bluuebunny/crossref_metadata_2025_split huggingface dataset:
Studies of the solution structure of the bleomycin‐A2– zinc complex by means of two‐dimensional NMR spectroscopy and distance geometry calculations
Authors: AKKERMAN Marcel A. J. , HAASNOOT Cornelis A. G. , HILBERS Cornelis W. 
Published: March 2005

Application of various two‐dimensional NMR techniques (SECSY, COSY and NOESY) enabled the complete assignment of the 1H‐NMR spectrum of the bleomycin‐A2–zinc complex in H2O and D2O at pH 6.7. The spectra were interpreted at 277 K as well as at 300 K. Identification of the resonances permitted a vicinal coupling constant analysis which revealed that the conformation around the Cα and Cβ bond of the β‐aminoalanine and β‐hydroxyhistidine residues is fixed. From this finding it was concluded that both amino functions of the β‐aminoalanine fragment and the amide and imidazole groups of the β‐hydroxyhistidine moiety are involved in zinc coordination. Also, for the mannose carbamoyl group and the pyrimidine ring active participation in zinc coordination could be established.NOE data together with the six coordination sites proposed above were used as interpoint distance constraints in distance geometry calculations for the bleomycin‐A2–zinc complex in H2O. Sets often structures, randomly chosen within the distance constraints, were calculated (with and without the zinc ion). The calculated structures were very similar but in case that the zinc ion was omitted some flexibility was observed, within the distance constraints, in the pyrimidine‐aminoalanine region. Because of the great overall similarity between the structures, a reliable representation of the solution conformation of the bleomycin‐zinc complex was reached. Surprisingly, no regular symmetry around the zinc ion was found to be present in the generated structures.
Purification and characterization of two lysozymes from rainbow trout (Salmo gairdneri)
Authors: GRINDE Bjørn , JOLLÈS Jacqueline , JOLLÈS Pierre 
Published: March 2005

Two different lysozymes, designated I and II, were purified from the kidney of rainbow trout. The enzymes had isoelectric points of approximately 9.5 and 9.65, and differed in their binding characteristics to a cation exchanger. Lysozyme II had the highest specific activity against Micrococcus luteus.
By sodium dodecyl sulphate gel electrophoresis, a molecular mass of 14.4 kDa was established for the two lysozymes.
For both type I and II enzymes, optimum pH under the present conditions was 5.5 and optimum temperature (at pH 6.2) around 45°C.
N‐terminal amino acid sequence determination indicated that the two trout lysozymes were c‐type lysozymes.
The fish lysozymes had a much higher rate of diffusion in agar than did the other lysozymes tested (possibly due to less interaction with agarose). This implies that a foreign lysozyme, such as hen egg white lysozyme, should not be used as a standard when assaying lysozyme activity with the lysoplate technique.
Active‐site‐specific zinc‐depleted and reconstituted cobalt(II) human‐liver alcohol dehydrogenase
Authors: SCHNEIDER‐BERNLÖHR Helga , FORMICKA‐KOZŁOWSKA Grażyna , BÜHLER Rolf , von WARTBURG Jean‐Pierre , ZEPPEZAUER Michael 
Published: March 2005

The active‐site zinc atom of the β1β1 isozyme of class I alcohol dehydrogenase (EC 1.1.1.1) from human liver was specifically removed by the chelating agent dipicolinic acid. From β1γ1 and γ1γ1 isozyme the active‐site zinc is extracted much more slowly than from β1β1 isozyme. Only partially active‐site metal‐depleted enzyme species were obtained from these isozymes.The active‐site‐specific reconstituted cobalt(II) derivative of the β1β1 isozyme shows spectroscopic properties comparable to those of the active‐site‐specific reconstituted cobalt(II) horse liver alcohol dehydrogenase. The coenzyme‐induced conformational change of the protein leads to a red shift of the d‐d band from 648 nm to 673 nm.The chromophoric substrate trans‐4‐(N,N‐dimethylamino)‐cinnamaldehyde forms ternary complexes with NADH and the different isozymes, in close analogy to horse liver alcohol dehydrogenase. The differences in the active sites between β1 and γ1 subunits (threonine‐48 instead of serine‐48) or between zinc and cobalt(II) are reflected in the visible absorption spectra of the metal‐bound chromophoric substrate.
H8Zn(c)2 and Zn(c)2Co(n)2 human liver alcohol dehydrogenase
Authors: FORMICKA‐KOZŁOWSKA Grażyna , SCHNEIDER‐BERNLÖHR Helga , von WARTBURG Jean‐Pierre , ZEPPEZAUER Michael 
Published: March 2005

