bluuebunny/crossref_metadata_2025_split huggingface dataset:
The Heat‐Shock Protein HslVU from Escherichia Coli is a Protein‐Activated ATPase as well as an ATP‐Dependent Proteinase
Authors: Seol Jae Hong , Yoo Soon Ji , Shin Dong Hun , Shim Yoon Kyung , Kang Man‐Sik , Goldberg Alfred L. , Chung Chin Ha 
Published: July 2004

HslVU in Escherichia coli a new two‐component ATP‐dependent protease composed of two heat‐shock proteins, the HslU ATPase and the HslV peptidase which is related to proteasome β‐type subunits. Here we show that the reconstituted HslVU enzyme degrades not only certain hydrophobic peptides but also various polypeptides, including insulin B‐chain, casein, and carboxymethylated lactalbumin. Maximal proteolytic activity was obtained with a 1:2 molar ratio of HslV (a 250‐kDa complex) to HslU (a 450‐kDa complex). By itself, HslV could slowly hydrolyze these polypeptides, but its activity was stimulated 20‐fold by HslU in the presence of ATP. The ATPase activity of HslU was stimulated up to 50% by the protein substrates, but not by nonhydrolyzed proteins, and this stimulation further increased 2–3‐fold in the presence of HslV. Concentrations of insulin B‐chain that maximally stimulated the ATPase allowed maximal rates of the B‐chain hydrolysis. Furthermore, addition of increasing amounts of ADP or N‐ethylmaleimide reduced ATP and protein or peptide hydrolysis in parallel. Thus, HslVU is a protein‐activated ATPase as well as an ATP‐dependent proteinase, and these processes appear linked.Surprisingly, the protein and peptide substrates do not compete with each other for hydrolysis. Lactacystin strongly inhibits protein degradation, but has little effect on peptide hydrolysis, while the peptide aldehydes are potent inhibitors of hydrolysis of small peptides, but have little effect on proteins. Thus, the functional requirements for ATP‐dependent hydrolysis of peptides and proteins appear different.
The Structure of the Carbohydrate Backbone of the Lipopolysaccharide from Acinetobacter Strain ATCC 17905
Authors: Vinogradov Evgeny V. , Bock Klaus , Petersen Bent O. , Holst Otto , Brade Helmut 
Published: July 2004

The structure of the carbohydrate backbone of the lipopolysaccharide from Acinetobacter strain ATCC 17905 was studied. After deacylation of the lipopolysaccharide, a mixture of two compounds (ratio approximately 2:1) was isolated by high‐performance anion‐exchange chromatography, the structures of which were determined by NMR spectroscopy and electrospray‐mass spectrometry as
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[Sug, 3‐deoxy‐d‐manno‐2‐octulopyranosonic acid (Kdo) in oligosaccharjde 1 (major portion) and d‐glyc‐ero‐d‐talo‐2‐octulopyranosonic acid (Ko) in oligosaccharide 2 (minor portion)]. All monosaccharide residues also possess the d‐configuration and are present in the pyranose form.
Seryl‐tRNA Synthetase from the Extreme Halophile Haloarcula marismortui
Authors: Taupin Claire M.‐J. , Härtlein Michael , Leberman Reuben 
Published: July 2004

The seryl‐tRNA synthetase from the extreme halophilic archaebacterium Haloarcula marismortul, belonging to the group Euryarchaeota, has been purified and its hyperhalophilic behavior demonstrated by activity and stability tests in KCl, NaCl and MgCl2, solutions. Although the natural external environment of this archaebacterium is rich in sodium ions and poor in potassium ions, the converse being the case in the bacterial cytosol, there is no large significant difference in activity and stability in vitro of the enzyme between solutions of NaCl and KCl. Low, but not high, concentrations of MgCl2, stabilize the enzyme. The enzyme aminoacylates tRNA from Escherichia coli even under the high salt conditions of the assay. A fluorescence study indicated that low salt denaturation of the hyperhalophilic enzyme is a biphasic process. The hyperhalophilic enzyme demonstrated immunological reactivity with antisera against the catalytic domain of the homologous E. coli enzyme.The gene coding for the H. marismortui enzyme has been isolated and sequenced. The derived amino acid sequence is the first of a hyperhalophilic aminoacyl‐tRNA synthetase. The wild‐type gene and a mutant gene with a deletion of the halophile‐specific insertion were expressed in E. coli using the T7 RNA polymerase and the ThiofusionTM expression systems. None of the expressed proteins were enzymically active. A structural model has been produced by comparison with other seryl‐tRNA synthetases which illustrates the high negative‐charge density of the surface of the hyperhalophilic enzyme.
Structural and Serological Characterisation of the O‐Specific Polysaccharide from Lipopolysaccharide of Acinetobacter calcoaceticus Strain 7 (DNA Group 1)
Authors: Vinogradov Evgeny V. , Pantophlet Ralph , Haseley Simon R. , Brade Lore , Holst Otto , Brade Helmut 
Published: July 2004

