bluuebunny/crossref_metadata_2025_split huggingface dataset:
The O18 antigens (lipopolysaccharides) of Escherichia coli
Authors: JANN Barbara , SHASHKOV Alexander S. , GUPTA Dhirendra S. , JANN Klaus 
Published: March 2005

The O‐specific polysaccharide moieties (PS) of the O18A, O18A1, O18B, and O18B1 antigens (lipopolysaccharides, LPS) consist of L‐rhamnose (Rha), N‐acetyl‐d‐glucosamine, D‐galactose, and D‐glucose in different molar ratios. By using chemical fragmentation, methylation, as well as one‐and two‐dimensional NMR spectroscopy, the structures of these polysaccharides were found to be

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In O18A‐PS and O18A1‐PS x= 2, whereas in O18B‐PS and in O18B11‐PS x= 3. In all four polysaccharides α‐d‐Galp (residue D) is substituted at O‐3. This substituent L (residue E) is β‐d‐GlcpNAc‐(1 in O18A‐PS and O18A1‐PS and it is α‐d‐Glcp‐(1 in O18B‐PS and O18B1‐PS. Whereas there is no further substituent on the main chain of the O18A and O18B polysaccharides, in O18A1‐PS and O18B1‐PS the α‐d‐GlcpNAc residue A is substituted with α‐Glcp‐(1 (residue F), which is linked to O‐6 in O18A1‐PS and to O‐4 in O18B1‐PS. These results show that the O18 antigen comprises a group of four related LPS (O18A and O18B, with their glucosylated forms O18A1 and O18B1). The results are discussed with respect to epitope definition and biochemical implications.
Substitution of Arg214 at the substrate‐binding site of p, ‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens
Authors: VAN BERKEL Willem , WESTPHAL Adrie , ESCHRICH Klaus , EPPINK Michel , DE KOK Arie 
Published: March 2005

The gene encoding p‐hydroxybenzoate hydroxylase from Pseudomonas fluorescens was cloned in Escherichia coli to provide DNA for mutagenesis studies on the protein product. A plasmid containing a 1.65‐kbp insert of P. fluorescens chromosomal DNA was obtained and its nucleotide sequence determined. The DNA‐derived amino acid sequence agrees completely with the chemically determined amino acid sequence of the isolated protein. The enzyme is strongly expressed under influence of the vector‐encoded lac promotor and is purified to homogeneity in a simple three‐step procedure.The relation between substrate binding, the effector role of substrate and hydroxylation efficiency was studied by use of site‐directed mutagenesis. Arg214, in ion‐pair interaction with the carboxy moiety of p‐hydroxybenzoate, was replaced with Lys, Gln and Ala, respectively. The affinity of the free enzymes for NADPH is unchanged, whereas the affinity for the aromatic substrate is strongly decreased. For enzymes Arg214→Ala and Arg214→Gln, the effector role of substrate is lost. For enzyme Arg214→Lys, binding of p‐hydroxybenzoate highly stimulates the rate of flavin reduction. In the presence of substrate or substrate analogues, the reduced enzyme Arg214→Lys fails to stabilize the 4a‐hydroperoxyflavin intermediate, essential for efficient hydroxylation. Like the wild‐type, enzyme Arg214→Lys is susceptible to substrate inhibition. From spectral and kinetic results it is suggested that secondary binding of the substrate occurs at the re side of the flavin, where the nicotinamide moiety of NADPH is supposed to bind.
Differences in the proteinase inhibition mechanism of human α2‐macroglobulin and pregnancy zone protein
Authors: JENSEN Poul Erik Hyldgaard , STIGBRAND Torgny 
Published: March 2005

