bluuebunny/crossref_metadata_2025 huggingface dataset:
The Interaction of Hevein with N‐acetylglucosamine‐containing Oligosaccharides
Authors: Asensio Juan Luis , Can̄ada Francisco Javier , Bruix Marta , Rodriguez‐Romero Adela , Jimenez‐Barbero Jesús
Published: March 2005

The three‐dimensional structure of hevein, a small protein isolated from the latex of Hevea brasiliensis (rubber tree), in water solution has been obtained by using 1H‐NMR spectroscopy and dynamic simulated annealing calculations. The average root‐mean‐square deviation (rmsd) of the best 20 refined structures generated using DIANA prior to simulated annealing was 0.092 nm for the backbone atoms and 0.163 nm for all heavy atoms (residues 3–41). The specific interaction of hevein with. N‐acetylglucosamine‐containing oligosaccharides has also been analyzed by 1H‐NMR. The association constants, Ka, for the binding of hevein to GlcNAc, chitobiose [GlcNAc‐β(1→4)‐GlcNAc], chitotriose [GlcNAc‐β(1→4)‐GlcNAc‐β(1→4)‐GlcNAc], and GlcNAc‐α(1→6)‐Man have been estimated from 1H‐NMR titration experiments. Since the measured Ka values for chitobiose binding are almost identical with and without calcium ions, it is shown that these cations are not required for sugar binding. The association increases in the order GlcNAc‐α(1→6)‐Man ≤ GlcNAc < chitobiose < chitotriose. The equilibrium thermodynamic parameters entropy and enthalpy of binding, S° and H°, for the hevein–chitobiose and hevein–chitotriose associations have been obtained from van't Hoff analysis of the temperature dependence of the Ka values between 25–40°C. The driving force for the binding process is provided by a negative H° which is partially compensated by a negative S°. These negative signs seem to indicate that hydrogen bonding and van der Waals forces are the major interactions stabilizing the complex. Protein–carbohydrate nuclear Overhauser enhancements have allowed a three‐dimensional model of the hevein‐chitobiose complex to be built. From inspection of this model, a hydrogen bond between Ser19 and the non‐reducing N‐acetyl carbonyl group is suggested, as well as between Tyr30 and HO‐3 of the same sugar residue. The N‐acetyl methyl group of the non‐reducing GlcNAc displays non‐polar contacts to the aromatic Tyr30 and Trp21 residues. In addition, the higher affinities deduced for the β‐linked oligosaccharides with respect to GlcNAc and GlcNAc‐α(1→6)‐Man can be explained by favourable stacking of the second β‐linked GlcNAc moiety and Trp21.
Phosphorylation Studies on Rat p53 Using the Baculovirus Expression System
Authors: Fuchs Bergund , Hecker Doris , Scheidtmann Karl Heinz
Published: March 2005

To elucidate the role of phosphorylation of p53 we used the baculovirus expression system to obtain high yields of protein eventually in distinct phosphorylation states. Initially, we obtained only marginal phosphorylation, despite high levels of expression. Two‐dimensional phosphopeptide maps exhibited the same pattern as known from rat cells although some sites were underrepresented. Coexpression of simian virus 40 (SV40) large T antigen or cyclin‐dependent kinases, cdc2 or cdk2, had only marginal effects on the phosphorylation state of p53. However, when we employed the phosphatase inhibitor okadaic acid, overall phosphorylation of p53 was drastically enhanced in a dose‐dependent manner and resembled that of p53 from SV40‐transformed rat cells. This hyperphosphorylation resulted in enhanced binding of a consensus oligonucleotide as revealed by electrophoretic mobility shift assays. To assess the role of individual phosphorylation sites, we generated a set of mutants at putative or identified sites. All mutants retained the ability to bind wild‐type conformation‐specific antibody Pab1620, to complex with SV40 large T antigen, and to bind to the consensus oligonucleotide. Moreover, most mutants exhibited enhanced DNA binding upon okadaic acid treatment, except for a mutant at the cdk site which failed to do so. These data show that: (a) insect cells contain all the protein kinases necessary for phosphorylation of a mammalian protein, p53; (b) in insect cells the ratio of kinase/phosphatase activities differs from that in mammalian cells so that underphosphorylation of recombinant proteins in this system may result from high phosphatase activities rather than saturation of kinases with recombinant substrate; (c) the system can be manipulated to obtain subpopulations of recombinant protein in a desired phosphorylation state, and (d) phosphorylation may regulate the DNA‐binding activity of p53.
Characterization of Monoclonal Antibodies that Strongly Inhibit Electrophorus Electricus Acetylcholinesterase
Authors: Remy Marie Hélène , Frobert Yveline , Grassi Jacques
Published: March 2005

