bluuebunny/crossref_metadata_2025_split huggingface dataset:
The lumazine synthase/riboflavin synthase complex of Bacillus subtilis
Authors: LADENSTEIN Rudolf , RITSERT Karl , HUBER Robert , RICHTER Gerald , BACHER Adelbert 
Published: March 2005

The lumazine synthase/riboflavin synthase complex of Bacillus subtilis consists of an icosahedral capsid of 60 β subunits enclosing a triplet of α subunits. An X‐ray structure of 0.32 nm resolution has been obtained for the icosahedral capsid of the native α3β60 complex [Ladenstein, R., Schneider, M., Huber, R., Bartunik, H. D., Wilson, K., Schott, K. & Bacher, A. (1988) J. Mol. Biol. 203, 1045–1070].β subunits were isolated after denaturation of the α3β60 complex and were subsequently reconstituted in a ligand‐driven reaction yielding artifactual, hollow β60 capsids with icosahedral symmetry. Hexagonal crystals (space group P6322) of the reconstituted capsids diffracted X‐rays to a resolution of 0.32 nm. Crystallographic intensity data were obtained using synchrotron radiation. Freeze‐etched electron‐microscopic images and rotation function calculations showed that the hexagonal crystal. forms of the artifactual β60 capsids and the native α3β60 complex are isomorphous. Orientation and translation parameters of the β‐subunit model were refined by XPLOR rigid‐body refinement. The electron‐density map was improved by cyclic icosahedral averaging and phase extension from 0.5–0.32 nm resolution. The β‐subunit structure was partially refined by energy minimization and crystallographic refinement (XPLOR) assuming strict icosahedral symmetry (final R factor 30.9% for data at 0.8–0.32 nm resolution).The topology and chain folding of the β subunits in the artifactual β60 capsid are similar to the native α3β60 enzyme. Structural features of the substrate‐binding site and the binding of the substrate‐analogous ligand 5‐nitro‐6‐ribitylamino‐2,4(1H,3H)‐pyrimidinedione are discussed. Ligand binding occurs at the pentamer interfaces and includes van der Waals' interactions and hydrogen bonding. The binding pocket shows a hydrophobic region which accomodates the pyrimidinedione ring and a hydrophilic region to which the ribityl side chain binds. Most amino acid residues involved in the active site are conserved as shown by sequence comparisons with the putative lumazine‐synthase genes of Escherichia coli and Photobacterium leiognathi. In the final electron‐density map, a residual density feature was tentatively assigned to a bound phosphate ion which mimics the binding of the second substrate, 3,4‐dihydroxy‐2‐butanone 4‐phosphate. This putative phosphate‐binding site involves a highly conserved amino acid sequence containing three basic residues.
Genuine and apparent cross‐reaction of polyclonal antibodies to proteins and peptides
Authors: LEDER Lukas , WENDT Hans , SCHWAB Christian , JELESAROV Ilian , BORNHAUSER Susanne , ACKERMANN Friederike , BOSSHARD Hans Rudolf 
Published: March 2005

Antiserum to a native protein may cross‐react with the corresponding denatured protein or with peptides. The cross‐reaction is either a genuine property of the antibodies or caused by antibodies produced against some unfolded protein contaminating the native protein used for immunization. Appropriate conformation‐sensitive immunoassays must be employed to distinguish a genuine from an apparent cross‐reaction.In the present study, we have analyzed critically the cross‐reaction of rabbit antisera against proteins and peptides. We have distinguished between genuine and apparent cross‐reaction with the help of the protein A antibody‐capture ELISA, a new conformation‐sensitive ELISA format. Three systems were analyzed: cross‐reaction of antisera to native yeast and horse cytochrome c with unfolded apo‐cytochrome c; cross‐reaction of antisera to a coiled‐coil leucine‐zipper peptide with a homologous random‐coil peptide obtained by introducing two proline residues into the leucine‐zipper sequence; cross‐reaction of antisera to two peptides that correspond to the N‐terminal and an internal sequence of ferredoxin:NADP+ reductase (FNR), with the native enzyme. The reaction of the anti‐(cytochrome c) sera was clearly due to antibodies produced against unfolded protein, it was an apparent and not a genuine cross‐reaction. Furthermore, the apparently cross‐reactive antibodies to horse cytochrome c did not discriminate against sequence‐related proteins from dog, beef, rabbit and pigeon. In contrast, antibodies to the leucine‐zipper peptide did cross‐react in a genuine way with the homologous random‐coil peptide, that is, the cross‐reactive antibodies do not seem to have been produced against the unfolded form of the leucine‐zipper peptide. Of the two anti‐peptide sera the one against the unstructured and highly accessible N‐terminal segment reacted strongly with the native protein. The second serum against a solvent‐accessible turn‐like sequence of FNR showed apparent cross‐reactivity: antibodies recognizing the native protein were directed against a minor conformational isoform of the free peptide and did not react with the principal form(s) of the free peptide.The generation of cross‐reactive antibodies depends on the conformational stability and intergrity of the immunogen and on the molecular form of its application, i.e., free, polymerized or carrier‐bound. The results clarify the different nature of cross‐reactivity of antisera to proteins and peptides. This knowledge is crucial if antisera are to be used as conformation‐specific probes.
Characterization of native laminin from bovine kidney and comparison with other laminin variants
Authors: LINDBLOM Anders , MARSH Tracey , FAUSER Charlotte , ENGEL Jürgen , PAULSSON Mats 
Published: March 2005