Different conformational states of human α2‐macroglobulin (α2M) and pregnancy zone protein (PZP) were investigated following modifications of the functional sites, i. e. the ‘bait’ regions and the thiol esters, by use of chymotrypsin, methylamine and dinitrophenylthiocyanate. Gel electrophoresis, mAb (7H11D6 and α 1:1) and in vivo plasma clearance were used to describe different molecular states in the proteinase inhibitors. In α2M, in which the thiol ester is broken by binding of methylamine and the ‘trap’ is closed, cyanylation of the liberated thiol group from the thiol ester modulates reopening of the ‘trap’ and the ‘bait’ regions become available for cleavage again. The trapping of proteinases in the cyanylated derivative indicates that the trap functions as in native α2M. In contrast, cyanylation has no effect on proteinase‐treated α2M. As demonstrated by binding to mAb, the methylamine and dinitrophenylthiocyanate‐treated α2M exposes the receptor‐recognition site, but the derivative is not cleared from the circulation in mice.The trap is not functional in PZP. In native PZP and PZP treated with methylamine, the conformational states seem similar. The receptor‐recognition sites are not exposed and removal from the circulation in vivo is not seen for these as for the PZP‐chymotrypsin complex. Tetramers are only formed when proteinases can be covalently bound to the PZP. Conformational changes are not detected in PZP derivatives in which the thiol ester is treated with methylamine and dinitrophenylthio‐cyanate.The results suggest that the conformational changes in α2M are generated by mechanisms different to these in PZP. The key structure gearing the conformational changes in α2M is the thiol ester, by which the events ‘trapping’ and exposure of the receptor‐recognition site can be separated. In PZP, the crucial step for the conformational changes is the cleavage of the ‘bait’ region, since cleavage of the thiol ester does not lead to any detectable conformational changes by the methods used.
Enzyme function in organic solvents
Authors: GUPTA Munishwar N. 
Published: March 2005

Enzyme catalysis in organic solvents is being increasingly used for a variety of applications. Of special interest are the cases in which the medium is predominantly non‐aqueous and contains little water. A display of enzyme activity, even in anhydrous solvents (water <0.02% by vol.), perhaps reflects that the minimum necessity for water is for forming bonds with polar amino acids on the enzyme surface.The rigidity of enzyme structure at such low water content results in novel substrate specificities, pH memory and the possibility of techniques such as molecular imprinting. Limited data indicates that, while enhanced thermal stability invariably results, the optimum temperature for catalysis may not change. If true in general, this enhanced thermostability would have extremely limited benefits.Medium engineering and biocatalyst engineering are relevant techniques to improve the efficiency and stability of enzymes in such low water systems. Most promising, as part of the latter, is the technique of protein engineering.Finally, this review provides illustrations of applications of such systems in the diverse areas of organic synthesis, analysis and polymer chemistry.
Fluorescent CMP‐sialic acids as a tool to study the specificity of the CMP‐sialic acid carrier and the glycoconjugate sialylation in permeabilized cells
Authors: GROSS Hans Jürgen 
Published: March 2005

The specificity of the Golgi carrier for CMP–sialic‐acids and the lumenal sialylation of glycoconjugates in mechanically permeabilized cells (semi‐intact CHO 15B cells) was studied with CMP‐activated fluorescent sialic acids as sensitive markers.Semi‐intact cells represent a well‐established cellular model for studies on the constitutive secretion pathway because the perforated plasma membrane allows membrane‐impermeable CMP–sialic‐acids to gain access to cellular organelles. The subcellular structures of semi‐intact cells remain morphologically intact and hence synthetic CMP–sialic‐acids can be assayed as substrates for the corresponding Golgi sugar‐nucleotide transporter.The results prove that the CMP–sialic‐acid carrier is able to translocate fluorescent CMP‐glycosides, despite the bulky fluoresceinyl residue located at position C5 or C9 of the sialic‐acid moiety; the data suggest a slightly higher affinity of the carrier for the C9‐substituted CMP‐glycoside, whereas the affinity of cellular sialyltransferases is fourfold higher for CMP‐5‐N‐fluoresceinyl‐aminoacetylneuraminic acid (5‐FTIUNeuAc; 5‐N‐fluoresceinylaminoneuraminic acid).Using CMP‐9‐fluoresceinylthioureido‐N‐acetylneuraminic acid (CMP‐9‐FTIUNeuAc), an easy and sensitive fluorometric assay was established for the lumenal sialylation in semi‐intact cells. Cellular proteins and gangliosides are both labelled by covalent incorporation of the fluorescent N‐acetylneuraminic acid analogue. The assay allows rapid screening for small biomolecules or proteins that influence cellular sialyl transport and sialyl transfer; the lumenal fluorescence incorporation does not require ATP or cytosolic compounds.The suitability of fluorescent CMP‐glycosides as markers for intracellular sialylation, proven in this paper, introduces the use of synthetic sialic acids for visualisation of cellular sialic acid pathways by fluorescence microscopy. Based on the data presented here, specific CMP‐N‐acetylneuraminic‐acid analogues can be produced and used for the characterization of the Golgi CMP‐sialic‐acid carrier.
Hydroperoxide metabolism in rat liver
Authors: HALLBRUCKER Christian , RITTER Markus , LANG Florian , GEROK Wolfgang , HÄUSSINGER Dieter 
Published: March 2005