The zinc ion in the noncatalytic site of human β1β1 and β1γ1 isozymes of class I alcohol dehydrogenases (EC 1.1.1.1) was specifically replaced by Co(II) ion. The absorption and CD spectra prove that these derivatives contain cobalt bound at the noncatalytic site to the same ligands and in the same coordination geometry as in the corresponding species obtained from the horse liver EE isozyme. These Zn(c)2Co(n)2 human liver alcohol dehydrogenases could be obtained in two ways: (a) by exchange dialysis, (b) by removal of the noncatalytic zinc and subsequent insertion of cobalt(II) ion into the empty site.The human isozymes differ from the horse liver EE enzyme in the possibility of forming stable species lacking the noncatalytic zinc ion. This difference in chemical reactivity of the noncatalytic zinc atom may be related to amino acid changes in the human isozymes, compared to horse liver alcohol dehydrogenase.
Purification and carbohydrate structure of natural murine interferon‐β
Authors: CIVAS AHMET , FOURNET Bernard , COULOMBEL Colette , LE ROSCOUET Danièle , HONVAULT Anne , PETEK Fahrettin , MONTREUIL Jean , DOLY Janine 
Published: March 2005

Mouse interferon‐β (Mu‐IFN‐β) induced in C‐243 cells with Newcastle disease virus was purified in four steps including ammonium sulfate fractionation, DEAE‐cellulose, monoclonal Mu‐IFN‐β antibody affinity and Mono‐S cation‐exchange chromatographies. Specific activity of the purified Mu‐IFN‐β ranged over 1.1–1.4 × 109 NIH units/mg protein. This preparation was submitted to pronase digestion and gel filtration on Fractogel TSK HW‐40. The permethylated and acetylated glycopeptide fraction was analyzed by chemical‐ionization (ammonia) mass spectrometry. The major glycopeptide is composed of Gal, Man, GlcNAc and NeuAc with a molar ratio of 2.0:3.6:3.4:0.5. The GLC pattern of methyl derivatives obtained by methanolysis and acetylation of fully methylated glycopeptide identified 2,3,4,6‐tetra‐O‐methylgalactose; 3,4,6‐tri‐O‐methyl‐mannose; 2,3,4‐and 2,4,6‐tri‐O‐methyl galactose; 2,4,di‐O‐methyl mannose and 3,6‐di‐O‐methylglucosamine.These results when compared with data on N‐glycans suggest the following structure for the carbohydrate moiety of Mu‐IFN‐β:

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One‐ and two‐dimensional NMR investigations of the heme pocket in free α(CO) chains from human hemoglobin
Authors: SCHAEFFER Corinne , CRAESCU Constantin T. , MISPELTER Joël , GAREL Marie‐Claude , ROSA Jean , LHOSTE Jean‐Marc 
Published: March 2005

Two‐dimensional nuclear magnetic resonance techniques were used to assign resonances corresponding to heme pocket residues of the isolated α(CO) subunits of the human adult hemoglobin (HbA). The assignment procedure was based on the partial identification of the amino acid spin system from the J‐correlated (COSY) spectrum and on the nuclear Overhauser effect connectivities (from NOESY spectra) with the heme substituents. We present here partial assignments corresponding to five amino acid residues: Leu‐86, Leu‐91, Val‐93, Leu‐101 and Leu‐136.Starting from the known crystallographic structure of the α subunit in the hemoglobin tetramer, we applied a dipolar model to compute the ring‐current shift of the protons from fifteen amino acid residues in the heme pocket. Comparison of the predicted and observed chemical shifts suggests that there is a very close similarity between the heme pocket tertiary structure of the α(CO) subunits in crystals of HbA(CO) and of the free α(CO) chains.The one‐dimensional NMR spectra were used to monitor the pH‐induced structural changes, the effects of chemical modification and of ligand substitution. Upon increasing the pH from 5.6 to 9.0 the structure of the heme environment appears to be invariant with the exception of some residues in the CD corner. The structure is also largely conserved when p‐chloromercuribenzoate is bound to Cys‐104. In contrast, the substitution of CO by O2 as ligand induces many large changes in the heme cavity which can be partially characterized by NMR spectroscopy.
Purification of the human O6‐methylguanine‐DNA methyltransferase and uracil‐DNA glycosylase, the latter to apparent homogeneity
Authors: MYRNES Bjørnar , WITTWER Christian Urs 
Published: March 2005

