bluuebunny/crossref_metadata_2025_split huggingface dataset:
Lipoamide dehydrogenase from Azotobacter vinelandii
Authors: WESTPHAL Adrie H. , de KOK Arie 
Published: March 2005

The gene encoding lipoamide dehydrogenase from Azotobacter vinelandii has been cloned in Escherichia coli. Fragments of 9–23 kb from Azotobacter vinelandii chromosomal DNA obtained by partial digestion with Sau3A were ligated into the BamHI site of plasmid pUC9. E. coli TG2 cells were transformed with the resulting recombinant plasmids. Screening for clones which produced A. vinelandii lipoamide dehydrogenase was performed with antibodies raised against the purified enzyme. A positive colony was found which produced complete chains of lipoamide dehydrogenase as concluded from SDS gel electrophoresis of the cell‐free extract, stained for protein or used for Western blotting. After subcloning of the 14.7‐kb insert of this plasmid the structural gene could be located on a 3.2‐kb DNA fragment. The nucleotide sequence of this subcloned fragment (3134 bp) has been determined.The protein‐coding sequence of the gene consists of 1434 bp (478 codons, including the AUG start codon and the UAA stop codon). It is preceded by an intracistronic region of 85 bp and the structural gene for succinyltransferase. A putative ribosome‐binding site and promoter sequence are given. The derived amino acid composition is in excellent agreement with that previously published for the isolated enzme. The predicted relative molecular mass is 50223, including the FAD. The overall homology with the E. coli enzyme is high with 40% conserved amino acid residues. From a comparison with the three‐dimensional structure of the related enzyme glutathione reductase [Rice, D. W., Schultz, G. E. & Guest, J. R. (1984) J. Mol. Biol. 174, 483–496], it appears that essential residues in all four domains have been conserved.The enzyme is strongly expressed, although expression does not depend on the vector‐encoded lacZ promoter. The cloned enzyme is, in all the respects tested, identical with the native enzyme.
8‐(2‐Carboxymethylthio)‐cGMP, a site‐1‐selective compound for cGMP‐dependent protein kinase
Authors: LANDGRAF Wolfgang , RACK Michael , HEIL Wolfgang G. , HOFMANN Franz 
Published: March 2005

The cGMP analogue 8‐(2‐carboxymethylthio)‐cGMP (CMT‐cGMP) was synthesized and its binding to cGMP‐dependent protein kinase (cGMP kinase) was studied. CMT‐cGMP bound at 4°C with an over 1400‐fold higher affinity to site 1 than to site 2 of the native enzyme with apparent Kd values of 4.1 nM and 5.9 μM, respectively. The apparent selectivity for site 1 was about threefold less with the autophosphorylated enzyme and about sixfold with the catalytically active fragment of cGMP kinase. The apparent selectivity was confirmed by determination of the dissociation of [3H]cGMP from cGMP kinase in the presence of 1 μM CMT‐cGMP at 4°C. The apparent site 1 selectivity was 250‐fold at 30°C under the conditions of the phosphotransferase assay. The apparent Kd values were 47 nM and 11.7 μM for site 1 and 2, respectively. CMT‐cGMP stimulated the phosphotransferase activity of native and autophosphorylated cGMP kinase with Km values of about 80 nM. About 60% of the total catalytic rate of cGMP kinase was obtained in the presence of 1 μM CMT‐cGMP and 0.13 mM Kemptide. The apparent Km values for ATP and Kemptide were not affected. However, CMT‐cGMP activated the enzyme to the same level as cGMP when 1.3 mM Kemptide was present. CMT‐cGMP and cGMP inhibited CAMP‐stimulated autophosphorylation of cGMP kinase with IC50 values of 0.7 μM and 2 μM, respectively. Neither compound stimulated autophosphorylation of cGMP kinase by itself.These results indicate that CMT‐cGMP binds with high preference to site 1 of cGMP kinase and that occupation of site 1 may lead to expression of a partial enzyme activity.
Control by pH of urea synthesis in isolated rat hepatocytes
Authors: BOON Louis , MEIJER Alfred J. 
Published: March 2005