Addition of t‐butylhydroperoxide (0.2 mM) to isolated perfused rat liver led to a net K+ release of 7.2 ± 0.2 μmol/g within 8 min and a net K+ reuptake of 6.6 ± 0.4 μmol/g following withdrawal of the hydroperoxide, in line with earlier findings by Sies et al. [Sies, H., Gerstenecker, C., Summer, K. H., Menzel, H. & Flohé, R. (1974) in Glutathione (Flohé, L., Benöhr, C., Sies, H., Waller, H. D., eds) pp. 261–276, G. Thieme Publ. Stuttgart]. Net K+ release roughly paralleled the amount of GSSG released from the liver under the influence of the hydroperoxide. The t‐butylhydroperoxide‐induced K+ efflux was inhibited by approximately 70% in the presence of Ba2+ (1 mM), by 30% in Ca2+‐free perfusions and was decreased by 50–60% when the intracellular Ca2+ stores were simultaneously depleted by repeated additions of phenylephrine. t‐Butylhydroperoxide‐induced K+ efflux was accompanied by a decrease of the intracellular water space by 58 ± 14 μl/g (n= 4), corresponding to a 10% cell shrinkage. The effect of t‐butylhydroperoxide on cell volume was inhibited by 70–80% in the presence of Ba2+.In isolated rat hepatocytes treatment with t‐butylhydroperoxide led to a slight hyperpolarization of the membrane at concentrations of 100 nM, but marked hyperpolarization occurred at t‐butylhydroperoxide concentrations above 10 μM. t‐Butylhydroperoxide (0.2 mM) transiently increased the portal‐perfusion pressure by 3.3 ± 0.6 cm H2O (n= 18), due to a slight stimulation of prostaglandin‐D2 release under the influence of the hydroperoxide. In the presence of Ba2+ (1 mM), t‐butylhydroperoxide increased the perfusion pressure by 12.7 ± 1.2 cm H2O (n= 9) and produced an approximately tenfold increase of prostaglandin‐D2 and thromboxane‐B2 release. Under these conditions, glucose output from the liver rose from 0.9 ± 0.03 to 2.9 ± 0.7 μmol · g−1· min−1 (n= 4) with a time course roughly resembling that of portal‐pressure increase and prostaglandin‐D2 overflow. These effects were largely abolished in the presence of ibuprofen or the thromboxane‐receptor‐antagonist BM 13.177.The t‐butylhydroperoxide effects on perfusion pressure, glucose and eicosanoid output were also enhanced in the presence of insulin or during hypotonic exposure; i.e. conditions known to swell hepatocytes, but not during hyperosmotic exposure.The data suggest that t‐butylhydroperoxide induces liver‐cell shrinkage and hyperpolarization of the plasma membrane due to activation of Ba2+‐sensitive K+ is apparently required for the t‐butylhydroperoxide‐induced K+ channel activation, and K+ efflux may be related to cellular thiol oxidation. t‐Butylhydroperoxide stimulates the formation of cyclooxygenase products. The data show that hydroperoxide effects on hepatic metabolism are more complex than previously thought; both, cell shrinkage and eicosanoid formation under the influence of t‐butylhydroperoxide may contribute to its known glycogenolytic effect.
Products of enzymatic reduction of benzoyl‐CoA, a key reaction in anaerobic aromatic metabolism
Authors: KOCH Jürgen , EISENREICH Wolfgang , BACHER Adelbert , FUCHS Georg 
Published: March 2005

