bluuebunny/crossref_metadata_2025_split huggingface dataset:
Specificities of Limulin and Wheat‐Germ Agglutinin Towards Some Derivatives of GM3 Gangliosides
Authors: MAGET‐DANA Régine , VEH Rüdiger W. , SANDER Michael , ROCHE Annie‐Claude , SCHAUER Roland , MONSIGNY Michel 
Published: July 2010

Lipid vesicles containing derivatives of GM3 ganglioside (II3‐N‐acetylneuraminosyllactosyl ceramide) were used to study the specificities of two lectins (limulin and wheat germ agglutinin) towards N‐acetyl neuraminic acid and N‐glycoloylneuraminic acid and some of their natural and chemically modified derivatives.The extent of the lectin binding to the gangliosides was related to the aggregation process of the lipid vesicles which was monitored as an absorbance increase.Limulin binds specifically to lipid vesicles containing N‐glycoloyl derivatives of GM3. The hydroxyl group at C‐4 and the carboxyl group of neuraminic acid have to be free for the binding to limulin. The side chain of neuraminic acid is not involved in the binding site of limulin.Wheat germ agglutinin binds to GM3 ganglioside only when the hydrophilic tail of the neuraminic group is cut off (C7 analogues). The acetamido group but not the carboxyl group is involved in the binding to wheat germ agglutinin. The wheat‐germ‐agglutinin‐induced aggregation of vesicles containing derivatives of GM3‐ganglioside is dependent on the pH, on the ionic strength and on the presence of Ca2+ ions. The dependence on ionic strength and Ca2+ is a consequence of the electrostatic repulsion of the vesicles.The wheat‐germ‐agglutinin‐induced aggregation process of vesicles containing any suitable GM3‐ganglioside derivative was reversed by the addition of N‐acetylglucosamine showing that the N‐acetylneuraminic acid derivatives bind to the N‐acetylglucosamine binding site.
A Cysteine‐Rich Collagenous Protein from Bovine Placenta
Authors: JANDER Renate , RAUTERBERG Jürgen , VOSS Bruno , von BASSEWITZ Dirk B. 
Published: July 2010

A high‐molecular‐weight collagenous protein was isolated, after limited pepsin digestion, from the endometrial villi of bovine placenta; it resembled the protein isolated from human aortas by Chung et al. [Chung, E., Rhodes, R. K. and Miller, E. J. (1976) Biochem. Biophys. Res. Commun. 71, 1167–1174] and from human placenta by Furuto and Miller [Furuto, D. K. and Miller, E. J. (1980) J. Biol. Chem. 255, 290–295]. After denaturation and reductive cleavage of disulfide bridges three chains with Mr of 40000–55000 were liberated. Two of the chains could be separated by ion‐exchange chromatography; the third chain aggregated under non‐denaturing conditions and could be isolated by sequential molecular‐sieve chromatography in denaturing and non‐denaturing buffers. Their amino acid composition resembled that of basement membrane collagens with respect to the high content of completely glycosylated hydroxylysine but differed with respect to their relatively low hydroxyproline content and high values of cysteine, tyrosine and glucosamine. A relatively low glycine content indicated regions of non‐collagenous structure. Reduction under non‐denaturing conditions and another pepsin treatment removed non‐collagenous regions containing mainly hydrophobic amino acid residues. The denaturation temperature of the component was considerably reduced by this treatment, indicating stabilization of triple‐helical structures by disulfide bridges in the native molecule.Fibrillar aggregates were precipitated with ATP which showed in the electron microscope a typical crossstriation pattern different to those of segment‐long‐spacing crystallites obtained from other collagen types. Immunofluorescence tests demonstrated the protein in blood vessels and interstitial areas of the renal medulla but not in basement membranes of glomeruli and tubuli.
Cathepsin D: Specificity of Peptide‐Bond Cleavage in Type‐I Collagen and Effects on Type‐III Collagen and Procollagen
Authors: SCOTT Paul G. , PEARSON Harold 
Published: July 2010