Uracil‐DNA glycosylase, the enzyme that catalyzes the release of free uracil from single‐stranded and double‐stranded DNA, has been purified 26 600‐fold from HeLa S3 cell extracts. The enzyme preparation was essentially homogeneous as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The native enzyme is a small monomeric protein of molecular mass 29 kDa. A minor uracil‐DNA glycosylase preparation was also obtained in the final chromatographic step. This preparation is homogeneous with a molecular mass of 29 kDa and may represent the mitochondrial enzyme.This report also presents a 700‐fold purification of HeLa S3 cell O6‐methylguanine‐DNA methyltransferase. The glycosylase and methyltransferase showed very similar chromatographic properties. The report indicates that the lability of the methyltransferase upon purification may be a consequence of the total separation of the two DNA repair enzymes or of the possibility that some other stabilizing factor is involved.
Characterization and specific assay for a galactoside β‐3‐galactosyltransferase of human kidney
Authors: BAILLY Pascal , Friedrich PILLER  , CARTRON Jean‐Pierre 
Published: March 2005

Two galactosyltransferases identified as UDP‐galactose: lactose (lactosylceramide) α‐4‐ and β‐3‐galactosyltransferases [Bailly P. et al. (1986) Biochem. Biophys. Res. Commun. 141, 84–91] have been characterized in human kidney microsomes. Using methyl β‐d‐galactoside as acceptor substrate, we have determined the experimental conditions (pH 5.0, 4 mM Cd2+) in which only the β‐3‐galactosyltransferase activity is detectable. The reaction product has been characterized by chemical methods and glycosidase studies. Under these experimental conditions, some of the enzyme properties have been further investigated. Apparent Km values are for UDP‐galactose, 0.170 mM; for lactose, 242 mM; and for lactosylceramide, 2.5 mM.Acceptor specificity studies suggest that the β‐3‐galactosyltransferase is specific for terminal Galβ1‐4Glc(NAc) residues and responsible for elongation of oligosaccharide chains in glycolipids. Competition studies with lactose and N‐acetylgalactosamine as acceptor substrates indicate that the transferase described here can be distinguished from the UDP‐galactose: 2‐acetamide‐2‐deoxy‐d‐galactose β‐3‐galactosyltransferase and therefore represents a novel enzyme capable of synthesizing unusual carbohydrate structures similar to those which accumulate in certain neurological diseases.
Subunits of Panulirus japonicus hemocyanin
Authors: MAKINO Nobuo 
Published: March 2005

Hexameric hemocyanin from a spiny lobster, Panulirus japonicus, comprises three major subunits (Ib, II and III) and one minor subunit (Ia), as reported in the preceding paper in this journal. It has previously been shown that the O2 equilibria of Panulirus hemocyanin can be described by a concerted model extended to three affinity states [Makino, N. (1986) Eur. J. Biochem. 154, 49–55].In this study the equilibrium binding of O2 to the reassociated subunits (Ib, II and III) was examined at various pH in the presence or absence of Ca2+ in order to test the applicability of the three‐state model to the homogeneous hexamers. The hexameric structure of the reassembled subunits was less stable than that of the native protein under the conditions examined. The model could be fitted to the O2‐binding isotherms of the homohexamers composed of the subunits II or III, if the molecular dissociation of the protein was taken into account. It was postulated that the monomeric hemocyanin has the same ligand affinity as that of the hexamer in the intermediate‐affinity state (S).The fitting of the model to the O2 binding of the subunit I was unsuccessful mainly because of the low cooperativity of the assembled subunits.
A functional five‐enzyme complex of chloroplasts involved in the Calvin cycle
Authors: GONTERO Brigitte , CÁRDENAS María Luz , RICARD Jacques 
Published: March 2005

A complex of five different enzymes: ribose‐phosphate isomerase, phosphoribulokinase, ribulose‐bisphosphate carboxylase/oxygenase, phosphoglycerate kinase and glyceraldehyde‐phosphate dehydrogenase, has been purified from spinach chloroplasts. These enzymes catalyse five consecutive reactions of the Calvin cycle, of which the two reactions catalysed by phosphoribulokinase and ribulose‐bisphosphate carboxylase/oxygenase are unique to this cycle. The five‐enzyme complex has been purified by successive chromatographies on DEAE‐Trisacryl, Sephadex G‐200 and hydroxyapatite. The homogeneity of the complex has been tested by analytical centrifugation and electrophoresis. Depending on the technique used to estimate its molecular mass, the value obtained varies between 520 kDa and 536 kDa. In addition to the five enzymes mentioned above, the complex contains a 65‐kDa polypeptide. The quaternary structure of these enzymes in the complex appears to be different from what has been described for the individual enzymes in the ‘noncomplexed state’.The five‐enzyme complex is functional as glyceraldehyde 3‐phosphate is formed from ribose 5‐phosphate. Preliminary experiments suggest that channelling of reaction intermediates occurs within the complex.
Aminopeptidase yscII of yeast
Authors: HIRSCH Hans H. , RENDUELES Paz SUÁREZ , ACHSTETTER Tilman , WOLF Dieter H. 
Published: March 2005

