bluuebunny/crossref_metadata_2025_split huggingface dataset:
Comparison of the carbohydrate‐binding specificities of seven N‐acetyl‐D‐galactosamine‐recognizing lectins
Authors: PILLER Véronique , PILLER Friedrich , CARTRON Jean‐Pierre 
Published: March 2005

Seven plant lectins, Dolichos biflorus agglutinin (DBA), Griffonia simplicifolia agglutinin (GSA, isolectin A4), Helix pomatia agglutinin (HPA), soybean (Glycine max) agglutinin (SBA), Salvia sclarea agglutinin (SSA), Vicia villosa agglutinin (VVA, isolectin B4) and Wistaria floribunda agglutinin (WFA), known to be specific for N‐acetyl‐D‐galactosamine‐(GalNAc) bearing glycoconjugates, have been compared by the binding of their radiolabelled derivatives, to eight well‐characterized synthetic oligosaccharides immobilized via a spacer on an inert silica matrix (Synsorb). The eight oligosaccharides included the Forssman, the blood group A and the T antigens, as well as αGalNAc coupled directly to the support (Tn antigen) and also structures with GalNAc linked α or β to positions 3 or 4 of an unsubstituted Gal. The binding studies clearly distinguished the lectins into αGalNAc‐specific agglutinins like DBA, GSA and SSA, and lectins which recognize α‐ as well as β‐linked GalNAc residues like HPA, VVA, WFA and SBA. HPA was the only lectin which bound to the βGall → 3αGalNAc‐Synsorb adsorbent (T antigen) indicating that it also recognizes internal GalNAc residues. Among the αGalNAc‐specific lectins, DBA strongly recognized blood group A structures while GSA displayed weaker recognition, and SSA bound only slightly to this affinity matrix. In addition, DBA and SSA were able to distinguish between GalNAc linked α1 → 3 and GalNAc linked α1 → 4, to the support, the latter being a much weaker ligand.These results were corroborated by the binding of the lectins to biological substrates as determined by their hemagglutination titers with native and enzyme‐treated red blood cells carrying known GalNAc determinants, e.g. blood group A, and the Cad and Tn antigens. For SSA, the binding to the αGalNAc matrix was inhibited by a number of glycopeptides and glycoproteins confirming the strong preference of this lectin for αGalNAc‐Ser/ Thr‐bearing glycoproteins.
Structural requirements for signal transduction of the insulin receptor
Authors: FLÖRKE Ralf‐Rüdiger , Klein Helmut W. , Reinauer Hans 
Published: March 2005

Structural requirements for signal processing by human placental insulin receptors have been examined. Insulin binding has been found to change the physico‐chemical properties of (αβ)2 receptors solubilized with Triton X‐100, indicating a marked alteration of the form, i.e. size and shape, of the molecular complex. (a) The Stokes radius decreases from about 9.5 nm to 7.9 nm, as determined by PAGE with Triton X‐100 in the buffer (Triton X‐100/PAGE), and from 9.1 nm to 8.7 nm, as assessed by gel filtration. (b) The sedimentation coefficient s2o,w rises from 10.1 S to 11.4 S. Upon dissociation of the receptor‐hormone complex, the alterations are reversed. After autophosphorylation of hormone‐bound (αβ)2‐insulin receptors, phosphate incorporation was found for 7.9‐nm receptor forms when receptor‐insulin complexes were crosslinked with disuccinimide suberate prior to Triton X‐100/PAGE. However, phosphate incorporation was demonstrated for the 9.5‐nm receptor forms when receptor‐insulin complexes were not prevented from dissociation. This strongly indicates that the (αβ)2) receptor is autophosphorylated after assuming its 7.9‐nm form upon insulin binding. Moreover, the insulin‐dependent structural alterations are not affected by autophosphorylation.In contrast to (αβ)2 receptors, the diffusion and the sedimentation behaviour of αβ receptors, which carry a dormant tyrosine kinase even in the hormone‐laden state, has been found to be insensitive to insulin binding.Different molecular properties of αβ and (αβ)2 receptors have also been detected by hormone binding studies. Insulin binding to (αβ)2 and αβ receptors differs markedly with respect to pH, ionic strength, and temperature. This might indicate that the structure of the hormone binding domain of αβ receptor changes on association into the (αβ)2 species. Alternatively, distinct hormone‐induced conformational alterations at the molecular level of αβ and (αβ)2 receptor species may lead to the different binding properties.Our data demonstrate that the (αβ)2‐insulin receptor undergoes extended conformational alterations upon insulin binding. This capacity for structural changes coincides with the hormone‐inducable enhancement of tyrosine autophosphorylation of the 7.9‐nm insulin‐bound receptor form. In contrast, αβ receptors appear to be looked in an inactive nonconvertable state. Thus, interaction between two αβ receptor units is required to allow extended conformational alterations. which are assumed to be the triggering event for augmented autophosphorylation.
Expression in Escherichia coli of ferredoxin: NADP+ reductase from spinach
Authors: ALIVERTI Alessandro , JANSEN Thomas , ZANETTI Giuliana , RONCHI Severino , HERRMANN Reinhold G. , CURTI Bruno 
Published: March 2005