Benzoyl‐coenzyme A is the most common central intermediate of anaerobic aromatic metabolism. Studies with whole cells of different bacteria and in vitro had shown that benzoyl‐CoA is reduced to alicyclic compounds, possibly via cyclohexadiene intermediates. This reaction is considered a ‘biological Birch reduction’. We have elucidated by NMR techniques the structures of six products of [ring‐13C6]benzoate reduction. The reaction is catalyzed by extracts from cells of a denitrifying Pseudomonas strain K172 anaerobically grown with benzoate and nitrate as sole carbon and energy sources. The assay mixture contained [ring‐13C6]benzoate plus traces of [U‐14C]benzoate, Mg2+, ATP, coenzyme A (CoA), and Ti(III) as reductant. The use of the multiply 13C‐labelled precursor increases the sensitivity of NMR detection and allows the analysis of crude product mixtures by two‐dimensional coherence transfer procedures such as total correlation 13C‐NMR spectroscopy and 13C‐filtered 1H‐NMR spectroscopy. The time course of product formation is consistent with the following order of events.Benzoyl‐CoA is formed from benzoate via benzoate‐CoA ligase. The first ring reduction product observed is cyclohex‐1,5‐diene‐1‐carboxyl‐CoA. The next intermediate is 6‐hydroxycyclohex‐1‐ene‐1‐carboxyl‐CoA which is derived from the diene by addition of water. Part of the diene seems to be reduced to cyclohex‐1‐ene‐1‐carboxyl‐CoA which becomes hydrated to trans‐2‐hydroxycyclohexane‐1‐carboxyl‐CoA; these two intermediates may be side products in vitro. The first non‐cyclic intermediate formed by β‐oxidation is 3‐hydroxypimelyl‐CoA. This aliphatic C7 dicarboxylic acid is proposed to be oxidized via glutaryl‐CoA and crotonyl‐CoA to three molecules of acetyl‐CoA and one molecule of CO2.A similar product pattern was observed in the benzoate‐degrading phototrophic bacterium Rhodopseudomonas palustris. This indicates that the enzymatic reduction of benzoyl‐CoA may be mechanistically similar in different anaerobes.
Purification, characterization and HPLC assay of Salmonella glucose‐1‐phosphate thymidylyltransferase from the cloned rfbA gene
Authors: LINDQUIST Lennart , KAISER Rudolf , REEVES Peter R. , LINDBERG Alf A. 
Published: March 2005

We here report on the purification and characterization of glucose‐1‐phosphate thymidylyl‐transferase, the first of four enzymes commited to biosynthesis of dTDP‐l‐rhamnose from Salmonella enterica strain LT2. The purification was greatly facilitated by the cloning of the rfbA gene encoding this enzyme. Pure enzyme was obtained by 109‐fold enrichment in three chromatography steps.The glucose‐1‐phosphate thymidylyltransferase catalyzes a reversible bimolecular group transfer reaction and kinetic measurements indicate that it acts by a ‘ping‐pong’ mechanism. The Km values for dTTP and α‐d‐glucose 1‐phosphate in the forward reaction are 0.020 mM and 0.11 mM, respectively. In the reverse reaction the Km values for dTDP‐d‐glucose and diphosphate are 0.083 mM and 0.15 mM, respectively. The enzyme also accepts UTP and UDP‐d‐glucose and α‐d‐glucosamine 1‐phosphate is accepted equally as well as α‐d‐glucose 1‐phosphate.The NH2‐terminal sequence of glucose‐1‐phosphate thymidylyltransferase agrees with the sequence predicted from the nucleotide sequence of the orf6.1 gene of the rfb gene cluster. The SDS/PAGE estimated subunit mass of 31 kDa agrees well with that calculated from the amino acid composition deduced from the nucleotide sequence of the orf6.1 gene (32453 Da).
RNA polymerase III
Authors: WILLIS Ian M. 
Published: March 2005