Control by pH of urea synthesis has been studied in isolated rat hepatocytes incubated with a physiological mixture of amino acids.Inhibition of urea synthesis by decreasing the pH of the medium was caused by diminished production of ammonia and not, as suggested in the literature, by inhibition of entry of ammonia into the ornithine cycle. The decrease by low pH of the rate of degradation of the added amino acids, that of alanine being quantitatively the most important, was accompanied by a decrease in their intracellular concentration.It is concluded that inhibited transport of amino acids across the plasma membrane of the hepatocyte is responsible, at least in part, for the fall in urea synthesis with decreasing pH.It is proposed that inhibition by low pH of other steps in the ureogenic pathway, distal to the production of ammonia, does not affect flux through the ornithine cycleper se, but rather contributes to the buffering of the intrahepatic concentration of ammonia.
Structural studies on the core and lipid A region of a 4‐amino‐L‐arabinose‐lacking Rc‐type mutant of Proteus mirabilis
Authors: RADZIEJEWSKA‐LEBRECHT Joanna , BHAT U. Ramadas , BRADE Helmut , MAYER Hubert 
Published: March 2005

The structure of the 4‐amino‐L‐arabinose‐lacking lipopolysaccharide of the Proteus mirabilis Rc‐type mutant R4, derived from wild‐type O28, was elucidated. The lipopolysaccharide core structure has previously been partially characterized. The linkage between heptose and deoxyoctulosonic acid(dOclA) is now reported, as well as the structure of the lipid A moiety of this mutant strain. Besides the tentative identification of an α‐linked glucosamine disaccharide in the lipid A backbone accompanying the usual β1 → 6‐linked glucosamine‐disaccharide, the only significant structural variation to previous studies was the lack of substitution of the C‐4′ phosphate by 4‐amino‐L‐arabinose. In addition, the substitution at C‐8 of one dOclA unit by 4‐amino‐L‐arabinose, previously reported for the R45 mutant of P. mirabilis 1959, is lacking in this R mutant. Also in addition to previous findings, the terminal unit of heptose was found to be substituted at C‐7 with phosphorylethanolamine (PEtN) and not only with phosphate, although this substitution is not complete as demonstrated by the relevant signals in 31P‐NMR. Additional studies with the wild‐type strain P. mirabilis O28 revealed the presence of 4‐amino‐L‐arabinose in both the core and the lipid A regions suggesting that the R4 mutant is defective in the biosynthesis of this amino sugar rather than in its transfer. Otherwise the lipid A regions of the mutant and the wild‐type strain show no structural differences.The following formula is proposed for the lipopolysaccharide of 4‐amino‐L‐arabinose‐lacking mutant R4/O28 P. mirabilis:
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The Paracoccus denitrificans cytochrome aa3 has a third subunit
Authors: HALTIA Tuomas , PUUSTINEN Anne , FINEL Moshe 
Published: March 2005

The presence of a third polypeptide subunit in Paracoccus cytochrome c oxidase is demonstrated. This protein (apparent molecular mass 23 kDa) binds dicyclohexylcarbodiimide in membranes of aerobically grown bacteria and in the purified enzyme.The N‐terminal amino‐acid sequence of this dicyclohexylcarbodiimide‐binding protein is identical to the deduced sequence of the COIII gene product [Raitio et al. (1987)EMBO J. 6, 2825–2833].We conclude that the aa3‐type oxidase in Paracoccus is composed of at least three subunits, which correspond to the three mitochondrially coded polypeptides in the eukaryotic enzyme.
Polyphthienoyl trehalose, glycolipids specific for virulent strains of the tubercle bacillus
Authors: DAFFÉ Mamadou , LACAVE Charlotte , LANÉELLE Marie‐Antoinette , GILLOIS Monique , LANÉELLE Gilbert 
Published: March 2005

Phthienoic acids constitute a family of dextro‐rotary odd‐numbered unsaturated fatty acids isolated exclusively from virulent strains of human and bovine tubercle bacilli. In the bacterial cell they are not free and a search for their linked form in complex wall lipids of Mycobacterium tuberculosis (strain Canetti) showed that they esterified trehalose.Structural elucidation of the major phthienoyl trehalose showed the occurrence of five acyl residues located at 2, 2′, 3′, 4 and 6′ positions of trehalose. The acyl substituents were mainly 2,4, 6‐trimethyl tetracos‐2‐enoic acid (C27 phthienoic acid) accompanied by its homologs. In addition to these branched fatty acids, straight‐chain C16 and C18 acyls composed about 20% of the substituents. The proposed structure is a new one, both for the mycobacterial‐specific glycolipid and for the substituted positions on trehalose.Other minor acyl trehaloses were detected in M. tuberculosis(strain Canetti), differing from the major component by the occurrence of an additional hydroxy fatty acid (3‐hydroxy‐2,4,6‐trimethyl tetracosanoic acid) or by the number of acyl substituents. The major glycolipid presented a weak activity in vitro on mitochondrial oxidative phosphorylation. These glycolipids and phthienoic acids could serve as virulence indicators.
Shape and location of eukaryotic initiation factor eIF‐2 on the 40s ribosomal subunit of rat liver
Authors: BOMMER Ulrich‐Axel , LUTSCH Gudrun , BEHLKE Joachim , STAHL Joachim , NESYTOVA Natalya , HENSKE Annemarie , BIELKA Heinz 
Published: March 2005

