bluuebunny/crossref_metadata_2025_split huggingface dataset:
Purification of a cytochrome b containing H2:heterodisulfide oxidoreductase complex from membranes of Methanosarcina barkeri
Authors: HEIDEN Stefanie , HEDDERICH Reiner , SETZKE Edgar , THAUER Rudolf K. 
Published: March 2005

The reduction of CoM‐S‐S‐HTP, the heterodisulfide of coenzyme M (H‐S‐CoM) and N‐7‐mercaptoheptanoylthreonine phosphate (H‐S‐HTP), with H2 is an energy‐conserving step in methanogenic archaea. We report here that in Methanosarcina barkeri this reaction is catalyzed by a membrane‐bound multienzyme complex, designated H2:heterodisulfide oxidoreductase complex, which was purified to apparent homogeneity. The preparation was found to be composed of nine polypeptides of apparent molecular masses 46 kDa, 39 kDa, 28 kDa, 25 kDa, 23 kDa, 21 kDa, 20 kDa, 16 kDa, and 15 kDa and to contain 3.2 nmol cytochrome b, 70 to 80 nmol non‐heme iron and acidlabile sulfur, 5 nmol Ni, and 0.6 nmol FAD per mg protein. The 23 kDa polypeptide possessed heme‐derived peroxidase activity indicating that this polypeptide is the cytochrome b.The purified H2:heterodisulfide oxidoreductase complex catalyzed the reduction of CoM‐S‐S‐HTP with H2 at a specific activity of 6 U/mg protein (1 U = 1 μmol · min−1), the reduction of benzylviologen with H2 at a specific activity of 66 U/mg protein and the reduction of CoM‐S‐S‐HTP with reduced benzylviologen at a specific activity of 24 U/mg protein. The complex did not mediate the reduction of coenzyme F420 with H2 nor the oxidation of reduced coenzyme F420 with CoM‐S‐S‐HTP. The reduced cytochrome b in the enzyme complex could be oxidized by CoM‐S‐S‐HTP and re‐reduced by H2. The specific rates of cytochrome oxidation and reduction were too high to be resolved under our experimental conditions.The findings suggest that the H2: heterodisulfide oxidoreductase complex is composed of a F420‐non‐reducing hydrogenase, a cytochrome b and heterodisulfide reductase and that cytochrome b is a redox carrier in the electron transport chain involved in CoM‐S‐S‐HTP reduction with H2.
Human 4‐hydroxyphenylpyruvate dioxygenase
Authors: RÜETSCHI Ulla , DELLSÉN Anita , SAHLIN Pelle , STENMAN Göran , RYMO Lars , LINDSTEDT Sven 
Published: March 2005

We report the primary structure of 4‐hydroxyphenylpyruvate dioxygenase [4‐hydroxyphenylpyruvate: oxygen oxidoreductase (hydroxylating, decarboxylating)]. The work is based on the isolation of cDNA clones from human liver λgt11 libraries. Several overlapping clones covering the coding sequence were characterized. In parallel, peptides from four different digests of the purified protein were analysed for their amino‐acid sequence. These peptide sequences covered 86% of the cDNA‐derived amino‐acid sequence. This gives the sequence for a polypeptide of 392 amino acids with a calculated molecular mass of 44.8 kDa.There is more than 80% identity between the human and the pig enzymes and also between these enzymes and the F antigen from rat and the two allelic forms of this antigen from mouse. The enzyme has 53% conserved amino acids and 27% identical amino acids in common with 4‐hydroxyphenylpyruvate dioxygenase from Pseudomonas sp. P. J. 874 and 52% conserved and 28% identical residues, with a protein from Shewanella colwelliana. At the C‐terminus there is 61% identity between the seven proteins. These results indicate that these proteins are all 4‐hydroxyphenylpyruvate dioxygenases. The identity of the C‐terminus makes this part of the molecule a candidate for a functional role in the catalytic process. At conserved positions in all seven enzymes, there are two tyrosine residues and three histidine residues, i. e. amino acids which have been implicated as ligands for iron in 2‐oxoacid‐dependent dioxygenases. The gene encoding the enzyme was localized to chromosome 12q14 → qter by Southern‐blot analysis of human‐rodent somatic‐cell hybrids.
Structure of the O‐specific polysaccharide containing pentitol phosphate, isolated from Hafnia alvei strain PCM 1191 lipopolysaccharide
Authors: GAMIAN Andrzej , ROMANOWSKA Elzbieta , DABROWSKI Ursula , DABROWSKI Janusz 
Published: March 2005