In this study, we describe three different monoclonal antibodies (mAbs Elec‐403, Elec‐408, and Elec‐410) directed against Electrophorus electricus acetylcholinesterase (AChE) which were selected as inhibitors for this enzyme. Two of these antibodies (Elec‐403 and Elec‐410), recognized overlapping but different epitopes, competed with snake venom toxin fasciculin for binding to the enzyme, and thus apparently recognized the peripheral site of AChE. In addition, the binding of Elec‐403 was antagonized by 1,5‐bis(4‐allyldimethylammoniumphenyl)pentan‐3‐one dibromide (BW284C51) and propidium, indicating that the corresponding epitope encompassed the anionic site involved in the binding of these low‐molecular‐mass inhibitors. The third mAb (Elec‐408), was clearly bound to another site on the AChE molecule, and its inhibitory effect was cumulative with those of Elec‐403, Elec‐410, and fasciculin. All mAbs bound AChE with high affinity and were as strong inhibitors with an apparent Ki value less than 0.1 nM. Elec‐403 was particularly efficient with an inhibitory activity similar to that of fasciculin. Inhibition was observed with both charged (acetylthiocholine) and neutral substrates (o ‐nitrophenyl acetate) and had the characteristics of a non‐competitive process. Elec‐403 and Elec‐410 probably exert their effect by triggering allosteric transitions from the peripheral site to the active site. The epitope recognized by mAb Elec‐408 has not been localized, but it may correspond to a new regulatory site on AChE.
K+‐H+ Exchange Activity in Brush‐Border Membrane Vesicles Isolated from Chick Small Intestine
Authors: Peral María J. , Cano Mercedes , Ilundáin Anunciación A.
Published: March 2005

The purpose of this study was to investigate the presence of a K+‐H+ exchanger in brush‐border membrane vesicles (BBMV) isolated from chick small intestine. 86Rb+, as a tracer for K+ transport, was used to probe for the exchange mechanism. An outwardly directed proton gradient (pH 5.5 inside, pH 7.5 outside) stimulated 86Rb+ uptake into voltage‐clamped BBMV. H+‐driven 86Rb+ uptake was only weakly inhibited by 5‐(N ‐ethyl‐N ‐isopropyl)amiloride, whereas this agent strongly inhibited H+‐driven Na+ uptake. At initial rates, proton‐driven 86Rb+ uptake was significantly reduced by external K+ but it was not significantly affected by external Na+. Conversely, extra vesicular Na+ inhibited proton‐driven Na+ uptake, whilst K+ had little effect. H+‐driven K+ uptake tended to saturate with increasing external K+ concentrations and Lineweaver‐Burk analysis of the data revealed a Km for external K+ of 2 mM. These findings are consistent with the presence of K+‐H+ exchange activity in the chicken jejunal brush‐border membrane.
Kinetic and Mechanistic Studies on the Reactions of 2‐Aminobenzoyl‐CoA Monooxygenase/Reductase
Authors: Langkau Bernd , Ghisla Sandro
Published: March 2005