The location of initiation factor eIF‐2 and of its subunits in quaternary initiation complexes (40s‐ribosomal‐subunit · eIF‐2 · GuoPP[CH2]P· Met‐tRNA1) was investigated by immunoelectron microscopy. Quaternary complexes were fixed with glutaraldehyde and reacted with affinity‐purified polyclonal antibodies against eIF‐2α, eIF‐2β or eIF‐2γ. The dimeric immune complexes obtained by sucrose gradient centrifugation were investigated electron microscopically after negative staining. Antibody‐binding sites were observed on the interface side of the 40s ribosomal subunit in the region between the ‘head’ and the ‘body’ (neck region) of the 40s ribosomal subunit. Within this region, eIF‐2α points to the rear side, whereas eIF‐2β and eIF‐2γ point to the frontal side of the 40s subunit indicating an elongated shape of eIF‐2 about 15 nm long.By analytical ultracentrifugation of isolated eIF‐2 the sedimentation and diffusion coefficients were determined to be 6.54 S and 4074 × 10−7 cm2/s respectively. From these data, a molar mass of 122.4kg/mol and a dry volume of 147.4nm3 were calculated. For the shape of eIF‐2 a prolate ellipsoid of revolution is assumed with a maximal length of about 15nm and with an axial ratio of about 1:3.5. This conclusion is further confirmed by a calculated frictional ratio of 1.37 and a Stokes radius of about 4.54 nm.
Biosynthesis and turnover of a 34‐kDa protein growth factor in human cytotrophoblasts
Authors: ROY‐CHOUDHURY Susanta , SEN‐MAJUMDAR Anis , MURTHY Uma , MISHRA Vishnu S. , KLIMAN Harvey J. , NESTLER John E. , STRAUSS Jerome F. , DAS Manjusri 
Published: March 2005

Recently we isolated a new protein growth factor of 34 kDa from synctial membranes of human placenta. In its polypeptide molecular mass, antigenic structure, receptor binding specificity and partial amino acid sequence, it is unlike several known growth factors, hormones and other proteins. Here we report studies on its biosynthesis and turnover in cultured cytotrophoblasts from term human placenta. Expression of the 34‐kDa protein in these cells was studied by immunoprecipitation and Western blot analyses using a highly specific antibody. The experiments have produced the following results.a) Immunostaining and Western blot analyses have demonstrated the presence of immunoreactive 34‐kDa protein in isolated cytotrophoblasts. The protein is present in both freshly isolated cells and in cells that have fused in culture to form multinuclear syncytiotrophoblasts.b) Trophoblastic biosynthesis of the protein has been demonstrated by in vitro translation of cellular mRNA and by metabolic labelling experiments with intact cells.c) Pulse‐chase experiments show that biosynthesis of the protein does not involve any detectable precursors of higher or lower molecular mass.d) Studies on turnover indicate that the synthesized protein is unusually stable with a half‐life of 50–70 h.
The conformation and stability of recombinant‐derived granulocyte‐macrophage colony stimulating factors
Authors: WINGFIELD Paul , GRABER Pierre , MOONEN Peter , CRAIG Stewart , PAIN Roger H. 
Published: March 2005

The conformation and stability of recombinant‐derived human and murine granulocyte‐macrophage colony stimulating factors produced in Escherichia coli have been investigated by analytical ultracentrifugation, urea‐gradient polyacrylamide gel electrophoresis and several spectroscopic methods.The proteins were demonstrated to be physically homogeneous monomeric proteins with compact globular shapes and shown to have similar secondary structures containing both α‐helix and β‐sheet structure.The intramolecular disulphide linkages of both proteins were shown to be essential for maintaining native conformation as reduction with dithiothreitol resulted in protein unfolding.Comparison of the human E. coli‐derived (non‐glycosylated) and mammalian cell culture‐derived (glyco‐sylated) proteins by urea‐gradient electrophoresis indicated that glycosylation had no major effect on the conformational stability and kinetics of urea induced unfolding and refolding.
Kinetics, anion binding and mechanism of Co(II)‐substituted bovine muscle carbonic anhydrase
Authors: REN Xilin , SANDSTRÖM Anders , LINDSKOG Sven 
Published: March 2005