S‐form lipopolysaccharide was isolated by phenol/water extraction from a strain of Acinetobacter calcoaceticus (DNA group 1). The structure of the O‐antigenic polysaccharide was determined by compositional analysis and NMR spectroscopy of the de‐O‐acylated lipopolysaccharide. The isolated polysaccharide obtained after hydrolysis of lipopolysaccharide in 0.01 M trifluoroacetic acid has the following structure:
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in which Pyr is pyruvate. The O‐acetyl substitution of D‐Gal was non‐stoichiometric. The O‐antigen was specifically recognised in western blots by polyclonal rabbit antisera.
Mitochondria1 Asparaginyl‐tRNA Synthetase Encoded by the Yeast Nuclear Gene YCR24c
Authors: Landrieu Isabelle , Vandenbol Micheline , Härtlein Michael , Portetelle Daniel 
Published: July 2004

One of the open reading frames located on yeast Saccharomyces cerevisiae chromosome III, YCR24c, appeared to code for a protein of unknown function, but the predicted sequence showed similarity with asparaginyl‐tRNA synthetase from Escherichiu coli, with 3896 amino acid identity. There is a putative mitochondrial targeting signal at the N‐terminus of the YCR24c product. Northern blot analysis of total RNA from a wild‐type strain Σ1278b confirmed that YCR24c was transcribed. Disruption of the chromosomal copy of YCR24c in a respiratory‐competent haploid cell induced a petite phenotype, but did not affect cell viability. This respiratory‐defective phenotype is typical for a mutation in a nuclear gene that induces a non‐functional mitochondrial protein synthesis system.The protein encoded by YCR24c was expressed in Escherichia coli in a histidine‐tagged form and isolated. The enzyme aminoacylated unfractionated Escherichiu coli tRNA with asparagine. These results identified YCR24c as the structural gene for yeast mitochondrial asparaginyl‐tRNA synthetase.
Bacillus Thermoamyloliquefaciens KP1071 α‐Glucosidase II is a Thermostable Mr 540000 Homohexameric α‐Glucosidase with both Exo‐α‐1,4‐Glucosidase and Oligo‐1,6‐Glucosidase Activities
Authors: Suzuki Yuzuru , Nobiki Masayoshi , Matsuda Michio , Sawai Takakazu 
Published: July 2004

