to give significant interactions with these P450s. A cyclic azatripeptide exhibiting a structure very similar to that of the tripeptide moiety of ergopeptides also interacted with P450s 3A with appearance of an intense type I difference spectrum. Very similar results were observed with two allelic forms of human liver P450 3A4, P450 NF25 and P450 hPCN1, produced in yeast. In both cases all the ergopeptides studied showed high affinities for the P450s (Ks 0.6–2.2 μM) and an intense shift from the low‐spin to the high‐spin state upon substrate binding (60–100% spin shift). Lysergic acid derivatives not bearing the tripeptide group of ergopeptides also completely failed to interact with P450s 3A4.Liver microsomes from rats pretreated with dexamethasone, a specific inducer of P450 3A, were found to be particularly active for the hydroxylation of bromocriptine, which occurs at the level of its tripeptide moiety. Human liver microsomes as well as P450 NF25 and P450 hPCN1 also exhibited a high activity for bromocriptine hydroxylation at this level.These results show that ergopeptides exhibit a particularly high affinity for P450s of the 3A subfamily. The tripeptide moiety of ergopeptides is essential for their recognition by P450s 3A and binds at a site close to P450 heme, producing type‐I difference spectra. Accordingly, at least one of the studied ergopeptides, bromocriptine, is hydroxylated by P450s 3A at the proline ring of the cyclopeptide moiety. As cyclosporine is known to be a good substrate of P450s 3A, these results suggest that P450s 3A may be especially prone in a general manner to recognize and oxidize peptides or pseudopeptides.
Cloning and expression of three abrin A‐chains and their mutants derived by site‐specific mutagenesisin Escherichia coli
Authors: HUNG Chih‐Hung , LEE Ming‐Chih , CHEN Jeen‐Kuan , LIN Jung‐Yaw 
Published: March 2005

DNAs encoding of three abrin A‐chains were obtained from the cDNA library of Abrus precatorius by polymerase chain reaction and ligated into the expression vector, pGEX‐2T. The mature A‐chains of abrins a, b and d have been expressed in the cytoplasm of Escherichia coli, and the yield of the soluble recombinant proteins was 7 mg/l induced culture. Three recombinant abrin A‐chains were purified to be homogeneity and their N‐glycosidase ability to inhibit protein biosynthesis in a cell‐free system and to depurinate 28S rRNA in rat liver ribosomes was demonstrated in vitro. The recombinant abrin‐a A‐chain had the highest N‐glycosidase activity among three recombinant abrin A‐chains while the recombinant abrin‐b A‐chain, the least.Three mutants, glutamic‐acid‐to‐alanine replacement (E164A), arginine to leucine (R167L) or double mutation (E164A and R167L) were constructed and expressed. The protein‐biosynthesis‐inhibitory activity of mutant (E164A), mutant (R167L) and the double mutant was found to be 25‐fold, 625‐fold and 1250‐fold lower than that of wild type, respectively. The results indicated that Arg167 was essential for abrin toxin A‐chain catalysis.
The derivation of the formyl‐group oxygen of chlorophyll b in higher plants from molecular oxygen
Authors: PORRA Robert J. , SCHÄFER Wolfram , CMIEL Edmund , KATHEDER Ingrid , SCHEER Hugo 
Published: March 2005

The mechanism of formation of the formyl group of chlorophyll b has long been obscure but, in this paper, the origin of the 7‐formyl‐group oxygen of chlorophyll b in higher plants was determined by greening etiolated maize leaves, excised from dark‐grown plants, by illumination under white light in the presence of either H218O or 18O2 and examining the newly synthesized chlorophylls by mass spectroscopy. To minimize the possible loss of 18O label from the 7‐formyl substituent by reversible formation of chlorophyll b‐71‐gem‐diol (hydrate) with unlabelled water in the cell, the formyl group was reduced to a hydroxymethyl group during extraction with methanol containing NaBH4: chlorophyll a remained unchanged during this rapid reductive extraction process.Mass spectra of chlorophyll a and [7‐hydroxymethyl]‐chlorophyll b extracted from leaves greened in the presence of either H218O or 18O2 revealed that 18O was incorporated only from molecular oxygen but into both chlorophylls: the mass spectra were consistent with molecular oxygen providing an oxygen atom not only for incorporation into the 7‐formyl group of chlorophyll b but also for the well‐documented incorporation into the 131‐oxo group of both chlorophylls a and b [see Walker, C. J., Mansfield, K. E., Smith, K. M. & Castelfranco, P. A. (1989) Biochem. J. 257, 599–602]. The incorporation of isotope led to as much as 77% enrichment of the 131‐oxo group of chlorophyll a: assuming identical incorporation into the 131 oxygen of chlorophyll b, then enrichment of the 7‐formyl oxygen was as much as 93%. Isotope dilution by re‐incorporation of photosynthetically produced oxygen from unlabelled water was negligible as shown by a greening experiment in the presence of 3‐(3,4‐dichlorophenyl)‐1,1‐dimethylurea.The high enrichment using 18O2, and the absence of labelling by H218O, unequivocally demonstrates that molecular oxygen is the sole precursor of the 7‐formyl oxygen of chlorophyll b in higher plants and strongly suggests a single pathway for the formation of the chlorophyll b formyl group involving the participation of an oxygenase‐type enzyme.
A Specific Association Between the Glyoxylic‐Acid‐Cycle Enzymes Isocitrate Lyase and Malate Synthase
Authors: Beeckmans Sonia , Khan A. Salam , Van Driessche Edilbert , Kanarek Louis 
Published: March 2005