The O‐specific polyaccharide of the lipopolysaccharide produced by Hafnia alvei strain PCM 1191 was shown by composition and methylation anlyses, periodate oxidation and one‐ and two‐dimensional 1H‐ and 31P‐NMR spectroscopy to be a polymer of a branched hexasaccharide repeating units having the structure:

image

where lAraol =l‐arabitolThe polysaccharide of 1191 strain has teichoic‐acid‐like character. Its peculiar feature is the presence of arabitol phosphate, a component observed for the first time in bacterial lipopolysaccharides.
Biochemical bases of the interaction of human basic fibroblast growth factor with glycosaminoglycans
Authors: COLTRINI Daniela , RUSNATI Marco , ZOPPETTI Giorgio , ORESTE Pasqua , ISACCHI Antonella , CACCIA Paolo , BERGONZONI Laura , PRESTA Marco 
Published: March 2005

In the present study we have attempted a characterization of the biochemical bases of the interaction of human basic fibroblast growth factor (bFGF) with glycosaminoglycans (GAGs) in solution. This interaction has been evidenced as the capacity of different GAGs and various sulfated compounds to protect bFGF and different bFGF mutants from tryptic cleavage.Heparin protects bFGF from trypsin digestion in a dose‐dependent fashion. Substitution by site‐directed mutagenesis of two or more basic residues with neutral glutamine residues in the aminoterminal region bFGF(27–32) or in the carboxyl‐terminal region bFGF(118–129) does not significantly affect the protective effect exerted by heparin. In contrast, heparin protection is abolished when the full region bFGF(27–32) is deleted.The capacity of different GAGs to protect bFGF from proteolytic cleavage decreases in the following order: heparin > heparan sulfate > dermatan sulfate = chondroitin sulfates A and C > hyaluronic acid = K5 polysaccharide, indicating that both the degree of sulfation and the backbone structure of GAG modulate its interaction with bFGF. This is confirmed by the different capacity of various sulfated compounds (including dextran sulfates, suramin, trypan blue, and sulfate ion) to protect bFGF from tryptic digestion. Moreover, tryptic digestion studies performed with various heparin molecules and dextran sulfates of different size, ranging from 2.0 kDa to 500 kDa, indicate that the number of bFGF molecules which interact with a single molecule of polysaccharide is related to the molecular mass of the GAG and that six hexose residues are sufficient to protect 1–2 molecules bFGF.In conclusion, our findings indicate that the capacity of GAGs to protect bFGF from tryptic cleavage depends upon their size, sulfation, distribution of the anionic sites along the chain, and structural requirements of the bFGF molecule. These studies will help to design synthetic oligosaccharides endowed with different bFGF agonist and/or antagonist activities.
Catalytic antibody activity elicited by active immunisation
Authors: GALLACHER Gerard , JACKSON Caroline S. , SEARCEY Mark , GOEL Rajiv , MELLOR Geoffrey W. , SMITH Carmen Z. , BROCKLEHURST Keith 
Published: March 2005

The hydrolytic activity of IgG purified from (a) 13 samples of ovine antiserum collected from three animals during a two‐year immunisation programme using a phosphate immunogen (comprising the amide conjugate bonded through the carboxy group of 4‐nitrophenyl 4‐carboxyme‐thylphenyl hydrogen phosphate and amino groups of keyhole‐limpet haemocyanin) and (b) a sample of ovine antiserum collected from another animal during an 18‐week immunisation programme using an analogous sulphone immunogen (comprising the amide conjugate bonded through the amino group of 4‐nitrobenzyl, 4‐(4‐aminobutoxy)benzyl sulphone and carboxyl groups of keyhole limpet haemocyanin) were evaluated kinetically by using 4‐nitrophenyl 4‐(3‐aza‐2‐oxoheptyl)phenylcarbonate and 4‐nitrophenyl 4‐(2‐hydroxyethoxy)phenyl carbonate as substrates.
Catalytic activity was found in all 13 samples of anti‐phosphate IgG but was absent in the sample of anti‐sulphone IgG as well as in all samples of IgG isolated from the serum of nonimmunised animals. These findings, taken together with the lack of catalytic activity of the antiphosphate IgG towards the 2‐nitrophenyl 4‐(3‐aza‐2‐oxoheptyl)phenyl carbonate, compel the view that the catalytic activity of the anti‐phosphate IgG preparation is entirely antibody‐mediated and is not due to contaminant hydrolytic enzymes. The fact that catalytic activity was found in all 13 samples of the anti‐phosphate IgG provides the first evidence that it is possible, as a routine, to elicit a catalytic antibody response in a host animal via active immunisation.
The nature of the, albeit small, variation in the catalytic characteristics of the anti‐phosphate IgG (increase in both kcat, the catalytic rate constant calculated as V/2[IgG] and kcat/Km, the apparent second‐order rate constant for the overall catalysed conversion of substrate to products, with increase in Km suggests simultaneous improvement in transition state binding and deterioration in substrate binding as predicted from immunogen design and the postulated general mechanistic basis of antibody catalysis.
This interpretation is supported by the difference in the values of the dissociation constant Ki for the competitive inhibition by the transition‐state analogue 4‐methylphenyl 4‐nitrophenyl hydrogen phosphate of reactions catalysed by two representative anti‐phosphate IgG samples: for the catalysis with Km= 4.5 μM, Ki= 9 nM and for that with Km= 1.3 μM, Ki= 80 nM.
4‐Nitrobenzyl 4‐(4‐aminobutoxy)benzyl sulphone, the hapten that was used to prepare the sulphone immunogen mentioned in (1), failed to inhibit the hydrolysis of 4‐nitrophenyl 4‐(3‐aza‐2‐oxoheptyl)phenyl carbonate catalysed by anti‐phosphate IgG. The sulphone moiety, therefore, does not appear to mimic adequately the carbonate in either ground‐state or transition‐state complexes.
Purification and photoaffinity labeling of fusicoccin receptors from maize
Authors: ADUCCI Patrizia , BALLIO Alessandro , FOGLIANO Vincenzo , FULLONE Maria Rosaria , MARRA Mauro , PROIETTI Noemi 
Published: March 2005