The kinetic mechanism of the flavoprotein 2‐aminobenzoyl‐CoA monooxygenase/reductase with its natural substrates 2‐aminobenzoyl‐CoA, NADH and O2 has been investigated using the stopped‐flow technique. Initial rate measurements indicate the formation of a ternary complex between oxidized enzyme and the two substrates 2‐aminobenzoyl‐CoA and NADH, a turnover number of ≈40 min−1 was found at pH 7.4 and 4°C. 2‐Aminobenzoyl‐CoA binds to oxidized enzyme to form a complex which is in a ≈1:1 equilibrium with a second, spectrophotometrically distinguishable one. Binding of 2‐amino benzoyl‐CoA to reduced enzyme is, in contrast, a simple second‐order process. Reduction of oxidized enzyme, both uncomplexed and in complex with 2‐aminobenzoyl‐CoA, by NADH is strongly biphasic. The first fast phase yields enzyme in which 50% of the total FAD is reduced to the FADH2 state. This rate is not affected by the presence of 2‐aminobenzoyl‐CoA. In contrast, 2‐aminobenzoyl‐CoA enhances ≈100‐fold the second phase, the reduction of the residual 50% FAD. This second phase of reduction (kobs= 2.0 s−1) is partially rate‐limiting in catalysis. The oxygen reaction of uncomplexed, reduced enzyme is also biphasic and no oxygenated intermediate was detected. Reoxidation of substrate‐complexed, reduced enzyme involves three spectroscopically distinguishable species. The first observable intermediate is highly fluorescent suggesting that it consists largely of flavin‐4a‐hydroxide. Thus, insertion of oxygen into 2‐aminobenzoyl‐CoA is essentially complete at this point and has a kobs≥80 s−1. The subsequent phase is accompanied by formation of the main product, 2‐amino‐5‐oxocyclohex‐1‐enecarboxyl CoA. This step consists in a hydrogenation of the primary, oxygenated and non‐aromatic CoA intermediate; it has a rate ≈1.3 s−1, which is thus the second rate‐limiting step in catalysis. As a side reaction of the oxidized enzyme and at low NADH concentrations the initially formed product disappears at a very slow rate (kobs≈ 0.05 s−1). This third ‘post‐catalytic’ process is not relevant for catalysis. The primary product 2‐amino‐5‐oxocyclohex‐1‐enecarboxyl‐CoA is dehydrogenated by the oxidized enzyme to yield the aromatic 2‐amino‐5‐hydroxybenzoyl‐CoA as secondary product. The reduced enzyme formed in this process is reoxidized by O2 to form H2O2. This explains the formation of different products depending on the actual concentration of NADH in the catalytic system, which has been reported previously [Buder, R., Ziegler, K., Fuchs, G., Langkau, B. & Ghisla, S. (1989) Eur. J. Biochem. 185, 637–643]. A kinetic mechanism is proposed based on the concept that aminobenzoyl‐CoA monooxygenase/reductase has two active sites which catalyze independently monooxygenation and hydrogenation of substrate or intermediate.
Fatty Acids Regulate Thy‐1 Antigen mRNA Stability in T Lymphocyte Precursors
Authors: Déglon Nicole , Wilson Anne , Desponds Chantal , Laurent Philippe , Bron Claude , Fasel Nicolas
Published: March 2005

In this study, we report the effect of fatty acids on the Thy‐1 antigen mRNA decay. Low serum and synthetic medium culture conditions were used to demonstrate that fatty acids, which are important metabolites involved as second messengers in signal transduction, also influence the steady‐state mRNA level. Detailed analysis demonstrated that polyunsaturated lipids attached to bovine serum albumin, such as linoleic, linolenic, and arachidonic acids, modulate gene expression specifically in the S1A T lymphoma cell line by inducing a 3–5‐fold increase in the steady‐state Thy‐1 mRNA level, concomitant with a twofold increase in cell surface expression. A similar modulation was observed in the immature CD4−CD8− T cell precursors but not in mature thymocytes. Nuclear run‐on and transfection experiments indicated that the observed Thy‐1 mRNA level is post‐transcriptionally regulated and that the presence of the coding region is sufficient for this adaptive response. A mechanism without a requirement for protein kinase C activation, but involving Ca2+ entry, could account for this difference in Thy‐1 mRNA stability.
A New Cationic Liposome Encapsulating Genetic Material
Authors: Puyal Christophe , Milhaud Pierre , Bienvenüe Alain , Philippot Jean R.
Published: March 2005

The endeavour to enhance gene therapy has led to increased research on the development of simple, efficient and safe delivery systems. This study deals with the use of an artificial cationic lipid on the encapsulation of genetic material in liposomes. The addition of a biologically degradable cationic phospholipid, dipalmitoyl‐L‐α‐phosphatidylethanolamine covalently coupled to L‐lysine, in a standard liposome formulation allowed us to obtain vesicles with high entrapment of various polynucleotides. Poly‐nucleotide degradation by nucleases is markedly prevented by these liposomes. The preparations were stable in both culture medium and human plasma. This latter finding is consistent with the weak binding of plasma proteins on the liposome surface. The efficiency of this new delivery system was demonstrated in antiviral assays. Finally, these liposomes displayed a relatively low cellular toxicity. All these findings indicate that these cationic vesicles are very suitable for genetic material vehiculation.
A Novel Approach to Introduce Site‐Directed Specific Cross‐Links Within RNA‐Protein Complexes
Authors: Zenkova Marina , Ehresmann Chantal , Caillet Joël , Springer Mathias , Karpova Galina , Ehresmann Bernard , Romby Pascale
Published: March 2005

We describe a methodology which allows the introduction of a photoactivatable azido group at specific internal positions of any RNA in order to identify the neighboring elements of an interacting protein. The first step involves site‐directed modification of the target RNA with an antisense oligodeoxyribonucleotide bearing, at its 3′ or 5′ phosphate, a 4‐[‐N ‐(2‐chloroethyl)‐N ‐methylamino]benzylmethylamino group. Position N7 of a guanine residue located in the close vicinity of the hybrid is the main target for alkylation. The antisense oligodeoxyribonucleotide is then removed by acidic pH treatment and a photoreactive reagent (2,4‐dinitro‐5‐fluorophenylazide) is condensed to the modified nucleotide.