A cDNA clone for the preprotein of spinach ferredoxin: NADP+ reductase has been modified to allow the expression in Escherichia coli of the mature flavoprotein form that lacks the transit peptide. An expression vector, pFNR1, was constructed by subcloning the fragment into the plasmid pDS12/RBSII, SphI. In the crude extracts of transformed cells after induction, two active holoproteins of 35 kDa and 32 kDa, respectively, were found. The 32‐kDa protein, purified by immunoaffinity chromatography, was found to lack the first 28 residues of the spinach protein sequence and to have a methionine as the N‐terminal residue instead of Val29. A new expression plasmid, pFNR2, was obtained by in vitro mutagenesis of the codon GTG for Val29 to the synonymous GTT; in this case, only the 35‐kDa protein was expressed by transformed cells.Both the 35‐kDa and 32‐kDa enzymes were purified and characterized. All the properties analyzed of the cloned 35‐kDa enzyme were very similar to those of the spinach flavoprotein. The 32‐kDa form showed the same catalytic efficiency of the spinach enzyme as a diaphorase but its interaction with oxidized ferredoxin was partially impaired.
Comparative structural and enzymatic properties of skeletal muscle myosin from neonatal and adult rabbits
Authors: HOUADJETO Maurice , BECHET Jean‐Jacques , d'ALBIS Anne 
Published: March 2005

Several structural and enzymatic properties of myosin from skeletal muscles of neonatal and adult rabbits were compared.Electrophoretic analyses and proteolysis experiments indicated that differences between the two myosin types could be attributed to their heavy subunits. Circular dichroism measurements of subfragment‐1 species, and trypsin‐digested derivatives showed that the neonatal protein contained less α‐helices than the adult form.The Mg2+ ‐ATPase activity of neonatal myosin was lower than that of adult myosin, especially in the presence of actin. In comparison with adult subfragment‐1, it was found that the binding of ATP analogues such as adenosine 5′‐[β,γ‐imino]triphosphate and PPi, or that of ATP (as deduced from the apparent KATPm) to neonatal subfragment‐1 in the presence of actin was enhanced, while that of ADP was decreased. On the other hand, the association of actin with the ADP ‐ neonatal‐subfragment‐1 complex was weaker. These features must be expressed in the cyclical actin‐myosin association/dissociation steps occurring in ATP hydrolysis, and more particularly in the reassociation of actin with the ATP‐hydrolysis‐products – myosin complex.
Escherichia coli DNA polymerase I: inherent exonuclease activities differentiate between monofunctional and bifunctional adducts of DNA and cis‐ or trans‐diamminedichloroplatinum(II)
Authors: BERNGES Frithjof , DÖRNER Gertrud , HOLLER Eggehard 
Published: March 2005

[3H]dGMP‐3′‐labelled, activated salmon testis DNA and [32P]dGMP‐5′‐labelled open circular M13 DNA were reacted with cis‐diamminedichloroplatinum(II), cis‐diamminechloroaquaplatinum(II), cis‐diamminediaquaplatinum(II) or trans‐diamminechloroaquaplatinum(II). The reaction was arrested after arbitrary times by adjustment to slightly alkaline solution conditions. The platinum‐containing DNA was digested with Escherichia coli DNA polymerase I. The progress of nucleotide release was measured by acid precipitation of undigested DNA. Solubilized nucleotides and adducts were analysed by HPLC.The 3′‐5′‐exonuclease activity liberated single‐coordinated dGMP‐platinum(II) adducts from both cis‐ and trans‐platinum(II) treated salmon testis DNA and a small fraction of adducts of cis‐platinum(II) that coordinated two molecules of dGMP. The bisadduct was derived from non‐neighboring guanine residues probably located at or close to 3′‐termini. This nuclease activity neither cut between nor after neighboring guanine residues crosslinked by cis‐platinum(II). No bisadduct was liberated for trans‐platinum(II).The 5′‐3′‐exonuclease activity did not liberate any nucleotide adducts from cis‐platinum(II)‐treated DNA. However, it removed single‐coordinated guanine adducts of trans‐diamminedichloroplatinum(II).From the kinetics of the appearance of dGMP monoadducts and the inhibition of digestion, a reaction scheme is formulated for the reaction of platinum(II) complexes with DNA that confirms and extends the previously published one [W. Schaller, H. Reisner & E. Holler (1987) Biochemistry 26, 943–950]. The longevity of the dGMP monoadduct intermediate is discussed in the context of the efficiency of cis‐diamminedichloroplatinum(II) as an antitumor drug.
Structure of the genes of two homologous intracellularly heterotopic isoenzymes
Authors: JURETIĆ Nenad , MATTES Urs , ZIAK Martin , CHRISTEN Philipp , JAUSSI Rolf 
Published: March 2005