Cathepsin D, purified from bovine thymus, has a limited proteolytic effect on types I and III bovine collagens. The α1 (I) chain was cleaved in native or denatured collagen only within the carboxy‐terminal extrahelical sequence, the major site being between residues C6 (Leu) and C7 (Ser). The α2 chain was unaffected in native collagen but was slowly cleaved between residues 782 (Phe) and 783 (Leu) in the denatured form. Cleavages, at 45° C, in type III collagen occur within the extra‐helical amino‐terminal sequence, on the carboxyterminal side of the lysine residue involved in intermolecular cross‐linking. All three sites of action are within sequences of general hydrophobic character.
The very restricted cleavage of peptide bonds in denatured collagens can be ascribed to the infrequent occurrence of groupings of more than two hydrophobic residues and to the high content of the conformation limiting residues proline and hydroxyproline.
The previously demonstrated failure of cathepsin D to solubilize a representative proportion of type III collagen from the fibres of bovine skin collagen (P. G. Scott and C. H. Pearson (1978) Biochem. Soc. Trans. 6, 1197–1199] may be explained by lack of ability of the enzyme to act on this collagen at 25° C, in such a manner as to separate molecules joined by intermolecular cross‐links involving the amino‐terminal extrahelical region of the molecule.
A Catalytic Mechanism for Benzylamine Oxidase from Pig Plasma
Authors: YADAV Kapil D. S. , KNOWLES Peter F. 
Published: July 2010

Ammonium ion is shown to decrease the rate constants for Schiff's base formation and formation of a reduced intermediate during the catalytic cycle of benzylamine oxidase from pig plasma. The rate constant for reoxidation of the reduced intermediate is also inhibited whilst the rate constant for conversion of the oxidised enzyme form back to native enzyme is stimulated by ammonium ion.
Ammonium ion changes the electron paramagnetic resonance spectrum of the cupric centres in the enzyme, indicating that ammonia binds to the copper.
A catalytic mechanism for benzylamine oxidase is proposed on the basis of these and other results. This mechanism includes a novel step in which a hydroxyl coordinated to copper acts as a nucleophyle to facilitate hydride ion transfer to oxygen during the reoxidation process.
Isolation and Characterization of Binder IIIA, a New Protein Which Binds Corticosteroid Anions
Authors: STEEGER Joseph R. , LITWACK Gerald 
Published: July 2010

A new protein binding corticosteroid metabolites has been purified over 300‐fold from liver cytosols of adrenalectomized rats, treated for 45 min in vivo with [1,2‐3H]cortisol. Purification was accomplished by column chromatography on Sephadex G‐25, DEAE‐Sephadex A‐50, Sephadex G‐75, and hydroxylapatite. The protein has a Stokes radius of 2.27 nm by gel filtration and an apparent sedimentation coefficient of 3.0 S by sucrose gradient centrifugation. The calculated molecular weight is 30700. The bound steroid was extracted and has been shown by Sephadex LH‐20 chromatography to be a monosulfate derivative of cortisol.Using liver cytosol from adrenalectomized rats pretreated in vivo for 45 min with [1,2‐3H]cortisol plus 1000‐fold excess competing steroid, cortisol derivatives and progesterone were shown to be the most active competitors. Testosterone and 17β‐estradiol were least active as competitors. The synthetic steroids, dexamethasone and triamcinolone, produced little or no competition. The protein has been named corticosteroid‐anion binder IIIA in keeping with its elution position from a DEAE‐Sephadex A‐50 column, compared to other binding proteins.Binder IIIA has been separated chromatographically from the glutathione S‐transferases (including ligandin) and protein z described by Arias [Levi, A. J., Gatmaitan, Z. and Arias, I. M. (1969) J. Clin. Invest. 48, 21856–21866], both of which have been shown to bind anionic metabolites. It has been resolved from the activities of transcortin, cortisone 5β‐reductase, and 3α‐hydroxysteroid dehydrogenase.
Molecular Properties of Two Mutant Species of the Elongation Factor Tu
Authors: VAN DER MEIDE Peter H. , Jan DUISTERWINKEL Feitse , DE GRAAF J. Martien , KRAAL Barend , BOSCH Leendert , DOUGLASS James , BLUMENTHAL Thomas 
Published: July 2010