This method was used to induce specific cross‐links between Escherichia coli threonyl‐tRNA synthetase and the leader region of threonyl‐tRNA synthetase mRNA, which is involved in translational feedback regulation. Control experiments revealed that the modification affects neither the structure of the mRNA nor the interaction with the enzyme. More than 50% of the modified mRNA complexed with threonyl‐tRNA synthetase can be cross‐linked to the enzyme, depending on the nucleotide modified.
The DNA‐binding Domain of Drosophila melanogaster c‐Myb Undergoes a Multistate Denaturation
Authors: Madan Anup , Radha P. K. , Srivastava Arvind , Padhy Lakshmi C. , Hosur Ramakrishna V.
Published: March 2005

The DNA‐binding domain of Drosophila c‐Myb protein has been studied using different spectroscopic probes, namely CD, fluorescence, acrylamide quenching and NMR, to determione the structure of some of its sub‐domains and their relative stabilities in aqueous solutions. While CD and fluorescence spectroscopy showed that the protein had completely lost its tertiary and secondary structures in approximately 3 M urea, solvent accessibility of the tryptophan residues was still partial, as determined by acrylamide quenching. This suggested the presence of significant amounts of residual structure which persisted until the urea concentration was raised to approximately 6.0 M. Thermal‐denaturation experiments also indicated the presence of an intermediate in the unfolding pathway. The experimental data could be fitted assuming a minimum of three states in both modes of denaturation. The thermodynamic parameters for the apparent three‐state transition have been determined. From the protein stability curve, we have determined that Drosophila melanogaster Myb R123 has maximal stability at 16°C and pH 7.0.
Binding of a Fluorescent Nucleotide Analog to Hsc70
Authors: Churchich Jorge E.
Published: March 2005

2′‐Deoxy‐3′‐anthraniloyladenosine‐5‐triphosphate(Ant‐dATP) was used as an environmentally sensitive probe of the nucleotide‐binding site of the molecular chaperone Hsc70.

When coordinated to the lanthanide ion Tb3+, Ant‐dATP is not hydrolyzed by Hsc70. The lanthanide ion acts as strong competitive inhibitor with respect to Mg2+ (Ki= 0.1 μM). Tb · Ant‐dATP recognizes the nucleotide site of Hsc70 as revealed by an increase in the emission anisotropy from 0.03 to 0.21 and by a change in the fluorescence‐decay time from 2.52 ns to 3.75 ns. Sensitized luminescence arising from resonance energy transfer from the anthraniloyl group to Tb3+ is substantially enhanced in the presence of Hsc70.

Binding of a 20‐amino‐acid‐residue peptide (Rnase‐S peptide) to Hsc70 causes a blue shift in the fluorescence spectrum of Ant‐dATP and enhances Tb3+ luminescence upon excitation at 330 nm. It is postulated that binding of the peptide to the COOH‐terminal domain of Hsc70 initiates domain movement and the structural changes might extend to the nucleotide‐binding site.
Induction of Terminal Enzymes for Heme Biosynthesis During Differentiation of Mouse Erythroleukemia Cells
Authors: Taketani Shigeru , Yoshinaga Takeo , Furukawa Takako , Kohno Hirao , Tokunaga Rikio , Nishimura Koichi , Inokuchi Hachiro
Published: March 2005