Crude soluble proteins from plasma membranes of maize shoots were purified (following the increase of fusicoccin‐binding specificity) by using an original multi‐step HPLC procedure. The method, based on a combination of adsorption, ion‐exchange and gel‐filtration chromatographies, is quick, efficient and does not damage the binding activity. It allows a 5000‐fold increase of specific activity; SDS/PAGE of purified fractions shows two doublets that correspond to proteins with apparent molecular masses of 90 kDa and 30 kDa.Crude or partially purified material was irradiated for various periods in the presence of a tritiated azido analogue of fusicoccin. The electrophoretic analysis of the irradiated material shows that with a short irradiation time only the 90‐kDa band is radiolabeled, whereas, as the irradiation time increases, a 30‐kDa band becomes radiolabeled and less radioactivity is detected in the 90‐kDa band. Irradiation of the crude material in the absence of the analogue results in a decrease of the binding capability of fusicoccin. The irradiated preparation also shows a decrease of photolabeling of the 90‐kDa band. Our data suggest that the 90‐kDa protien is the functional fusicoccin receptor. This conclusion is at variance with results of other authors who suggest the 30‐kDa protein as the true receptor.
Trimethylphosphine binding to horse‐heart and sperm‐whale myoglobins
Authors: BRUNEL Christian , BONDON Arnaud , SIMONNEAUX Gérard 
Published: March 2005

Two‐dimensional nuclear magnetic resonance techniques have been used to assign resonances corresponding to the heme pocket and several other residues of horse heart and sperm whale myoglobins ligated by trimethylphosphine. The assignment procedure was based mainly on the nuclear Overhauser effect connectivities with the ligand and the heme substituents. For quantitative measurements of Overhauser effects, application of truncated driven techniques between a proton from distal residues and methyl groups from the ligand was used to determine internuclear distances. These new results have permitted us to map the heme pockets and to investigate the conformational differences in the heme pockets between horse heart and sperm whale myoglobins. The interproton distances between distal amino acid residues and trimethylphosphine were found to be longer in horse heart myoglobin relative to those in sperm whale myoglobin. This result suggests that the size of the heme pocket is larger in horse heart myoglobin.Association and dissociation rate constants were measured for trimethylphosphine binding to myoglobins. Both values were four times larger for horse heart myoglobin than those for sperm whale myoglobin. This observation confirms the structural results obtained with NMR studies and is rationalized by a greater stabilization of a larger pocket in horse heart myoglobin relative to sperm whale myoglobin.
A novel cAMP‐dependent regulatory region including a sequence like the cAMP‐responsive element, far upstream of the human CYP21A2 gene
Authors: WATANABE Nobuaki , KITAZUME Masaru , FUJISAWA Jun‐ichi , YOSHIDA Mitsuaki , FUJII‐KURIYAMA Yoshiaki 
Published: March 2005