The molecular properties of two mutant species of the elongation factor Tu (EF‐Tu), derived from either tuf A or tufB, have been studied. One, designated EF‐TuAR, is the product of a kirromycin‐resistant tufA gene. The other designated EF‐TuBo is a tufB product and is present in a kirromycin‐resistant mutant of Escherichia coli (LBE 2012) also harbouring the EF‐TuAR species. EF‐TuAR has been isolated in homogeneous form as a single gene product from the mutant strain LBE 2045, in which the tufB gene has been inactivated by an insertion of the bacteriophage Mu. EF‐TuBo has been isolated from LBE 2012 together with EF‐TuAR in a 1:1 mixture. Fractionation of this mixture on DEAF‐Sephadex A‐50 resulted in an enrichment of EF‐TuBo of about 80%. The properties of EF‐TuAR and EF‐TuBo have been compared to those of a kirromycin‐sensitive species designated EF‐TuAS, which was isolated from LBE2045 by transduction of wild‐type tufA.We show here that all three EF‐Tu species are fully competent to sustain polypeptide synthesis. All also appear to interact normally with guanine nucleotides and EF‐Ts. Only in the presence of the antibiotic do the following differences appear. (a) Kirromycin causes EF‐TuAS (wild‐type tufA gene product) to be retained on, and thus block, the ribosome. (b) EF‐TuAR fails to bind the antibiotic and thus is capable of protein synthesis in its presence. (c) EFTuBo fails to sustain polypeptide synthesis upon binding of kirromycin. It does not, however, block the ribosome, so the strain harbouring both this protein and EF‐TuAR (LBE2012) is kirromycin resistant.
Protein‐Bound Polymeric and Monomeric ADP‐Ribose Residues in Hepatic Tissues
Authors: WIELCKENS Klaus , BREDEHORST Reinhard , ADAMIETZ Peter , HILZ Helmuth 
Published: July 2010

Determination of poly(ADP‐ribose) levels was performed by a new procedure involving covalent chromatography of released polymer, degradation to phosphoribosyl AMP and quantification of this specific derivative by a radioimmunoassay. In adult rat liver, about 85 pmol polymeric ADP‐ribose residues/g tissue was found. Similar values were obtained when the determination was carried out by an independent procedure not involving boronate chromatography or sedimentation of DNA.In adult rat liver, polymeric ADP‐ribose residues amounted to about 1/200 of total monomeric ADP‐ribose residues. Most of the polymeric ADP‐ribose residues were linked to proteins by NH2OH‐sensitive bonds, while mono(ADP‐ribose)‐protein conjugates consisted of about equal amounts of NH2OH‐sensitive and NH2OH‐resistant subfractions.Poly(ADP‐ribose) levels immediately after birth were similar to the adult status. They decreased, however, by a factor of three at the time of most rapid post‐natal liver growth (day 17). A comparison with the protein‐bound monomeric ADP‐ribose residues indicated independent changes, and therefore presumably independent functions of these monomeric ADP‐ribose residues.
Kinetics of Thyroglobulin Iodination and Thyroid Hormone Synthesis Catalyzed by Peroxidases: The Role of H2O2
Authors: VIRION Alain , POMMIER Jacques , DEME Danièle , NUNEZ Jacques 
Published: July 2010

When thyroglobulin iodination by thyroid peroxidase or lactoperoxidase is studied with optimal concentrations of H2O2‐generating system, thyroid hormone synthesis (coupling reaction) begins after a constant lag period which is independent of the concentration of iodide, thyroglobulin or enzyme. Increasing the concentration of the H2O2‐generating system inhibited the iodination reaction whereas the length of the lag period was decreased. When thyroglobulin containing iodinated tyrosine residues but no hormone was incubated with increasing concentrations of iodide and limiting amounts of the H2O2‐generating system the iodination reaction was stimulated and the coupling reaction was inhibited.
To analyze whether the iodination and coupling reactions require different enzyme‐H2O2 species the iodination and coupling reactions and the absorbance at 430 nm (compound II) were measured simultaneously with lactoperoxidase. Under these conditions: (a) the coupling reaction occurred only in the presence of sufficient amounts of H2O2 whereas the level of iodination was the same; (b) an increase in absorbance at 430 nm was observed only when the iodination reaction leveled off, i.e. just before the beginning of the coupling reaction; (c) during the lag period, i.e. the iodination step, the only enzyme species seen in the Soret region was the native enzyme; and (d) adding iodinated thyroglobulin containing no hormone to preformed compound II or III resulted in a prompt coupling reaction. These data suggest that the iodination and coupling reactions are catalyzed by different enzyme‐H2O2 species.
When thyroid peroxidase or lactoperoxidase were preincubated for varying periods of time in the presence of the H2O2‐generating system their activity towards guaiacol decreased progressively: with thyroid peroxidase the Km for guaiacol was increased 10‐fold after 5 min of preincubation and 40‐fold with lactoperoxidase. The activity of the preincubated enzymes was restored by iodide. Preincubated lactoperoxidase had the Soret spectrum of compound III. Similarly, the rate of iodination of thyroglobulin was inhibited with the preincubated enzyme, but increasing the iodide concentration restored the iodinating activity.
The proposal is made that different enzyme‐H2O2 species are the active agents in the catalysis of the iodination and coupling reaction respectively.
Isolation of Component C4 of Human Complement and Its Polypeptide Chains
Authors: LUNDWALL Åke , MALMHEDEN Ingrid , SJÖQUIST John , STÅLENHEIM Gunnemar 
Published: July 2010