A comprehensive characterization of laminin isoforms requires access to native preparations of laminins of a defined subunit composition. For this purpose an optimized isolation procedure was developed and shown to be broadly applicable to normal mammalian tissues. The protocol does in addition yield side fractions highly enriched in collagens XII and XIV.The major laminin purified from bovine kidney is indistinguishable from mouse Engelbreth‐Holm‐Swarm (EHS) tumor laminin in electron microscopy, but contains an A chain that migrates in a position intermediate to the Ae and the Am chains on SDS/PAGE. Antisera raised against mouse EHS‐tumor laminin crossreact with B chains, but not with the A chain, of kidney laminin. Further, this A chain is not recognized by antisera raised against the Am chain. Laminins from heart and kidney both contain a significant subpopulation with a 190‐kDa polypeptide identified as the B1s chain. The Am‐containing laminins from heart and placenta differ morphologically from the Ae‐containing EHS laminin in having one short arm that does not have the characteristic globule‐rod‐globule appearance. Further, the Am‐containing laminins show a significantly higher thermal stability of the coiled‐coil α‐helical region in the long arm than does Ae‐containing EHS laminin, indicating that certain combinations of laminin chains interact more strongly than others.
Purification, characterization and inhibition of dihydropyrimidinase from rat liver
Authors: KIKUGAWA Mariko , KANEKO Masae , FUJIMOTO‐SAKATA Shigeko , MAEDA Mitsuko , KAWASAKI Koichi , TAKAGI Tatsuya , TAMAKI Nanaya 
Published: March 2005

Dihydropyrimidinase (DHPase) was purified 564‐fold over the initial rat liver extract, using heat, ammonium sulfate fractionation, DEAE‐Sepharose CL‐6B, carboxymethyl‐Sepharose CL‐6B, hydroxyapatite and Sephacryl S‐300 chromatography. The purified enzyme was shown to be homogeneous by gel electrophoresis both in the presence and absence of SDS. Its molecular mass, determined by gel filtration, was 215 kDa and the subunit mass was 54 kDa. DHPase catalyzed the reversible cyclization of 5,6‐dihydrouracil (H2Ura) to N‐carbamoyl‐β‐alanine or 5,6‐dihydrothymine (H2Thy) to N‐carbamoyl‐β‐aminoisobutyric acid.Authentic 5‐bromo‐5,6‐dihydrouracil (BrH2Ura) and commercially available H2Thy were racemic. However, these 5‐substituted 5,6‐dihydropyrimidines were hydrolyzed by over 96% and 98%, respectively, by DHPase. These results suggest that dihydropyrimidinase has no stereo specificities for 5‐substituents of H2Ura. The addition of H2Ura and H2Thy competitively inhibited the enzyme activity against BrH2Ura. However, the addition of N‐carbamoyl‐β‐alanine or N‐carbamoyl‐β‐aminoisobutyric acid showed hyperbolic mixed‐type inhibition, when BrH2Ura was used as the substrate. The values of the dissociation constants of BrH2Ura, N‐carbamoyl‐β‐alanine and N‐carbamoyl‐β‐aminoisobutyric acid were 17 μM, 0.38 mM and 0.38 mM, respectively.DHPase from the rat liver contains 4 mol Zn2+/mol active enzyme, presumably one atom/subunit. Zn2+ also inhibited the hydrolysis of BrH2Ura by the enzyme. The Ki for Zn2+ as an inhibitor of DHPase was 23 μM, and the maximum rate of inactivation was 0.057 min−1 at 37°C. H2Ura and H2Thy protected the enzyme activity from Zn2+ inactivation.
Nitric oxide reductase from Pseudomonas stutzeri
Authors: ZUMFT Walter G. , BRAUN Cornelia , CUYPERS Heinrich 
Published: March 2005