Mutant strains of the yeast Saccharomyces cerevisiae defective in aminopeptidase yscII were isolated by screening for reduced external activity against the chromogenic substrate lysine β‐naphthylamide. One of the selected mutant strains analyzed in detail showed wild‐type staining activity when tested at 23°C but mutant activity after exposure to 37°C, suggesting a temperature‐sensitive mutation. Electrophoretic separation of mutant crude extracts on non‐denaturing polyacrylamide gels and subsequent activity staining using lysine and leucine β‐naphthylamides as substrates revealed that in all strains isolated the same distinct activity band was affected, which corresponded to the aminopeptidase activity identified previously as aminopeptidase yscII [Achstetter, T., Ehmann, C. & Wolf, D. H. (1983) Arch. Biochem. Biophys. 226, 292–305].All mutants strains isolated fell into the same complementation group. Tetrad dissection of sporulated diploids heterozygous for the wild‐type and mutant allele resulted in a 2:2 segregation of mutant and wild‐type phenotype indicating a single gene mutation. The characteristics of the mutations analyzed point to the gene which we called APE2 as the structural gene of aminopeptidase yscII. No vital consequences of aminopeptidase yscII deficiency on cell life and differentiation could be detected. However, the enzyme seems to be involved in the cellular supply of leucine from externally offered leucine‐containing dipeptide substrates.
Membrane‐potential‐dependent changes in the stoichiometry of charge translocation by the mitochondrial electron transport chain
Authors: MURPHY Michael P. , BRAND Martin D. 
Published: March 2005

The charge/oxygen (q+/O) stoichiometry of mitochondria respiring on succinate was measured under conditions of high membrane potential (ΔΨ). The technique used was a variation of the steady‐state method of Al‐Shawi and Brand [(1981) Biochem. J. 200, 539–546]. We show that q+/O was about 2.7 at high values of ΔΨ (170 mV). As ΔΨ was lowered from 170 mV to 85 mV with the respiratory inhibitor malonate the q+/O stoichiometry increased to 6.0.A number of artefacts which could have led to an underestimation of the q+/O stoichiometry were eliminated. These included effects of any rapid change in mitochondrial volume, internal pH, activity of the endogenous K+/H+ exchanger or in H+ conductance due to changes in ΔΨ after the addition of inhibitor. The experiments presented here are the first direct demonstration that the stoichiometry of proton pumping by the mitochondrial respiratory chain changes as ΔΨ is varied.
The effect of poly(ADP‐ribose) on interactions of DNA with histones H1, H3 and H4
Authors: WĘSIERSKA‐GA̧DEK Józefa , SAUERMANN Georg 
Published: March 2005

The competition between poly(ADP‐ribose) and DNA for binding of the histones H1, H3 and H4 was studied, using a membrane filter‐binding test. Poly(ADP‐ribose) differently affected the interaction between DNA and the individual histones. While poly(ADP‐ribose) effectively competed with DNA for binding of histone H4, it equally competed with DNA for binding of histone H3 and only inefficiently competed with DNA for binding of histone H1. Moreover, preformed complexes were correspondingly affected by the addition of competing polynucleotides, thereby also indicating the reversibility of complex formation. The competition capacity of DNA for histone H4 binding did not depend on DNA size. Competition experiments with poly(A) also indicated that poly(ADP‐ribose) preferentially affected DNA‐histone H4 interaction.The significance of the differing binding properties is discussed with regard to the possible molecular function of poly(ADP‐ribose), especially with regard to its potential effect on nucleosome structure.
Identification and sequence determination of a cDNA clone for tomato pectin esterase
Authors: RAY John , KNAPP Julie , GRIERSON Don , BIRD Colin , SCHUCH Wolfgang 
Published: March 2005

Cell wall softening during tomato fruit ripening is brought about through the action of a number of pectolytic enzymes. We have reported previously the cloning and characterisation of the cDNA for the major cell wall softening (degrading) enzyme, polygalacturonase [Grierson, D., Tucker, G. A., Keen, J., Ray, J., Bird, C. R. and Schuch, W. (1986) Nucleic Acids Res. 14, 8595–8603]. We have now isolated a cDNA clone for tomato pectin esterase, an enzyme also implicated in cell wall softening. Here we report the structure of this cDNA and compare it with the structure of pectin esterase derived from amino acid sequence experiments [Markovic, O. and Jornvall, H. (1986) Eur. J. Biochem. 158, 455–462].We have used the pectin esterase cDNA clone to analyse pectin esterase gene expression during development and ripening of normal and mutant fruit.
Respiratory nitrate reductase from Paracoccus denitrificans
Authors: BALLARD Anna L. , FERGUSON Stuart J. 
Published: March 2005