α‐Glucosidase II of the facultative thermophile Bacillus thermoamyloliquefaciens KP1071 (FERM‐P8477; growth over 30–66°C) was purified to a homogeneous state. Its Mr was estimated as 90000 by SDS/PAGE. However, the enzyme behaved as an active Mr 540000 protein on gel filtration with each of two gels of different matrices as well as on gel electrophoresis under native conditions. The enzyme was not glycosylated. Its isoelectric point was estimated as 5.7. The N‐terminal sequence of 20 residues was determined as Ala1‐Ile‐Gln‐Pro‐Glu‐Gln‐Asp‐Asp‐Lys‐Thr‐Gln‐Glu‐Asp‐Gly‐Tyr‐Ile‐Asp‐Ile‐Gly‐Asn20. The sequence did not resemble those of procaryotic and eucaryotic proteins hitherto reported including the monomeric exo‐α‐1,4‐glucosidase and the monomeric oligo‐1,6‐glucosidase from the same microorganism. The α‐glucosidase II had no antigenic group shared with the latter two enzymes.Analysis of substrate specificity showed that the α‐glucosidase II has dual activity towards oligo‐1,6‐glucosidases and exo‐α‐1,4‐glucosidases, but its preference is for non‐reducing terminal α‐1,4 glucosidic bonds in substrates. Kinetic studies proved that both activities are attributed to the same catalytic site. The enzyme was most active at 81 °C and pH 7.0. Its half‐life at pH 6.8 was 10 min at 81 °C, and 5 h at 55 °C in 6.4 M urea, 26% ethanol or 2.5% SDS.We suggest that the α‐glucosidase II is a thermostable, homohexameric enzyme of origin distinct from the exo‐α‐1,4‐glucosidase and the oligo‐1,6‐glucosidase present in the same strain.
cDNA Cloning and Expression of Chicken Aminopeptidase H, Possessing Endopeptidase as well as Aminopeptidase Activity
Authors: Adachi Hideki , Tsujimoto Masafumi , Fukasawa Masayoshi , Sato Yuji , Arai Hiroyuki , Inoue Keizo , Nishimura Toshihide 
Published: July 2004

Chicken aminopeptidase H is a cysteine protease possessing endopeptidase as well as aminopeptidase activity [Rhyu, M. R., Nishimura, T., Kato, Y., Okitani, A. & Kato, H. (1992)Eur. J. Biochem. 208, 53–59]. This enzyme exhibits molecular masses of 400 kDa on gel filtration and 52 kDa on SDS/PAGE, indicating that it consists of eight subunits with the same molecular mass.In the current study, we cloned the cDNA for the catalytic subunit of chicken aminopeptidase H. The open reading frame of the aminopeptidase H gene consists of 1362 base pairs encoding a 52‐kDa protein consistent with the molecular mass determined on SDS/PAGE; the deduced amino acid sequence contains all the partial sequences determined for the purified enzyme. The sequence is similar to that of the bleomycin hydrolase of rabbit lung, which has been partially determined. The recombinant 52‐kDa protein expressed in COS7 cells exhibited both aminopeptidase and endopeptidase activities, which were inhibited by monoiodoacetic acid. Furthermore, the expression of aminopeptidase H in COS7 cells was also recognized on immunoblotting. This gene is the first one for aminopeptidase H in an animal tissue whose sequence has been completely determined.
Structural and Serological Characterization of the O‐Antigenic Polysaccharide of the Lipopolysaccharide from Acinetobacter Strain 90 Belonging to DNA Group 10
Authors: Haseley Simon R. , Holst Otto , Brade Helmut 
Published: July 2004

Water‐soluble lipopolysaccharide (phenol/water extraction) isolated from Acinetobacter strain 90, which belongs to DNA group 10, was hydrolysed with 1 % acetic acid, ultracentrifuged, and water‐soluble products finally eluted from a Sephadex G‐50 column. The major fraction, a polysaccharide, contained D‐Gal, D‐GlcNAc, D‐GalNAc, and 4,6‐dideoxy‐4‐[(R)‐3‐hydroxybutyramido]‐D‐galactose (Fuc4NBuOH). The polysaccharide was characterised by means of monosaccharide analyses, Smith‐degradation, N‐deacetylation/deamination, and NMR studies, and was shown to have a branched pentasaccharide repeating unit.
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This structure was specifically recognized in western blots and enzyme immunoassays by polyclonal rabbit antisera.
Structural Characterization of Bovine Collectin‐43
Authors: Rothmann Annette B. , Mortensen Hanne D. , Holmskov Uffe , Højrup Peter 
Published: July 2004