Nitric oxide (NO) reductase is an integral membrane component of the anaerobic respiratory chain of Pseudomonas stutzeri that transforms nitrate to dinitrogen (denitrification). The enzyme catalyzes the reduction of NO to nitrous oxide. The structural genes for the NO reductase complex, norC and norB, were sequenced and their organization established by primer extension and Northern blot analysis. The norCB genes encoding the cytochrome c and cytochrome b subunits of the enzyme are contiguous and transcribed as a single 2.0‐kb transcript. The promoter region has a canonical recognition motif for the transcriptional activator protein Fnr, centered at –40.5 nucleotides from the initiation site of transcription. No similarity of the derived gene products to known cytochromes of b‐ or c‐type was found in a data bank search.Post‐translational processing of the two subunits was limited to the removal of the terminal methionine to leave an N‐terminal serine in either subunit. The mature cytochrome c subunit (16508Da, 145 residues) is predicted to be a bitopic protein with a single membrane anchor. The mature cytochrome b subunit (53006Da, 473 residues) is a putatively polytopic, strongly hydrophobic membrane‐bound protein with 12 potential transmembrane segments. Several histidine and proline residues were identified with potentially structural and/or functional importance.Mutational inactivation of NO reductase by deletion of norB or the norCB genes affected strongly the in vivo activity of respiratory nitrite reductase (cytochrome cd1), but to a much lesser extent the expression level of this enzyme. In turn, mutational inactivation of the structural gene for cytochrome cd1, nirS, or loss of in vivo nitrite reduction by mutation of the nirT gene, encoding a presumed tetraheme cytochrome, lowered the expression level of NO reductase to 5–20%, but hardly its catalytic activity. The cellular concentration of NO reductase increased again on restoration of nitrite reduction in the nirS::Tn5 mutant MK202 by complementation with nirS or with the heterologous nirK gene, encoding the Cu‐containing nitrite reductase from Pseudomonas aureofaciens. Thus, NO may be required as an inducer for its own reductase. Our results show that the nitrite‐reducing system and the NO‐reducing system are not operating independently from each other but are interlaced by activity modulation and regulation of enzyme synthesis.
Distinct Signaling Pathways Mediate Phorbol‐Ester‐Induced and Cytokine‐Induced Inhibition of Erythropoietin Gene Expression
Authors: Fandrey Joachim , Huwiler Andrea , Frede Stilla , Pfeilschifter Josef , Jelkmann Wolfgang 
Published: March 2005

Hypoxia‐induced erythropoietin (Epo) production in vitro is suppressed by interleukin 1β (IL‐ 1β), tumor necrosis factor α (TNF) and phorbol esters. Herein, the Epo‐synthesizing human hepatoma cell line HepG2 was used to investigate whether protein kinase C (PKC) is involved in the inhibitory action of the cytokines. Within 1 h after the onset of hypoxia, Epo mRNA levels were markedly increased in untreated HepG2 cells as quantitated by competitive reverse transcription PCR. The cytokines IL‐1β and TNF prevented this hypoxia‐induced increase in Epo mRNA levels. In phorbol‐ester‐treated cells first inhibitory effects on Epo mRNA levels were observed only after 3 h. Western blot analyses revealed the presence of four isoenzymes of PKC in HepG2 cells. None of these isoenzymes was translocated in response to TNF or IL‐1β, suggesting that the cytokines do not activate PKC in HepG2 cells. In contrast, phorbol esters translocated and, upon prolonged exposure, down‐regulated PKC isoenzymes α and ɛ. Activation of protein kinase A by dibutyryl‐cAMP partially antagonized the cytokine‐dependent inhibition of Epo production but did not influence the inhibitory effect of phorbol esters. Endogenous cAMP levels in HepG2 cells were unchanged by cytokine treatment.Obviously, at least two signaling pathways exist that can confer inhibition of Epo production in HepG2 cells. One of these may be mediated by down‐regulation of the PKC α or ɛ isoenzyme. The other pathway, however, which is triggered by IL‐1β and TNF, is independent of PKC.
N5‐Methyltetrahydromethanopterin:Coenzyme M Methyltransferase from Methanobacterium thermoautotrophicum
Authors: Gärtner Peter , Weiss David S. , Harms Ulrike , Thauer Rudolf K. 
Published: March 2005