Recent studies on RNA polymerase III (pol III) gene transcription have provided a new awareness of the molecular complexity of this process. Fortunately, while the number of transcription components has been increasing, fundamental similarities have emerged regarding the function of eukaryotic promoter elements and the factors that bind them to form preinitiation complexes. Among these, the ability of transcription factor IIIB (TFIIIB) and pol III to transcribe the Saccharomyces cerevisiae U6 gene suggests that the concept of a minimal pol II promoter comprising a TATA box and an initiator region has a parallel in the pol III system. Furthermore, for each of the three classes of eukaryotic RNA polymerase, the assembly of transcription preinitiation complexes and, to some extent, the nature of these complexes appears to be more similar than was previously anticipated.This work highlights the novel functions and transcriptional properties of newly identified pol III genes, discusses the diversity of pol III promoter structures and presents the notion that the exclusive use of extragenic promoters by some pol III genes (so‐called type‐3 genes) may have evolved since the divergence of yeast and higher eukaryotes. Additionally, recent progress is reviewed on the identification and cloning of subunits for TFIIIC and TFIIIB. Particular emphasis is given to two components of TFIIIB, the TATA‐binding protein and a protein with TFIIB homology (PCF4), since the properties of these molecules suggest a model whereby the polymerase specificity of transcription complexes is determined.
An allosterically insensitive class of cyclohexadienyl dehydrogenase from Zymomonas mobilis
Authors: ZHAO Genshi , XIA Tianhui , INGRAM Lonnie O. , JENSEN Roy A. 
Published: March 2005

The key enzyme of tyrosine biosynthesis in many Gram‐negative prokaryotes is cyclohexadienyl dehydrogenase. The Zymomonas mobilis gene (tyrC) coding for this enzyme was cloned in Escherichia coli by complementation of a tyrosine auxotroph. The tyrC gene was 882 bp long, encoding a protein with a calculated molecular mass of 32086 Da. The Z. mobilis cyclohexadienyl dehydrogenase expressed in E. coli was purified to electrophoretic homogeneity. The subunit molecular mass of the purified enzyme was 32 kDa as determined by SDS/PAGE. The ratio of the activity of arogenate dehydrogenase to that of prephenate dehydrogenase (approximately 3: 1) remained constant throughout purification, and the two activities were therefore inseparable. The genetic and biochemical data obtained demonstrated a single enzyme protein capable of catalyzing either of two reactions. Km values of 0.25 mM and 0.18 mM were obtained for prephenate and L‐arogenate, respectively. The Km value obtained for NAD+ (0.09 mM) was the same regardless of whether the enzyme was assayed as arogenate dehdrogenase or as prephenate dehydrogenase. Unlike the corresponding enzyme of Pseudomonas aeruginosa or E. coli, the cyclohexadienyl dehydrogenase of Z. mobilis lacks sensitivity to feedback inhibition by L‐tyrosine. A typical NAD+‐binding domain was found to be located at the N‐terminus of the protein.Although the deduced amino‐acid sequence of the Z. mobilis cyclohexadienyl dehydrogenase showed relatively low identity (19–32%) with the prephenate dehydrogenases of Bacillus subtilis and Saccharomyces cerevisiae, as well as with the cyclohexadienyl dehydrogenase components of the bifunctional T‐proteins of E. coli and Erwinia herbicola, a presumptive motif was identified which may correspond to critical residues of the binding site for cyclohexadienyl substrate molecules.Immediately upstream of tyrC a portion of a gene was sequenced and found to exhibit clearcut homology of the deduced amino‐acid sequence with the B. subtilis hisH gene product. Thus, the Zymomonas gene organization is reminiscent of the linkage of genes encoding a tyrosine‐pathway dehydrogenase and a histidine‐pathway aminotransferase in B. subtilis.
Evidence for a common structure for a class of membrane channels
Authors: HOLZENBURG Andreas , JONES Phil C. , FRANKLIN Tim , PALI Tibor , HEIMBURG Thomas , MARSH Derek , FINDLAY John B. C. , FINBOW Malcolm E. 
Published: March 2005