Bovine collectin‐43 (CL‐43), the most recently disclosed member of the collectin group, has been characterized structurally at the protein level by a combination of mass spectrometry and protein sequencing. The molecular mass of reduced CL‐43 was determined by the use of mass spectrometry to be 33.6±0.1 kDa. Furthermore, the mass spectrum showed the presence of a truncated version of the poly‐peptide, which has also previously been shown by SDS/PAGE and N‐terminal sequencing. N‐terminal Edman degradation of peptides from a tryptic digestion of native CL‐43 verified the published sequence derived from cDNA studies and partial protein sequencing [Lim, B.‐L., Willis, A. C., Reid, K. B. M., Lu, J., Lauersen, S. B., Jensenius, J. C. & Holmskov, U. (1994) J. Biol. Chem. 269, 11820–118241 with two exceptions. Using mass spectrometry and N‐terminal sequencing, a large number of post‐translational modifications were found in the collagen‐like region (repetitive Gly‐Xaa‐Yaa sequence). All proline residues located in the Yaa‐position in the collagen‐like region were found to be partially hydroxylated while all lysine residues in the Yaa position were fully hydroxylated and glycosylated. The glycosylation was determined as glycosyl‐galactosyl O‐linked to a hydroxylated lysine residue. Mass spectrometric analysis of a peptic digest of the N‐terminal tryptic peptide revealed that the three polypeptide chains were disulphide linked in a rather surprising pattern. The cysteine residues were inter‐chain disulphide linked by Cys15 in polypeptide chain 1 to Cys15 in polypeptide chain 2, Cys20 in chain 2 to Cys20 in chain 3 and Cys20 in chain 1 to Cys15 in chain 3.The four cysteine residues at the C‐terminus were intra‐chain disulphide linked, Cys204 to Cys299 and Cys277 to Cys291, as expected for a C‐type lectin.
13C‐NMR Studies of Transmembrane Electron Transfer to Extracellular Ferricyanide in Human Erythrocytes
Authors: Himmelreich Uwe , Kuchel Philip W. 
Published: July 2004

Human erythrocytes are known to reduce ferricyanide (hexacyanoferrate) [Fe(CN)6]3‐ to ferrocyanide [Fe(CN)6]2‐ in an extracellular reaction that involves the transmembrane transfer of reducing equivalents; potentially these could be either electrons from NADH, formed in glycolysis inside the cells or transmembrane exchange of reduced solutes. The 13C‐NMR resonance of [Fe(13CN)6]3 (which was synthesised in our laboratory) was seen to be very broad while that of ferrocyanide was narrow. This phenomenon formed the basis of a simple non‐invasive procedure to study ferricyanide reduction in high‐haematocrit suspensions of erythrocytes. The method should be directly applicable to other cell types.In a series of experiments, erythrocyte metabolism was studied in the presence of ferricyanide, using 1H, 13C, and 31P NMR spectroscopy. Incubating the cells with 13C‐labelled glucose enabled the rate of ferricyanide reduction, glucose utilisation, and lactate and bicarbonate production to be measured simultaneously. Various metabolic states were imposed as follows: glycolysis was inhibited with F− and iodoacetate; glucose transport was inhibited with phloretin and cytochalasin B; and anion transport was inhibited with dinitrostilbene 2,2′‐disulfonate and p‐chloromercuriphenyl sulfonate. Earlier work was confirmed, showing that ascorbate is intimately involved in the reduction reaction; but its main action appears not to be mediated by membrane transport but in a membrane‐associated redox‐protein complex that is functionally linked to glycolysis. Also, large differences (factors of three) in the rate of the reduction reaction were recorded in erythrocytes from different, apparently healthy, donors.
Spectroscopic Probes of the Individual and Combined Effects of Triton X‐100 and Chloroform on Serum Albumins and Serum‐Albumin · Bilirubin Complexes
Authors: Patra Samir Kumar , Pal Medini Kanta 
Published: July 2004