The binding of N−3 to Co(II)‐substituted bovine carbonic anhydrase III was measured at various pH values by spectrophotometric titrations. The apparent Ki values were found to increase with pH in the studied range between pH 5.8 and 8.9. The inhibition of CO2 hydration by N−3 was found to be essentially uncompetitive at all investigated pH values (pH 6.3–8.9). The Ki values for the inhibition of kcat are much smaller than those obtained in the spectrophotometric titrations indicating that an enzyme form with a high affinity for N−3, presumably having a metal‐bound H2O, accumulates in the steady state at saturating CO2 concentrations. Assuming that the low pH limit of Ki= 9 μM for the inhibition of kcat represents the affinity of N−3 for the Co(II)‐OH2 form, a pKa value near 5 can be estimated for Co(II)‐bound water from the pH dependence of N− binding in the absence of CO2.Measurements of time‐resolved absorption spectra during CO2 hydration in the presence of a low N−3 concentration showed the transient appearance of the characteristic spectrum of the enzyme‐N−3 adduct clearly demonstrating the accumulation in the steady state of an enzyme form with a high affinity for N−3. In similar experiments without inhibitor the transient formation of a spectral form corresponding to a Co(II)‐OH2 species has been demonstrated. This spectral form is rather featureless lacking the absorption maxima at 618 nm and 640 nm characteristic of the Co(II)‐OH− species. Our results strongly support the hypothesis that the rate‐limiting step in CO2 hydration catalyzed by carbonic anhydrase III is the protolysis of metal‐bound water.
Regulation of collagen metabolism and cell growth by epidermal growth factor and ascorbate in cultured human skin fibroblasts
Authors: HATA Ryu‐ichiro , SUNADA Hironobu , ARAI Katsuhiko , SATO Toshiaki , NINOMIYA Yoshifumi , NAGAI Yutaka , SENOO Haruki 
Published: March 2005

Epidermal growth factor (2–50 ng/ml), prepared from mouse submaxillary glands, stimulated growth and the synthesis of non‐collagenous proteins and hyaluronic acid, but inhibited collagen synthesis in cultured human skin fibroblasts, both stimulation and inhibition being dose‐dependent. All these effects may be intrinsic functions of the epidermal growth factor molecule, because these effects were cancelled by the co‐presence of antiserum specific for epidermal growth factor and because they were also observed following the addition of human epidermal growth factor produced urogastrone cDNA. On the other hand, l‐ascorbate (vitamin C) stimulated growth and collagen synthesis, as well as synthesis of non‐collagenous proteins, with no significant effect on hyaluronic acid synthesis.Co‐presence of epidermal growth factor and ascorbate gave additive effects on growth and protein synthesis of the cells. These results suggest that the two growth‐promoting factors, epidermal growth factor and l‐ascorbate, modulate metabolism of extracellular matrix components as well as cell growth in a quite different manner in human skin fibroblasts.
Purification and characterization of dihydropyridine receptor from rabbit skeletal muscle
Authors: SOLDATOV Nikolai M. 
Published: March 2005

Digitonin and 3‐[(3‐cholamidopropyl)dimethylammonio]‐2‐hydroxy‐1‐propane sulfonate (Chapso) were used to solubilize the receptor of dihydropyridine calcium antagonists from the transverse tubule membranes of rabbit skeletal muscle. The receptor retained the ability for selective adsorption from either detergent extract by dihydropyridine‐Sepharose. Incubation of the affinity resin with nitrendipine resulted in the elution of the receptor protein composed of two main polypeptides with molecular masses of 160 kDa and 53 kDa, as shown by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Only these two subunits were found in the receptor preparation purified to a specific dihydropyridine‐binding activity of 2500–2800 pmol/mg protein (60–70% purity) from digitonin‐solubilized membranes by a combination of wheat‐germ‐agglutinin–Sepharose, anion‐exchange and dihydropyridine‐Sepharose chromatography steps.The individual subunits were isolated in dodecyl‐sulfate‐denatured form from the preparation of the receptor, enriched by a two‐step large‐scale procedure applied to Chapso‐solubilized membranes. The 160‐kDa subunit slowly changed its apparent molecular mass to 125 kDa upon disulfide bond reduction without formation of novel peptides. This finding implies that 160‐Da subunit is cross‐linked by intramolecular S‐S bridge(s). Chemical deglycosylation with trifluoromethanesulfonic acid showed that the carbohydrate content of large and small subunits accounted for 7.5% and 6.6% by mass, respectively. The dihydropyridine receptor subunits are glycosylated through N‐glycoside bonds only. In their ratio of polar to hydrophobic amino acid residues in the amino acid composition of the receptor subunits, these polypeptides behave rather as peripheral proteins. It is suggested that the main portion of polypeptide chains is located outside the membrane in contact with solvent.
Characterisation of binding of the methoxyacrylate inhibitors to mitochondrial cytochrome c reductase
Authors: BRANDT Ulrich , SCHÄGGER Hermann , von JAGOW Gebhard 
Published: March 2005