The b‐type haem centres of the three (α, β and γ) subunit nitrate reductase from Paracoccus denitrificans have been analysed by redox potentiometry. Two components were identified with mid‐point potentials +95 mV and +210 mV.
Washing, in the absence of Mg2+ ions, of cytoplasmic membrane vesicles from P. denitrificans promoted selective release of nitrate reductase activity. The released enzyme was purified by chromatography and shown to contain α and β but not γ polypeptides. A haem spectrum was absent, consistent with the lack of the α subunit. The α and β polypeptides of the water‐soluble nitrate reductase had molecular masses that were identical to those of the detergent‐purified enzyme and also of the nitrate reductase in cytoplasmic membranes. This observation, together with the failure of protease inhibitors to prevent release from the membrane, indicates that the release is not related to limited proteolysis of the α and/or β polypeptides. The relative molecular mass of the water‐soluble αβ enzyme was estimated to be approximately 200000.
The water‐soluble nitrate reductase was released from intact inverted cytoplasmic membrane vesicles as jugded by loss of NADH‐NO−3 reductase activity and retention by the vesicles after washing of uncoupler‐sensitive NADH‐oxidase activity. These observations show that α and β polypeptides, and therefore the active site for nitrate reduction, are located on the cytoplasmic side of the membrane.
Attempts to reverse the nitrate reductase activity of the enzyme, using nitrite as reductant plus ferricyanide or chlorate as tested oxidants, were unsuccessful. The implications for the mechanism of the enzyme are discussed.
Existence of malfunctioning proα2(I) collagen genes in a patient with a proα2(I)‐chain‐defective variant of Ehlers‐Danlos syndrome
Authors: HATA Ryu‐Ichiro , KURATA Shun‐ichi , SHINKAI Hiroshi 
Published: March 2005

Collagen synthesis was examined in skin fibroblasts from a patient with a variant of Ehlers‐Danlos syndrome.The relative rate of collagen synthesis to total protein synthesis in the patient's fibroblasts was always one‐half of that in fibroblasts from normal controls. Total collagen synthesis, as assessed by quantification of total hydroxyproline, was also significantly lower than that of controls, indicating that the rate of collagen synthesis by the patient's fibroblasts was decreased compared with that by normal fibroblasts. Analysis of procollagen and collagen components showed the absence of the proα2(I) chain and its derivatives. Dot‐blot and Northern‐blot analyses showed the patient's fibroblasts to contain less than 10% of the mRNAs for proα2(I) found in control fibroblasts. In spite of these results, Southern blot analysis of genomic DNA indicated the presence of the same number of genes for the proα2(I) collagen chain in the patient's fibroblasts as in control fibroblasts, suggesting malfunctioning proα2(I) collagen genes as the cause for failure of the patient's fibroblasts to synthesize proα2(I) collagen chains.
Plastid‐localised seed acyl‐carrier protein of Brassica napus is encoded by a distinct, nuclear multigene family
Authors: SAFFORD Richard , WINDUST John H. C. , LUCAS Clare , DE SILVA Jacquie , JAMES Christopher M. , HELLYER Amanda , SMITH Colin G. , SLABAS Antoni R. , HUGHES Stephen G. 
Published: March 2005

Acyl‐carrier protein (ACP) is a key component involved in the regulation of fatty acid biosynthesis in plants. cDNA clones encoding ACP from Brassica napus (oil seed rape) embryos have been isolated using oligonucleotide probes derived from heterologous ACPs. Analysis of the DNA sequence data, in conjunction with N‐terminal amino acid sequence data, revealed ACP to be synthesized from nuclear DNA as a precursor containing a 51‐amino‐acid N‐terminal extension.Immunocytochemical studies showed ACP to be localised solely within the plastids of B. napus seed tissue and it would therefore appear that the N‐terminal extension functions as a transit peptide to direct ACP into these organelles. Analysis of several cDNA clones revealed sequence heterogeneity and thus evidence for an ACP multigene family. From ten cDNA clones, six unique genes, encoding five different mature ACP polypeptides, were identified. Northern blot hybridisation studies provide evidence that the seed and leaf forms of rape ACP are encoded by structurally distinct gene sets.