The effects of the non‐ionic surfactant Triton X‐100 on the biphasic induced CD spectra of bilirubin complexes of human and bovine serum albumins (HSA and BSA) are divergent. While Triton X‐100 inverts the induced CD spectrum of HSA · bilirubin, this surfactant enhances the ellipticity values of induced CD of BSA · bilirubin without inversion. The effect of Triton X‐100 on the characteristic ultraviolet‐CD spectra of the albumins are similar; both the albumins are denatured from their native globular structures. The anionic surfactant SDS, unlike non‐ionic Triton X‐100, dislodges the ligand from its protein complexes, indicating that both electrostatic and hydrophobic forces are involved in binding of bilirubin to the albumins.The aprotic solvent chloroform inverts the biphasic induced CD spectra of HSA · bilirubin and BSA · bilirubin, whereas CHCl3 has relatively little effect on the ultraviolet CD spectra of the albumins. The combined effect of Triton X‐100 and CHCl3 shows that the effect of CHCl3 predominates over that of Triton X‐100. The perturbing effects of Triton X‐100 and CHCl3 on the CD or induced CD spectra of the proteins or their bilirubin complexes are reversible, and independent of the order in which components were added. The observations suggest that the denaturation of the albumins by Triton X‐100 or solvation of CHCl3 within albumins markedly alter the internal topography or dynamics of the receptor sites, triggering alterations of the chirality of the bound pigment in sign and/or magnitude.
Man9‐Mannosidase from Pig Liver is a Type‐II Membrane Protein That Resides in the Endoplasmic Reticulum
Authors: Bieberich Erhard , Treml Kornelia , Völker Christof , Rolfs Andreas , Kalz‐Füller Burga , Bause Ernst 
Published: July 2004

Man9‐mannosidase, one of three different α1,2‐exo‐mannosidases known to be involved in N‐linked oligosaccharide processing, has been cloned in λgt10, using a mixed‐primed pig liver cDNA library. Three clones were isolated which allowed the reconstruction of a 2731‐bp full‐length cDNA. The cDNA construct contained a single open reading frame of 1977 bp, encoding a 659‐residue polypeptide with a molecular mass of ≈73 kDa. The Man9‐mannosidase specificity of the cDNA construct was verified by the observation that all peptide sequences derived from a previously purified, catalytically active 49‐kDa fragment were found within the coding region. The N‐terminus of the 49‐kDa fragment aligns with amino acid 175 of the translated cDNA, indicating that the catalytic activity is associated with the C‐terminus. Transfection of COS 1 cells with the Man9‐mannosidase cDNA gave rise to a > 30‐fold over‐expression of a 73‐kDa protein whose catalytic properties, including substrate specificity, susceptibility towards α‐mannosidase inhibitors and metal ion requirements, were similar to those of the 49‐kDa enzyme fragment. Thus deletion of 174 N‐terminal amino acids in the 73‐kDa protein appears to have only marginal influence on the catalytic properties.Structural and hydrophobicity analysis of the coding region, as well as the results from tryptic degradation studies, point to pig liver Man9‐mannosidase being a non‐glycosylated type‐II transmembrane protein. This protein contains a 48‐residue cytosolic tail followed by a 22‐residue membrane anchor (which probably functions as internal and non‐cleavable signal sequence), a lumenal ≈100‐residue‐stem region and a large 49‐kDa C‐terminal catalytic domain. As shown by immuno‐fluorescence microscopy, the pig liver enzyme expressed in COS 1 cells, is resident in the endoplasmic reticulum, in contrast to COS 1 Man9‐mannosidase from human kidney which is Golgi‐located [Bieberich, E. & Bause, E. (1995)Eur. J. Biochem. 233, 644–649]. Localization of the porcine enzyme in the endoplasmic reticulum is consistent with immunoelectron‐microscopic studies using pig hepatocytes. The different intracellular distribution of pig liver and human kidney Man9‐mannosidase is, therefore, enzyme‐specific rather than a COS‐1‐cell‐typical phenomenon. Since we oberserve ≈81% sequence similarity between the two α‐mannosidases, we deduce that the localization in either endoplasmic reticulum or Golgi is likely to be sequence‐dependent.
Comparative Biochemical Characterization of the Iron‐Only Nitrogenase and the Molybdenum Nitrogenase from Rhodobacter Capsulatus
Authors: Schneider Klaus , Gollan Ute , Dröttboom Melanie , Selsemeier‐Voigt Sabine , Müller Achim 
Published: July 2004