Deletion mutants in the 5′ upstream sequence of the human CYP21A2 gene demonstrated a novel regulatory DNA element responsible for cAMP‐dependent expression of the gene in the transient expression system using Y‐1 cells (mouse adrenocortical tumor cell line). This regulatory element (‐2574 to ‐2489 bp) was also found to exhibit a strong enhancer activity through heterologous promoters in response to cAMP and to contain a sequence like the cAMP‐responsive element (CRE) and a CAAT‐like sequence. The CRE‐like sequence has a five‐base motif (5′‐TGACG‐3′) of the palindromic CRE consensus (TGACGTCA).Competitive gel mobility shift assays using nuclear extracts of bovine adrenal cortex with sequences typical of the binding sites for the binding proteins of CRE and the CAAT‐like sequence revealed that these binding proteins, or related factors, bound to their cognate DNA binding sites in the upstream enhancer region of the CYP21A2 gene. These two enhancer elements and their cognate binding factors cooperate with previously identified tissue‐specific enhancers (adrenal‐specific protein factor and Ad4‐like sequences) and their binding factors to express a high level of cAMP‐responsive expression of the CYP21A2 gene.
Tissue‐specific molecular diversity of amidating enzymes (peptidylglycine α‐hydroxylating monooxygenase and peptidylhydroxyglycine N‐C lyase) in Xenopus laevis
Authors: IWASAKI Yasuno , SHIMOI Hiroko , SAIKI Hiromasa , NISHIKAWA Yoshiki 
Published: March 2005

We investigated the molecular diversity of the paired enzymes, peptidylglycine α‐hydroxylating monooxygenase (PHM) and peptidylhydroxyglycine N‐C lyase (PHL), involved in peptide C‐terminal amidation. Three kinds of amidating enzyme (AE) cDNAs (AE‐I, AE‐II and AE‐III) have previously been isolated from Xenopus laevis skin. While AE‐I cDNA encodes only PHM, AE‐III cDNA encodes a protein containing both PHM and PHL sequences and a transmembrane domain. On the other hand, the translated product of AE‐II has not been detected yet. Endoproteolytic cleavage of the AE‐III protein generates separated forms of PHM and PHL that are purified from X. laevis skin. Expression of AE‐III in insect cells using a baculovirus expression vector system indicated that PHM and PHL exist as a membrane‐associated, bifunctional enzyme without endoproteolysis in insect cells.Both PHM and PHL activities were detected in all the X. laevis tissues examined. Particularly, the highest levels of both activities were found in skin, brain and heart. We identified basically three types of enzymes in X. laevis; soluble PHM, soluble PHL and a membrane‐associated, bifunctional enzyme that has both PHM and PHL domains. While the skin contained soluble types of PHM and PHL, the brain and heart predominantly contained the membrane‐associated, bifunctional type. Analysis of mRNA levels by the reverse‐transcript polymerase chain reaction method and Western blot analysis using PHM‐specific antibody revealed that such molecular diversity of PHM and PHL among the tissues are produced by changing the ratio of AE‐I mRNA/AE‐III mRNA, and by endoproteolytic processing of the membrane associated precursor protein.
Detection of the in vivo incorporation of a metal cluster into a protein
Authors: GOLLAN Ute , SCHNEIDER Klaus , MÜLLER Achim , SCHÜDDEKOPF Kerstin , KLIPP Werner 
Published: March 2005

The photosynthetic bacterium Rhodobacter capsulatus has, in addition to the Mo nitrogenase, a second Mo‐independent nitrogen‐fixing system, an ‘iron‐only’ nitrogenase which is strongly repressed by molybdate. The MoO2− concentration causing 50% repression of the alternative nitrogenase in nifHDK− cells was 6 nM. If MoO42− was added to a growing nifHDK− culture which had already expressed the alternative nitrogenase, the production of ethane from acetylene, by whole cells, was stimulated dramatically. In spite of the fact that C2H6 formation decreased continously during the duration of the experiment (3 days), the total C2H6 production increased about twofold within the first 24 h, whereas the relative yeild of C2H6 increased from 2% (C2H6/C2H4X100) in the absence of MoO42−, to a maximal value of 69% in the presence of MoO42− (1 mM) after 72 h incubation. This ‘Mo effect’ appeared to be stronger the higher the MoO42− concentration in the medium and the longer the incubation time. In the presence of ReO4−, WO42− or VO43−, a similar effect did not occur.The ‘Mo effect’ was not observed in a nifHDK−nifE− double mutant which is unable to synthesize the FeMo cofactor and was diminished in a nifHDK−nifQ− mutant.Crude extracts from nifHDK− cells cultivated in the presence of MoO42−, also showed enhanced production of ethane. Component 1, purified from those extracts, displayed an S= 3/2 EPR signal which was relatively weak but characteristic for the FeMoco. These results strongly support the suggestion that the ‘Mo effect’ is a consequence of the formation of a hybrid enzyme consisting of the apoprotein of the alternative nitrogenase and the FeMo cofactor of the conventional nitrogenase.The ‘Mo effect’ was not influenced by the addition of chloramphenicol to the cultures. The occurrence of the ‘Mo effect’ appeared, therefore, to be independent of de‐novo protein synthesis. The analysis of nifE‐lacZ and nifN‐lacZ fusions proved that both genes necessary for the FeMo cofactor synthesis are also expressed under conditions of MoO42− deficiency.The possible explanations for incorporation of the FeMoco into component 1 of the alternative nitrogenase are discussed.
Biosynthesis and secretion of human interleukin 2 glycoprotein variants from baculovirus‐infected Sf21 cells
Authors: GRABENHORST Eckart , HOFER Bernd , NIMTZ Manfred , JÄGER Volker , CONRADT Harald S. 
Published: March 2005