There is accumulating evidence that metabolic pathways are organized in vivo as multienzyme clusters or metabolons. To assess interactions between consecutive enzymes of a pathway in vitro, it is usually essential to modify the physical properties of water around the enzymes, e.g. by immobilizing the latter onto a solid support. Such immobilized enzyme preparations can be embedded in agarose gels and used for affinity electrophoresis [Beeckmans, S., Van Driessche, E. & Kanarek, L. (1989) Eur. J. Biochem. 183, 449–454; Beeckmans, S., Van Driessche, E. & Kanarek, L. (1990) J. Cell. Biochem. 43, 297–306].In this study we use the aforementioned technique to investigate the association between two plant glyoxylic acid cycle enzymes, i.e. isocitrate lyase and malate synthase. A specific histochemical staining technique is described for both enzymes. Affinity electrophoresis using either isocitrate lyase or malate synthase as the immobilized enzyme clearly shows that associations are formed between both enzymes. Moreover, experiments with metabolically unrelated enzymes prove that the observed interaction is specific.
Interdomain Loops and Conformational Changes of Glutamate Synthase as Detected by Limited Proteolysis
Authors: Vanoni Maria A. , Mazzoni Alessandra , Fumagalli Prisca , Negri Armando , Zanetti Giuliana , Curti Bruno 
Published: March 2005

Azospirillum brasilense glutamate synthase, a complex iron‐sulfur flavoprotein, was subjected to limited proteolysis using trypsin and chymotrypsin, in the absence or presence of its substrates or their analogs. Time‐dependent degradation of glutamate synthase α and β subunits, to yield several fragments of different stability, was observed, the a subunit being more sensitive than the β to proteolytic attack. The main sites of proteolytic cleavage were determined by densitometric analysis of the electrophoretic patterns obtained under denaturing conditions and by N‐terminal sequencing of the major proteolytic products. These analyses showed that most of the peptide bonds sensitive to the proteases are clustered in two regions of the α subunit, outside the proposed substrate and cofactor binding regions of glutamate synthase [Pelanda, R., Vanoni, M. A., Perego, M., Piubelli, L., Galizzi, A., Curti, B. & Zanetti, G. (1993) J. Biol. Chem. 268, 3099–3106]. Therefore, these protease‐sensitive sites can be identified as flexible loops, exposed to solvent, connecting adjacent domains of the protein. The presence of the enzyme substrates or their analogs caused significant changes in the proteolytic patterns. NADP+ protected the C‐terminal region of glutamate synthase β subunit from tryptic cleavage, supporting the proposal that it contains the pyridinenucleotide‐binding site. Furthermore, NADP+, and to a lesser extent the glutamine analog l‐methionine sulfone, which binds presumably to the N‐terminal region of the a subunit, altered the sensitivity to proteolysis of the sites of the a subunit proposed to be part of links between domains of glutamate synthase. These results show that long‐range conformational changes of glutamate synthase occur on binding of its substrates.The study of several NADPH‐dependent diaphorase activities of glutamate synthase was also undertaken in order to test if proteolytic fragments of the enzyme retained their ability to transfer electrons from NADPH to synthetic electron acceptors. Although proteolysis yielded partial loss of all enzyme NADPH‐dependent reactions, the kinetic analysis showed that the rates of reduction of iodonitrotetrazolium, ferricyanide and dichlorophenolindophenol were at least twofold faster than the rate of the physiological glutamate synthase reaction. These results indicate that enzyme reduction and intramolecular electron transfer are not rate limiting during catalysis of the physiological glutamate synthase reaction.
Transcription of Immune Genes in the Giant Silkmoth, Hyalophora Cecropia, is Augmented by H2O2 and Diminished by Thiol Reagents
Authors: Sun Shao‐Cong , Faye Ingrid 
Published: March 2005

Insects have an effective humoral immune system, residing in immune proteins that are synthesized largely in the fatbody. κB‐like motifs upstream of the immune protein genes bind the Cecropia immunore‐sponsive factor (CIF), and confer high levels of inducible expression.We have extended our studies and sought evidence that oxygen‐derived active species might modulate the expression of the immune protein genes and the activation of CIF after the administration of different inducers. Mitogens, like arachidonic acid, phorbol esters, phytohemagglutinin, bacteria, and components of microbial cell envelopes stimulate expression of the attacin genes, both in vivo and in vitro. A general stimulant of oxidative stress, H2O2, stimulated expression of these genes and the weak immune response to wounding was greatly augmented by administration of H2O2. These responses were largely or entirely inhibited by dithiothreitol and by N‐acetylcysteine. Nonspecific responses were excluded since immune genes failed to respond to albumin or starch, and the expression of a non‐immunoresponsive gene was not affected. Electrophoretic mobility‐shift assays showed that H2O2 and bacteria, when administered in vitro, could activate CIF in fatbody cells and that dithiothreitol and N‐acetylcysteine prevented this process. Our data suggest that the induction of the immune protein genes is mediated through the activation of CIF, contingent upon thiol oxidation induced by oxidative stress.