The genes of mitochondrial and cytosolic aspartate aminotransferase of chicken were cloned and sequenced. In both genes nine exons encode the mature enzyme. The additional exon for the N‐terminal presequence that directs mitochondrial aspartate aminotransferase into the mitochondria is separated by the largest intron from the rest of the gene. A comparison of the two genes of chicken with the aspartate aminotransferase genes of mouse [Tsuzuki, T., Obaru, K., Setoyama, C. & Shimada, K. (1987) J. Mol. Biol. 198, 21–31; Obaru, K., Tsuzuki, T., Setoyama, C. & Shimada, K. (1988) J. Mol. Biol. 200, 13–22] reveals closely similar structures: in the gene of both the mitochondrial and the cytosolic isoenzyme all but one intron positions are conserved in the two species and five introns out of nine are placed at the same positions in all four genes indicating that the introns were in place before the genes of the two isoenzymes diverged.The variant consensus sequence (T/C)11T(C/T)AG at the 3′ splice site of the introns of the genes for nuclearencoded mitochondrial proteins, which had been deduced from a total of 34 introns [Juretić, N., Jaussi, R., Mattes, U. & Christen, P. (1987) Nucleic Acids Res. 15, 10083–10086], was confirmed by including an additional 22 introns into the comparison. The position −4 at the 3′ splice site is occupied by base T in 43% of the total 56 introns and appears to be subject to a special evolutionary constraint in this particular group of genes.The following course of evolution of the aspartate aminotransferase genes is proposed. Originating from a common ancestor, the genes of the two isoenzymes intermediarily evolved in separate lineages, i.e. the ancestor eukaryotic and ancestor endosymbiontic cells. When endosymbiosis was established, part of the endosymbiontic genome, including the aspartate aminotransferase gene, was transferred to the nucleus. This process probably led to the conservation of certain splicing factors specific for nuclear‐encoded mitochondrial proteins. The presequence for the mitochondrial isoenzyme was acquired by DNA rearrangement. In the eukaryotic lineage, the mitochondrial isoenzyme evolved more slowly than its cytosolic counterpart.
Kinetics and specificities of two closely related β‐glucosidases secreted by Schizophyllum commune
Authors: LO Amy C. , BARBIER Jean‐René , WILLICK Gordon E. 
Published: March 2005

Two β‐glucosidases (I and II) were isolated from Schizophyllum commune, and their physical and chemical properties studied. The two enzymes have very similar sequences, as shown by HPLC analysis of tryptic digests and partial amino acid sequencing. As judged by their circular dichroism spectra, they have almost identical secondary structure. The estimates for α‐helix, β‐sheet, and other structures were 21%, 40% and 39%, respectively, for β‐glucosidase I and 27%, 32% and 41% for β‐glucosidase II. Their near‐ultraviolet spectra were identical. β‐Glucosidase I was more highly glycosylated than β‐glucosidase II, having 2 mol N‐acetylglucosamine/mol enzyme 36, mol mannose/mol enzyme and 1.2 mol glucose/mol enzyme vs 1.2, 17 and 3 mol/mol, respectively, in β‐glucosidase II. The native glycosylated form of β‐glucosidase I had a molecular mass of 102 kDa, and that of β‐glucosidase II, 96 kDa. As estimated from sensitivity to N‐glycanase, β‐glucosidase II sugars were mainly asparagine linked, but much of the sugar in β‐glucosidase I was not removed by this treatment and was apparently serine or threonine linked.Kinetic analysis showed that both forms had similar Km values (0.3–2.1 mM) for oligosaccharides of 2–6 residues, but the kcat values of β‐glucosidase II were lower by 30–75% than those of β‐glucosidase I. The substrate dependence of kcat/Km indicated that both enzymes had binding sites for three glucose residues. The pH optimum of β‐glucosidase I was higher than that of β‐glucosidase II (5.8 vs 5.1). Both had similar specificities for several (R)‐β‐D‐glucosides tested. Both enzymes were competitively inhibited by their glucose product, but β‐glucosidase II was consistently less inhibited than β‐glucosidase I. Cellobiase activity was much more markedly inhibited than the activity with higher oligosaccharides, and the result of this, plus the lower hydrolytic rate with cellobiose, resulted in an accumulation of cellobiose as higher oligosaccharides were digested. Glucono‐δ‐lactone inhibited both enzymes and the hydrolysis of all oligosaccharide substrates similarly (Ki= 4 μM). We conclude that the catalytic site is identical in both enzymes, but subtle structural differences are reflected in a differential activity on the higher oligosaccharides and in the differential effects of the glucose product as an inhibitor. Furthermore, ethanol had a stimulatory effect on β‐glucosidase I but inhibited β‐glucosidase II, which presumably reflects differential effects of ethanol on the conformations of the two species.
Heat capacity and entropy changes of troponin C from bullfrog skeletal muscle induced by calcium binding
Authors: IMAIZUMI Masamoto , TANOKURA Masaru 
Published: March 2005