The component proteins of the iron‐only nitrogenase were isolated from Rhodobacter capsulatus (ΔnifHDK, μModABCD strain) and purified in a one‐day procedure that included only one column‐chromatography step (DEAE‐Sephacel). This procedure yielded component 1 (FeFe protein, Rc1Fe), which was more than 95% pure, and an approximately 80 % pure component 2 (Fe protein, Rc2Fe). The highest specific activities, which were achieved at an Rc2Fe/Rc1Fe molar ratio of 40:1, were 260 (C2H4 from C2H2), 350 (NH3 formation), and 2400 (H2 evolution) nmol product formed min−1 mg protein−1. The purified FeFe protein contained 26 ± 4 Fe atoms; it did not contain Mo, V, or any other heterometal atom.The most significant catalytic property of the iron‐only nitrogenase is its high H2‐producing activity, which is much less inhibited by competitive substrates than the activity of the conventional molybdenum nitrogenase. Under optimal conditions for N2 reduction, the activity ratios (mol N2 reduced/mol H2 produced) obtained were 1:1 (molybdenum nitrogenase) and 1:7.5 (iron nitrogenase). The Rc1Fe protein has only a very low affinity for C2H2. The Km value determined (12.5 kPa), was about ninefold higher than the Km for Rc1Mo (1.4kPa). The proportion of ethane produced from acetylene (catalyzed by the iron nitrogenase), was strictly pH dependent. It corresponded to 5.5% of the amount of ethylene at pH 6.5 and was almost zero at pH values greater than 8.5.In complementation experiments, component 1 proteins coupled very poorly with the ‘wrong’ component 2. Rc1Fe, if complemented with Rc2Mo, showed only 10–15% of the maximally possible activity. Cross‐reaction experiments with isolated polyclonal antibodies revealed that Rc1Fe and Rc1Mo are immunologically not related.The most active Rc1Fe samples appeared to be EPR‐silent in the Na2S2O4‐reduced state. However, on partial oxidation with K3[Fe(CN)6] or thionine several signals occurred. The most significant signal appears to be the one at g= 2.27 and 2.06 which deviates from all signals so far described for P clusters. It is a transient signal that appears and disappears reversibly in a redox potential region between ‐100 mV and +150 mV. Another novel EPR signal (g= 1.96, 1.92, 1.77) occurred on further reduction of Rc1Fe by using turnover conditions in the presence of a substrate (N2, C2H2, H+).
A Simple Mathematical Model and Practical Approach for Evaluating Citric Acid Cycle Fluxes in Perfused Rat Hearts by 13C‐NMR and 1H‐NMR Spectroscopy
Authors: Tran‐Dinh S. , Hoerter J. A. , Mateo P. , Bouet F. , Herve M. 
Published: July 2004

We propose a simple mathematical model and a practical approach for evaluating the flux constant and the absolute value of flux in the citric acid cycle in perfused organs by 13C‐NMR and 1H‐NMR spectroscopy. We demonstrate that 13C‐NMR glutamate spectra are independent of the relative sizes of the mitochondrial and cytosolic compartments and the exchange rates of glutamates, unless there is a difference in 13C chemical shifts of glutamate carbons between the two compartments.Wistar rat hearts (five beating and four KCl‐arrested hearts) were aerobically perfused with 100% enriched [2‐13C]acetate and the kinetics of glutamate carbon labeling from perchloric acid extracts were studied at various perfusion times. Under our experimental conditions, the citric acid cycle flux constant, which represents the fraction of glutamate in exchange with the citric acid cycle per unit time, is about 0.350 ± 0.003 min−1 for beating hearts and 0.074 ± 0.004 min−1 for KCl‐arrested hearts. The absolute values of the citric acid flux for beating hearts and for KCl‐arrested hearts are 1.06 ± 0.06 μmol · min−1· mg−1 and 0.21 ± 0.02 μmol · min−1· g−1, respectively. The fraction of unlabeled acetate determined from the proton signal of the methyl group is small and essentially the same in beating and arrested hearts (7.4 ± 1.7% and 8.8 ± 2.1%, respectively). Thus, the large difference in the Glu C2/C4 between beating and arrested hearts is not due to the important contribution from anaplerotic sources in arrested hearts but simply to a substantial difference in citric acid cycle fluxes. Our model fits the experimental data well, indicating a fast exchange between 2‐oxoglutarate and glutamate in the mitochondria of rat hearts. Analysis of the flux constant, calculated from the half‐time of glutamate C4 labeling given in the literature, allows for a comparison of the citric acid flux for various working conditions in different animal species.