The three E‐β‐methoxyacrylate (MOA) inhibitors oudemansin A, strobilurin A and MOA stilbene [3‐methoxy‐2(2‐styrylphenyl)propenic acid‐methylester], which differ by more than one order of magnitude in their binding affinity to the mitochondrial ubihydroquinone: cytochrome c oxidoreductase (bc1 complex), bind to a site that is not identical to the binding site for ubihydroquinone, the substrate of the outer ubiquinone reaction site (Qo centre). Although the ubihydroquinone molecule is still bound in the presence of the MOA inhibitors, its electrons cannot be transferred to the iron‐sulfur centre. A shift of the relative position of the ubihydroquinone molecule in the reaction centre due to a conformational distortion of cytochrome b induced by the binding of the MOA inhibitor seems to be the reason for the blocked electron transfer.Further analysis shows that ubihydroquinone affects the Kd values of all three MOA inhibitors tested: the values are raised by a constant factor of two, although the inhibitors bind with quite different affinity. The iron‐sulfur protein is not involved in the binding of the MOA inhibitors.These results have direct implications for the proper use of MOA inhibitors in experiments designed to analyse the structure/mechanism relationship in cytochrome c reductase. In particular, point mutations recently described in MOA‐inhibitor‐resistant mutants can no longer be taken to affect necessarily the ubihydroquinone binding site.
Involvement of various organs in the initial plasma clearance of differently glycosylated rat liver secretory proteins
Authors: GROSS Volker , HEINRICH Peter C. , vom BERG Daniela , STEUBE Klaus , ANDUS Tilo , TRAN‐THI Thuy‐Anh , DECKER Karl , GEROK Wolfgang 
Published: March 2005

The initial plasma clearance and organ distribution of α1‐acid glycoprotein and α2‐macroglobulin carrying different types of oligosaccharide, side chains was studied in rats. The differently glycosylated proteins were synthesized by rat hepatocytes in culture in the presence of tunicamycin (unglycosylated form), swainsonine (hybrid type), or 1‐deoxymannojirimycin (high‐mannose type). Deglycosylated glycoproteins (Asn‐GlcNAc) were obtained by endoglucosaminidase H treatment of high‐mannose‐type glycoproteins.Ten minutes after intravenous injection 3% of complex type, 26% of hybrid type, 84% of high‐mannose type. 64% of unglycosylated and 80% of deglycosylated α1‐acid glycoprotein disappeared from the plasma. The respective values for α2‐macroglobulin were 26%, 42%, 59% and 67%. When the clearance of total hepatic secretory proteins was examined, major differences between glycosylated and unglycosylated (glyco)proteins were found, particularly in the case of low‐molecular‐mass polypeptides.Whereas complex‐type α1‐acid glycoprotein and α2‐macroglobulin showed no accumulation in various organs, hybrid‐type α1‐acid glycoprotein and α2‐macroglobulin were present in spleen and liver. High‐mannose‐type α1‐acid glycoprotein and α2‐macroglobulin also accumulated mainly in spleen and liver. Spleen had the highest specific activity; liver, due to its larger organ mass, represented the major organ for the uptake of high‐mannose‐type glycoproteins. Competition experiments with mannan and GlcNAc–bovine‐serum‐albumin showed a mannose/GlcNAc receptor‐mediated removal. Whereas unglycosylated α1‐acid glycoprotein was taken up by the kidney, unglycosylated α2‐macroglobulin was found in the spleen. Deglycosylated glycoproteins (Asn‐GlcNAc) were removed from the plasma via two different mechanisms: firstly, clearance by the kidney similar to the unglycosylated glycoproteins; secondly, clearance by a mannose/GlcNAc receptor‐mediated uptake mainly into the spleen.We conclude that N‐linked oligosaccharide side chains are important for the plasma survival of hepatic secretory glycoproteins and that unphysiologically glycosylated forms are cleared by different mechanisms.