In order to elucidate the structural changes of bullfrog skeletal muscle troponin C (TnC) caused by Ca2+ binding, microcalorimetric titrations were performed in both the absence and presence of Mg2+, at pH 7.0, and at 5°, 15°, and 25°C. The results indicate that, in the absence of Mg2+, Ca2+ binding to sites 1 and 2 gives rise to a large hydrophobic effect, a sequestering of nonpolar groups on the surface of molecule to the interior, and a tightening of the molecular structure as a whole. In contrast, hydrophobic groups were exposed from the interior to the surface of molecule and the molecular mobility was increased, upon Ca2+ binding to site 3. Ca2+ binding to site 4 induced much less change in the conformation of TnC molecule than that to the other sites. The presence of 5 mM Mg2+ dramatically reduced the magnitude of the conformational changes in TnC on Ca2+ binding to sites 1 and 2. On the other hand, the conformational changes by Ca2+ binding to sites 3 and 4 were not affected by Mg2+.Ca2+ binding to site 3 of TnC, thus, causes the conformational changes distinctly different from those upon Ca2+ binding to the other sites. The conformational changes upon Ca2+ binding to site 3 of TnC are also clearly distinct from the Ca2+ ‐induced conformational changes of parvalbumins, but are similar to those of calmodulin. Site 3 of TnC may, therefore, be the regulatory site in muscle contraction, and the exposure or generation of hydrophobic groups on the surface of molecule as well as the increase in molecular mobility may be the common characteristics of the regulation by Ca2+ ‐binding proteins.
Fructose 1‐phosphate and the regulation of glucokinase activity in isolated hepatocytes
Authors: DAVIES Dewi R. , DETHEUX Michel , VAN SCHAFTINGEN Emile 
Published: March 2005

Fructose 1‐phosphate kinase was partially purified from Clostridium difficile and used to develop specific assays of fructose 1‐phosphate and fructose.The concentration of fructose 1‐phosphate was below the detection limit of the assay (25 pmol/mg protein) in hepatocytes incubated in the presence of glucose as sole carbohydrate. Addition of fructose (0.05–1 mM) caused a concentration‐dependent and transient increase in the fructose 1‐phosphate content. Glucagon (1 μM) and ethanol (10 mM) caused a severalfold decrease in the concentration of fructose 1‐phosphate in cells incubated with fructose, whereas the addition of 0.1 μM vasopressin or 10 mM glycerone, or raising the concentration of glucose from 5 mM to 20 mM had the opposite effect. All these agents caused changes in the concentration of triose phosphates that almost paralleled those of the fructose 1‐phosphate concentration.Sorbitol had a similar effect to fructose in causing the formation of fructose 1‐phosphate. D‐Glyceraldehyde was much less potent in this respect than the ketose and its effect disappeared earlier. The effect of D‐glyceraldehyde was reinforced by an increase in the glucose concentration and decreased by glucagon.Both fructose and D‐glyceraldehyde stimulated the phosphorylation of glucose as estimated by the release of 3H2O from [2‐3H]glucose, but the triose was less potent in this respect than fructose and its effect disappeared earlier. Glucagon and ethanol antagonised the effect of low concentrations of fructose or D‐glyceraldehyde on the detritiation of glucose. These results support the proposal that fructose 1‐phosphate mediates the effects of fructose, D‐glyceraldehyde and sorbitol by relieving the inhibition exerted on glucokinase by a regulatory protein [Van Schaftingen, E. (1989) Eur. J. Biochem. 179, 179–184].
Conformational preferences of [Leu5]enkephalin in biomimetic media
Authors: PICONE Delia , D'URSI Annamaria , MOTTA Andrea , TANCREDI Teodorico , TEMUSSI Piero A. 
Published: March 2005