Component C4 of human complement was purified from fresh frozen plasma with a yield of 25%, using an initial batch separation with quaternary diethyl‐(2‐hydroxypropyl)aminoethyl—Sephadex followed by column chromatography on DEAE‐cellulose and gel filtration in Sephadex G‐200.The final product was homogenous according to polyacrylamide gel electrophoresis and immunochemical methods. Low‐speed sedimentation‐equilibrium analyses revealed a molecular weight of 189000, using a value of 0.736 ml/g for the partial specific volume.The polypeptide chains of reduced and alkylated C4 were separated on DEAE‐Sepharose in the presence of 8 M urea. Gel filtration in Sepharose 4B in 6 M guanidine hydrochloride revealed molecular weights of 88000, 72000 and 32000 for the α, β and γ chain respectively.The amino acid compositions of component C4 and its constitutive chains were also determined.
A Small‐Angle X‐Ray Scattering Study of the Complex Formation between Elongation Factor. Tu · GTP and Valyl‐tRNAVal1 from Escherichia coli
Authors: ÖSTERBERG Ragnar , SJÖBERG Bo , LIGAARDEN Roar , ELIAS Per 
Published: July 2010

The complex formation between elongation factor Tu (EF‐Tu) · GTP and valyl‐tRNAVal1 has bin investigated using the small‐angle X‐ray scattering titration technique. The main species observed is a 1:1 complex with a stability constant log K ≥ 6. The corresponding interaction between EF‐Tu · GTP and non‐aminoacylated tRNA appears to be much weaker with an estimated log K∼4.The radius of gyration determined for the EF‐Tu · GTP—valyl‐tRNAVal1 complex is larger (R = 3.6 nm) than that of EF‐Tu · GTP (R = 2.5 nm). Likewise, the maximum distance within this complex is larger (Dmax= 12.5 nm) than the one within EF‐Tu · GTP (Dmax= 8.5 nm). These data as well as the p(r) curve are consistent with a multiellipsoid model for the complex. From this model it is indicated that the acceptor stem of tRNA is attached to EF‐Tu and that the anticodon stem and loop protrude into the solution.
Sidedness of e− Donation and Stoichiometry of H+ Pumps at Sites II + III in Mitochondria from Rat Liver
Authors: DI VIRGILIO Francesco , POZZAN Marina , AZZONE Giovanni Felice 
Published: July 2010

The stoichiometry of the redox H+ pumps at site II and site II+III of the respiratory chain of rat‐liver mitochondria has been determined with three different e− donors: succinate, duroquinol and NADH with duroquinone by‐pass of rotenone inhibition. Succinate provides reducing equivalents to the cytochrome b‐c1 complex from the matrix side. Duroquinol, either added from the outside or obtained from the reduction of exogenous duroquinone by the NADH dehydrogenase, presumably is an intramembrane e− donor to cytochrome b or to the endogenous ubiquinone.The stoichiometries were determined at steady‐state rates of ion translocation and e− transfer. The ion translocation was followed on the basis of the rates of H+ extrusion and of K+ and Ca2+ uptake. The e− transfer was followed either with the polarographic or with the spectrophotometric technique, i. e. absorbance changes during oxidation of duroquinol to duroquinone. In this latter case N‐ethylmaleimide was used to inhibit the NADH dehydrogenase activity responsible for the re‐reduction of the duroquinone formed during duroquinol oxidation. During operation of sites II+III, with oxygen as e− acceptor, all three substrates tested gave H+/2e− and charge/2e− ratios close to 8. 0. During operation of site II, with ferricyanide as e− acceptor, the H+/2e− ratios were about 4. 0 and the charge/2e− ratios close to 2. 0. The present results support previous reports from our laboratory on the stoichiometries of the redox H+ pumps at sites II and III. Furthermore the equality of the H+ and charge stoichiometry, with substrates having different sides of interaction with complex III suggests that e− donors located on the matrix side or intramembrane (and possibly on the cytoplasmic side) share the same pathway in order to feed electrons into the H+ pump at site II.
Binding of Spermine to tRNATyr Stabilizes the Conformation of the Anticodon Loop and Creates Strong Binding Sites for Divalent Cations
Authors: NÖTHIG‐LASLO Vesna , ŽIVKOVIĆ Tomislav , KUĆAN Željko , WEYGAND‐DURAŠEVIĆ Ivana 
Published: July 2010