N
5‐Methyltetrahydromethanopterin:coenzyme M methyltransferase from methanogenic Archaea is a membrane associated, corrinoidcontaming enzyme complex which uses a methyl‐transfer reaction to drive an energy‐conserving sodium ion pump. The purified methyltransferase from Methanobacterium thermoautotrophicum (strain Marburg) exhibited a rhombic EPR signal indicative of a base–on cob(II)amide. In this form, the enzyme was almost completely inactive. Upon addition of Ti(III)citrate, which is a one–electron reductant known to reduce corrinoids to the cob(I)amide form, the EPR signal was completely quenched. In the reduced form, the enzyme was active. When the purified complex was incubated in the presence of both Ti(III) and N5‐methyltetrahydromethanopterin (CH3‐H4MPT), enzyme‐bound Co‐methyl‐5′‐hydroxybenzimidazolyl cob(III)amide was formed. Upon incubation of the methylated enzyme with either tetrahydromethanopterin or coenzyme M, the enzyme was demethylated with the concomitant formation of CH3–H4MPT and methyl‐coenzyme M, respectively. Enzyme demethylation, in contrast to enzyme methylation, was not dependent on the presence of Ti(III). Methyl transfer from the methylated enzyme to coenzyme M was essentially irreversible. These results are interpreted to that the purified enzyme complex is active only when the enzyme–bound corrinoid is in the reduced cob(I)amide form, and that methyl transfer from CH3‐H4MPT to coenzyme M proceeds via nucleophilic attack of the cobalt(I) on the N5‐methyl substituent of CH3‐H4MPT, forming an enzyme‐bound CH3‐corrinoid as intermediate.Methyl–coenzyme M formation from CH3‐H4MPT and coenzyme M, as catalyzed by the purified methyltransferase, was stimulated by sodium ions, half‐maximal activity being obtained at approximately 50 μM Na+. We therefore infer that the methyltransferase, as isolated, is capable of vectorial sodium ion translocation.
Further Evidence for Multiple Pathways in Soluble Methane‐Monooxygenase‐Catalysed Oxidations from the Measurement of Deuterium Kinetic Isotope Effects
Authors: Wilkins Patricia C. , Dalton Howard , Samuel Christopher J. , Green Jeffrey 
Published: March 2005

The data from the deuterium isotope experiments in this study show that the primary kinetic isotope effect for methane oxidation catalysed by soluble methane monooxygenase from Methylococcus capsulatus (Bath) is very small, <2. In contrast, the primary kinetic isotope effect for –CH3 group oxidation in toluene is large, >7. A mechanistic pathway in which a substrate radical is formed from hydrogen atom abstraction by a ferryl species is believed to operate for CH4, the toluene –CH3 group and similar alkanes. Direct oxygen atom addition, rather than H atom abstraction, is indicated for aromatic ring oxidations in benzene and toluene and for styrene oxide formation from styrene. Thus, more than one mechanistic pathway appears to operate in soluble methane‐monooxygenase‐catalysed reactions and, in some cases, the pathway chosen may be dictated by the substrate.In the soluble methane‐monooxygenase‐catalysed oxidation of toluene the rates of: (a) substrate dissociation from the enzyme‐substrate complex, (b) product formation and (c) product release (benzyl alcohol and p ‐cresol) from the enzyme‐product complex are comparable in magnitude. Therefore all three of these steps are partially rate–determining in the soluble methane monooxygenase catalytic cycle for toluene oxidation.
Phosphorylation of Inositol 1,4,5–Trisphosphate Analogues by 3‐Kinase and Dephosphorylation of Inositol 1,3,4,5‐Tetrakisphosphate Analogues by 5‐Phosphatase
Authors: Van Duken Peter , Lammers Aleida A. , Ozaki Shoichiro , Potter Barry V. L. , Erneux Christophe , Van Haastert Peter J. M. 
Published: March 2005