Electron microscopic analysis of gap‐junction‐like structures isolated from an arthropod (Nephrops norvegicus) and composed of a 16‐kDa polypeptide, show the functional unit to be a star‐shaped hexamer of protein arranged around a central channel which runs perpendicular to the plane of the membrane. Estimations of the molecular volume carried out on an averaged projection are consistent with a subunit mass of 16–18 kDa. Fourier transform infrared spectroscopy indicates a high α‐helical content for the protein, supporting secondary‐structure predictions of four transmembrane α helices/monomer. The averaged projection shows a close resemblance to a hexamer of the 16‐kDa protein built on the basis of a four α‐helical bundle [Finbow, M. E., Eliopoulos, E. E., Jackson, P. J., Keen, J. N., Meagher, L., Thompson, P., Jones, P. C. & Findlay, J. B. C. (1992) Protein Eng. 5, 7–15]. The reconstructed image is also similar to that obtained for gap‐junction‐like channels isolated from a related arthropod [Homarus americanus; Sikerwar, S. S., Downing, K. H. & Glaeser, R. M. (1991) J. Struct. Biol. 106, 255–263] whose protein content was unknown but which we demonstrate may be composed of a related 16‐kDa protein.Previous studies have shown a high sequence identity of the Nephrops 16‐kDa protein with the 16‐kDa proteolipid subunit c of the vacuolar H+‐ATPase, both of which in turn bear similarity to the 8‐kDa proteolipid subunit of the F1F0‐ATP synthase. Expression of cDNA coding for the Nephrops 16‐kDa protein in Saccharomyces cerevisiae, in which the endogenous gene coding for the V‐ATPase proteolipid has been inactivated, restores V‐ATPase activity and cell growth.
Characterization and photoaffinity labeling of the ATP binding site of the ryanodine receptor from skeletal muscle
Authors: ZARKA Aliza , SHOSHAN‐BARMATZ Varda 
Published: March 2005

The photoaffinity analog of ATP, 3′‐O‐(4‐benzoyl)benzoyl‐adenosine 5′‐triphosphate (Bz2ATP) was used to covalently label and to identify the ATP binding site of the skeletal muscle ryanodine receptor. Like ATP, Bz2ATP stimulates up to fivefold the binding of ryanodine to its receptor. Photoactivation by ultraviolet light of the benzophenone group in the [α‐32P]Bz2ATP results in covalent binding of [α‐32P]Bz2ATP to the 450‐kDa polypeptide, the ryanodine receptor's subunit. An apparent molar stiochiometry of Bz2ATP to the tetrameric ryanodine receptor complex of 1.146±0.087 (n= 2) was estimated. The covalent binding of [α‐32P]Bz2ATP was inhibited by ATP and analogous compounds in the order: ATP = AdoPP[CH2]P= ADP = Ado = cAMP > AMP > ITP = GTP. Similar specificity was obtained for the stimulation of ryanodine binding by these nucleotides. ATP increased the ryanodine binding affinity by about sixfold.The polycationic dye ruthenium red, known as an inhibitor of Ca2+ release and ryanodine binding, inhibited the labeling of the ryanodine receptor by [α‐32P]Bz2ATP. Tryptic digestion of the ryanodine receptor revealed a [α‐32P]Bz2ATP‐labeled 76‐kDa tryptic fragment. Digestion of either the [α‐32P]Bz2ATP‐labeled 450‐kDa or the 76‐kDa polypeptides with S. aureus resulted in the appearance of four labeled fragments of 39, 33, 27 and 13 kDa, where the 39‐kDa fragment is the precursor of the 27‐kDa and 13‐kDa fragments. The results suggest that the regulation of Ca2+ release by ATP involves an ATP binding site(s) located on the 27‐kDa and 13‐kDa fragments of the ryanodine receptor protein.