Electron spin resonance (ESR) spectra of yeast tRNATyr, spin‐labelled in the isopentenyladenosine residue adjacent to the anticodon, were measured as a function of temperature at various spermine/tRNA ratios. The critical temperature, at which a change in the activation energy for spin‐label motion takes place, changes abruptly by almost 10°C upon the addition of the fifth spermine molecule/tRNATyr molecule, indicating a marked stabilization of the anticodon region.Scatchard plots for Mn2+ binding to tRNATyr in the presence of spermine do not follow theoretically predicted curves for electrostatic type of interaction, assuming that four negative charges on tRNA are neutralized by each spermine molecule. It was estimated that two to three new binding sites for divalent canons are created upon the binding of spermine to tRNATyr.
Modification of the Spectral Properties of Cytochrome b in Mutants of Saccharomyces cerevisiae Resistant to 3‐(3,4‐Dichlorophenyl)‐1,1‐dimethylurea
Authors: BRIQUET Michel , GOFFEAU André 
Published: July 2010

The effects of five inhibitors of the cytochrome bc1 complex: 3‐(3.4‐dichlorophenyl)‐1,1‐dimethylurea (diuron), 2‐n‐heptyl‐4‐hydroxyqttinoline‐N‐oxide (HpHOQnO), antimycin A, funiculosin and mucidin were measured in submitochondrial particles of strains of the yeast Saccharomyces cerevisiae belonging to two classes of diuronresistant mutants Diu 1 and Diu 2 which are modified in different exons of the split mitochondrial gene of cytochrome b.
The oxidation of NADH and of cytochronic b‐561 exhibits a similar resistance to diuron and HpHOQnO in Diu I and Diu2 mutants.
No extra reduction of cytochrome b‐561 and cytochrome b‐565 is observed in the presence of diuron and HpHOQnO.
Both Diu 1 and Diu 2 mutants exhibit the red shift of cytochrome b‐561 induced by concentrations of HpHOQnO 2‐3‐times higher than those required in the parental strain.
The spectral and respiratory effects of antimycin A, funiculosin and mucidin are generally similar in the diuron‐resistant mutants and in their parental strains. However a cross‐resistance between diuron and antimycin A is indicated in one Diu 2 mutant.
From the combined genetic and biochemical data it is concluded that the interaction of diuron and HpHOQnO with cytochrome b is mediated by at least two specific amino acids located apart in tile central region of tile apocytochrome b peptide coded by mitochondrial DNA. These two amino acids control tightly the extra reduction of cytochromes b‐565 and b‐561 as well as tile flow of electrons through the bc1 complex. However the binding of HpHOQnO required for the expression of the red shift of cytochrome b‐561 is only slightly affected by the diu‐l and diu‐2 mutations.
Displacement of the Human Apoprotein A‐I by the Human Apoprotein A‐II from Complexes of (Apoprotein A‐I)‐Phosphatidylcholine‐Cholesterol
Authors: ROSSENEU Maryvonne , VAN TORNOUT Philippe , LIEVENS Marie‐José , ASSMANN Gerd 
Published: July 2010

Reassembly experiments, involving isolated human apoproteins A‐I and A‐II and (dimyristoylglycerophosphocholine)‐cholesterol vesicles were performed with apoprotein mixtures at apoprotein A‐I/A‐II molar ratios varying between 0 and 3. The apoproteins were incubated at 24°C, 28°C and 32°C with either pure dimyristoylglycerophosphocholine vesicles or with dimyristoylglycerophosphocholine cholesterol vesicles containing 0, 5, 10, 15 mol/100 mol cholesterol. The kinetics of association were followed by measuring the increase of the fluorescence polarization ratio after label ing the lipids with diphenyl hexatriene. The complexes were separated from the free protein by gradient ultracentrifugation. Total protein was assayed and the apoproteins A‐I and A‐II were quantified separately by immunonephelometry. The content of apoprotein A‐I was also monitored by measuring the intrinsic tryptophan fluorescence.The results suggest that apoprotein A‐II has a greater affinity than apoprotein A‐I for the phospholipidcholesterol vesicles and that apoprotein A‐II is able to quantitatively displace apoprotein A‐I from the lipidprotein complexes. The content of apoprotein A‐II in the complexes increases proportionally to the concentration of apoprotein A‐II in the incubation mixture until saturation is reached. At saturation the dimyristoylglycerophosphocholine/apoprotein A‐II ratio in the complex is dependent upon the cholesterol content of the original vesicles and increases from 60 to 275 mol/mol between 0 and 15 mol/100 mol cholesterol. From these experiments one can calculate that 1 mol human apoprotein A‐I is displaced by 2 mol human apoprotein A‐II.