The conformation of [Leu5]enkephalin has been studied by 1H‐NMR spectroscopy in media more like the actual environment in which the agonist‐receptor interaction takes place than water, i.e. in three cryoprotective mixtures (dimethylformamide/water, methanol/water and ethylene glycol/water), in aqueous SDS and in two neat solvents, dimethylformamide and acetonitrile, whose dielectric constants (36.7 and 37.5) are intermediate between that of water and that of the lipid phase. In all cases examined, contrary to the studies in water or dimethylsulfoxide, we were able to detect numerous nuclear Overhauser effects, indicating that the media employed favour well‐defined structures and/or reduce the internal motions of the peptide.Data from both organic solvents and cryoprotective mixtures suggest a 4 → 1 β turn as the most probable structure of [Leu5]enkephalin in solution, whereas in SDS/H2O micelles the structural picture appears completely different, suggesting the presence of a 5 → 2 β turn. The existence of two different preferred conformations of enkephalins may possibly be related to their ability to be effective towards both μ and δ opioid receptors.
Sequence of proton abstraction and stereochemistry of the reaction catalyzed by naphthoate synthase, an enzyme involved in menaquinone (vitamin K2) biosynthesis
Authors: IGBAVBOA Urule , LEISTNER Eckhard 
Published: March 2005

The enzymic conversion of the coenzyme A ester of 4‐(2′‐carboxyphenyl)‐4‐oxobutyric acid (i. e.o‐succinylbenzoic acid) to 1,4‐dihydroxy‐2‐naphthoic acid is a cyclization reaction which is part of menaquinone (vitamin K2) biosynthesis. This conversion, which is probably a two‐step process, was investigated using chirally labelled samples of the coenzyme A ester of 4‐(2′‐carboxyphenyl)‐4‐oxobutyric acid. To synthesize these, the following enzymes were employed: isocitrate: NADP+ oxidoreductase (EC 1.1.1.42), isocitrate glyoxylate‐lyase (EC 4.1.3.1), 2‐oxoglutarate dehydrogenase complex (which includes EC 1.2.4.2), 4‐(2′‐carboxyphenyl)‐4‐oxobutyrate synthase system and 4‐(2′‐carboxyphenyl)‐4‐oxobutyrate: CoA ligase.Isocitrate: NADP+ oxidoreductase was employed to generate the two enantiometric samples of 2‐oxoglutarate enantiotopically labelled at C‐3. These samples were converted enzymically to succinate with retention of configuration at C‐2 and C‐3, and to 4‐(2′‐carboxyphenyl)‐4‐oxobutyric acid with retention of configuration at C‐3. Isocitrate glyoxylate‐lyase and isocitrate NADP+ oxidoreductase were employed to generate samples of 2‐oxoglutarate enantiotopically tritiated at C‐4 or at C‐3 and C‐4. The four variously labelled samples of 2‐oxoglutarate were enzymically converted to the coenzyme A ester of 4‐(2′‐carboxyphenyl)‐4‐oxobutyric acid.The resulting variously labelled coenyzme A esters were incubated with naphthoate synthase to investigate the ring closure reaction. In the first step the 2HRc atom of the oxobutvric moiety of the coenzyme A ester is equilibrated with solvent protons in a fast and reversible reaction. Subsequently the 2HSi and 3HSi atoms are removed whereas the 3HRe atom becomes the proton at C‐3 of 1,4‐dihydroxy‐2‐naphthoic acid. The second step in this ring closure reaction is the rate‐limiting step.
Internalization of the cytotoxic molecules of T101 F(ab′)2— (ricin‐A‐chain) immunotoxin into human T‐leukemic cells
Authors: RAVEL Sophie , CASELLAS Pierre 
Published: March 2005

We have investigated the internalization step of an immunotoxin and its relationship with cytotoxicity, with the F(ab′)2—T101(ricin‐A‐chain) immunotoxin, directed against the CD5 antigen expressed on leukemic CEM cells.We first demonstrated that the biological action of the conjugate was related to its entry into the cell by an energy‐dependent endocytotic process. We also found that during the first hours of cell intoxication, internalization is not the rate‐limiting step of immunotoxin cytotoxicity. Internalization becomes limiting in cell intoxication only when the entry rate is low.Lastly we show that ammonium chloride, which strongly enhances immunotoxin potency, acts on internalized molecules for a very short time, suggesting that this enhancer affects an early intracellular step.