Human interleukin 2 (IL‐2) and human IL‐2 mutant proteins, with artificially introduced N‐glycosylation or O‐glycosylation sites, have been expressed in a lepidopteran cell line (Sf21, Spodoptera frugiperda) using recombinant baculovirus vectors. Only approximately 25% of the total recombinant IL‐2 protein synthesized by Sf21 cells was secreted into the culture medium. Significant N‐terminal truncations were detected in the secreted polypeptides (up to 85% of the molecules). Alanine and proline were absent in the major truncated forms; the first 3–5 amino acids were also absent in a small proportion of the purified proteins.The introduction of potential artificial O‐glycosylation peptide sequences (¨GGKAPTPPPK¨), to the C‐terminus or between positions 80 and 81 of the IL‐2 polypeptide chain, resulted in the secretion of unglycosylated and O‐glycosylated variant forms. Fast atom bombardment mass spectrometry, compositional analysis and methylation analysis, of the tryptic glycopeptide APTPPPK, revealed the presence of either GalNAc or the disaccharide Gal(β1–3)GalNAc as the only carbohydrate constituents attached exclusively to Thr in this peptide, in a specific ratio for each individual IL‐2 mutant protein. The Gal(β1–3)GalNAc protein forms could be partially altered in vitro to mammalian‐type glycoforms by porcine liver β‐galactoside α‐2,3‐sialyltransferase in the presence of CMP‐N‐acetylneuraminic acid.An IL‐2 mutant form, with an 11‐amino‐acid peptide of human interferon‐β at position 4, which includes its only N‐glycosylation site, had exclusively truncated proximally fucosylated oligomannosidic glycans; Man3GlcNAc[Fuc(α1–6)]GlcNAc or Man2GlcNAc[Fuc(α1–6)]GlcNAc structures, in a ratio of 3:1, were detected in the secreted proteins. No evidence was obtained for the presence of secreted proteins with complex oligosaccharide chains, irrespective of the cell‐culture conditions used or the harvesting time, for infected cells with recombinant baculovirus constructs.
Docking the mitochondrial inhibitor protein IF1 to a membrane receptor different from the F1‐ATPase β subunit
Authors: LOPEZ‐MEDIAVILLA Casilda , VIGNY Hélène , GODINOT Catherine 
Published: March 2005

Monoclonal antibodies reacting with the inhibitor protein (IF1) of the mitochondrial ATPase/ATP synthase complex did not modify the IF1‐induced inhibition of soluble F1 ATPase activity. On the contrary, they increased the ATPase activity of inverted electron‐transport particles without inducing a significant release of IF1 from these particles. This suggested that IF1 could be linked to a membrane protein when it was not inhibiting the ATPase activity.IF1 antibodies have been used to show that IF1 can bind not only to the β subunit of F1‐ATPase [Klein, G., Satre, M., Dianoux, A. C. & Vignais, P. V. (1981) Biochemistry 20, 1339–1344] but also to a protein present in the inner‐mitochondrial membrane. The cross‐linking of IF1 to this membrane protein gave a product of Mr 15000–16000 that migrated differently from IF1 and from the dimer of IF1 using SDS/PAGE. When the cross‐linked product was obtained by using a cleavable cross‐linking reagent, the complex between IF1 and the docking protein was partly dissociated and free IF1 was recovered. Considering the molecular mass of IF1, this docking protein for IF1 has apparent Mr 5000–6000.The complex between IF1 and this receptor protein can be detected in low amounts by antibodies against IF1 in the absence of cross‐linking reagent. Since this complex remained in the pellet after treatment of the membrane with Triton X‐100, it should be a membrane protein. Therefore, IF1 can bind not only to its inhibitory‐binding site at the β subunit of F1, but also to a non inhibitory site which is a membrane protein of approximate Mr 5000–6000.