A series of 32P‐labeled d‐myo ‐inositol 1,3,4,5‐tetrakisphosphate [Ins(1,3,4,5)P4] analogues was enzymically prepared from the corresponding d‐myo–inositol 1,4,5‐trisphosphate [Ins(1,4,5)P3] analogues using recombinant rat brain Ins(1,4,5)P3 3‐kinase and [γ‐32P]ATP. Ins(1,4,5)P3 analogues with bulky groups at the 2‐OH position, substitutions of phosphates by thiophosphates and d‐6‐deoxy‐myo ‐Ins(1,4,5)P3 were tested. Using [3H]Ins(1,4,5)P3 and ATPγS, a [3H]Ins(1,3,4,5)P4 analogue with a thiophosphate at the D‐3 position was prepared. The D‐4 and/or D‐5 phosphate group seemed to be important for 3‐kinase activity, while the OH group at position 6 was not crucial. The addition of bulky groups at the 2‐OH position did not prevent phosphorylation.The labeled Ins(1,3,4,5)P4 analogues were purified and their degradation by type‐I Ins(1,4,5)P3/Ins(1,3,4,5)P4 5‐phosphatase was compared with the degradation of Ins(1,3,4,5)P4. Substitution of the phosphate group at positions 1 or 3 by a thiophosphate, or the addition of bulky groups at the 2‐OH position did not prevent degradation. d‐6–Deoxy‐myo ‐inositol 1,3,4,5‐tetrakisphosphate could not be degraded by the 5‐phosphatase, indicating the importance of the 6‐OH group for 5‐phosphatase action. d‐6‐Deoxy‐myo–inositol 1,3,4,5‐tetrakisphosphate could be an important tool in elucidating the cellular functions of Ins(1,3,4,5)P4
Synthesis, Three‐Dimensional Structure, and Specific 15N‐Labelling of the Streptococcal Protein G B1‐Domain
Authors: Boutillon Christophe , Wintjens René , Lippens Guy , Drobecq Hervé , Tartar André 
Published: March 2005

The 55‐amino‐acid B1‐domain of the streptococcal protein G shows a high binding affinity to IgG isolated from a wide range of mammalian species. Since the B1‐domain forms an extremely stable globular folding unit containing the major secondary structure elements and is devoid of proline residues and disulfide bridges, it is also a useful tool for protein folding and stability studies. Its small size makes this protein an ideal candidate for production by chemical synthesis, allowing incorporation of non‐natural amino acids with the possibility of assessing the influence of such residues on both the functional and structural characteristics of proteins.In this study, we enployed three successive chemical syntheses of the B1‐domain in order to define the optimal conditions of coupling and protection. The stepwise solid‐phase methodology using the tert ‐butyloxycarbonyl/benzyl strategy was used for this purpose.First, the sequence assembly difficulties were evaluated. After analyzing of the problems found during assembly, a second optimized synthesis was performed leading to formation of a synthetic B1‐domain with a higher yield; the synthetic B1‐domain was completely functional in its binding properties to IgG. Three orthogonal purification steps (gel‐permeation, reverse‐phase and ion‐exchange HPLC) were required to obtain a sample suitable for structural analysis by high‐resolution NMR. This study led to the conclusion that the synthetic B1‐domain adopts a three‐dimensional structure identical to that of the molecule obtained by recombinant techniques [Gronenborn, A. M., Filpula, D. R., Essig, N. Z., Achari, A., Whitlow, M., Wingfield, P. T. & Clore, G. M. (1991) Science 253, 657–661].To demonstrate the usefulness of the chemical approach for the specific introduction of labelled amino acids in the primary structure, fourteen α‐15N‐labelled amino acids were incorporated at selected critical positions during the third synthesis. This analog is the first in a series of molecules planned to study in detail the folding dynamics of the B1‐domain.
Mutational Effects on the Spectroscopic Properties and Biological Activities of Oxidized Bovine Adrenodoxin, and Their Structural Implications
Authors: Beckert Vita , Schrauber Hannelore , Bernhardt Rita , Van Dijk Alard A. , Kakoschke Christel , Wray Victor 
Published: March 2005

Of the aromatic 1H‐NMR signals of oxidized bovine adrenodoxin only those of His56 showed intrinsic chemical shift changes upon replacement of Tyr82 by Ser or Leu, that must arise from a loss of a through‐space ring‐current effect of the tyrosine ring in these mutants. Thus, of the three His residues contained in adrenodoxin, His56 is closest to Tyr82, and hence to the highly acidic determinant region of adrenodoxin that is the interaction site for adrenodoxin reductase and P‐450. The strong dependence of the fluorescence intensity of Tyr82 on the residue in position 56 supported this observation.As a consequence of this, the effects of replacement of His56 by Gln or Thr on cytochrome c reduction and cytochromes P‐45O11β (CYP11B1)‐dependent and P ‐450scc (CYP11A1)‐dependent substrate conversions were studied. No influence on Vmax values was observed for all reactions mediated by the mutants, implying His56 does not play a decisive role in the intramolecular or intermolecular electron transfer. In contrast, the Km values were increased, as was the Ks value for binding of CYP11A1 to the [H56T]adrenodoxin.The secondary structure deduced from further NMR data of adrenodoxin was compared with that of other ferredoxins. Tyr82 is in a region of the molecule containing no secondary‐structure elements. The data for Tyr82 are in keeping with the biological activities and suggests it is in a flexible, solvent‐exposed region of the molecule.