To examine the induction of terminal enzymes of the heme‐biosynthetic pathway during erythroid differentiation, mouse protoporphyrinogen oxidase (PPO) cDNA has been cloned. The deduced amino acid sequence derived from the nucleotide sequence revealed that mouse PPO consists of 477 amino acid residues, without the leader peptide, which is imported into mitochondria. Comparison of the amino terminus of the deduced amino acid sequence of mouse PPO cDNA with that of purified bovine PPO provided conclusive evidence for lack of the leader peptide in the former. The amino acid sequence has 86% and 28% identity with human PPO and Bacillus subtilis HemY, respectively. When mouse erythroleukemia (MEL) cells were induced with dimethylsulfoxide, PPO mRNA was induced within 12 h of treatment, and with further incubation, reached a plateau. mRNAs for coproporphyrinogen oxidase (CPO) and ferrochelatase (FEC) were induced within 12 h, and continued to increase with time up to 48 h. The activities of CPO and FEC markedly increased with time up to 72 h, while PPO activity increased 1.8‐fold within 12 h and remained unchanged thereafter. Immunoblot analysis showed that levels of PPO, CPO and FEC paralleled their corresponding activities. The magnitude of PPO induction was less than that of CPO and FEC. Thus, induction of three terminal enzymes of the heme‐biosynthetic pathway is an early event in MEL cell differentiation. The concomitant induction may play an important role in producing large amounts of heme during erythroid differentiation.
1H and 15N Assignment of NMR Spectrum, Secondary Structure and Global Folding of the Immunophilin‐Like Domain of the 59‐kDa FK506‐Binding Protein
Authors: Rouvière‐Fourmy Nathalie , Craescu Constantin T. , Mispelter Joël , Lebeau Marie‐Claire , Baulieu Etienne‐Emile
Published: March 2005

FKBP59, a 59‐kDa FK506 binding protein, was discovered in heterooligomeric complexes containing nontransformed, non‐DNA binding, steroid receptors. Sequence similarity search and secondary structure prediction suggested that the protein has a multi‐domain organization, the N‐terminal domain having a great similarity to human FKBP12 (12‐kDa FK506‐binding protein). FKBP59 binds immunosuppressant FK506 and has peptidylprolyl cis‐trans ‐isomerase activity, both properties being localized in the N‐terminal domain (FKBP59‐I). In order to characterize its conformational features and to better understand its biological significance, we overexpressed and 15N‐labeled this domain (149 amino acids) in Escherichia coli and initiated an NMR structural study in solution. Almost complete sequence‐specific assignment of the 1H and 15N resonances was achieved using two‐dimensional and three‐dimensional homonuclear and heteronuclear experiments. Localization of the secondary structure elements was derived essentially from CαH chemical shift distribution along the sequence, the short‐range and medium‐range NOE connectivities and exchange kinetics of amide protons. The domain has a structured part comprising six β‐strands and a three‐turn α‐helix between K87 and M96. The first 17 residues are highly flexible and show no regular secondary structure. The β‐sheet structure, derived from long‐range connectivities between backbone protons, consists of six β‐strands defined as follows: B1, V22–124; B2, V32–K37; B3, D50–L61; B4, T64–S68 and F76–L80; B5, E100–K107; B6, L127–F137. They are organized in an antiparallel β‐sheet with the connecting topology +1, +3, +1, ‐3, +1. The α‐helix connects strand B4 to strand B5. Globally, the structure of FKBP59‐I, derived from the present work, is similar to the NMR‐derived structures of uncomplexed FKBP12. However, several conformational differences were noted at this level of structural analysis. The β‐sheet of the FKBP59 domain has an additional strand at the N‐terminal and the α‐helix is longer by about one helical turn. In addition, strand B4 has two components, separated by a large bulge (seven residues); the first component was observed in the X‐ray or NMR structures of complexed FKBP12 but not in the NMR‐derived, uncomplexed structure.
Coenzyme F420‐Dependent N5,N10‐Methylenetetrahydromethanopterin Reductase (Mer) from Methanobacterium Thermoautotrophicum Strain Marburg
Authors: Vaupel Martin , Thauer Rudolf K.
Published: March 2005

The gene encoding the F420‐dependent N5,N10‐methylenetetrahydromethanopterin reductase (Mer), which catalyzes an intermediate step in methanogensis, was cloned and sequenced from the thermophilic Methanobacterium thermoautotrophicum strain Marburg. The gene was identified on a 3.8‐kbp BamHI fragment of M. thermoautotrophicum genomic DNA using a homologous probe. The mer gene encoded an acidic protein of 321 amino acids, corresponding to a calculated molecular mass of 33 492 Da. Sequence analysis revealed the presence of a ribosome binding site, a putative promoter, and a possible terminator structure. The size of the mer mRNA was estimated as 1 kb indicating monocistronic transcription. The mer gene was expressed in Escherichia coli yielding an active enzyme of 36 kDa consistent with the apparent molecular mass described for the enzyme from M. thermoautotrophicum. Sequence comparisons revealed similarities between the F420‐dependent N5,N10‐methylenetetrahydromefhanopterin reductase and a F420‐dependent reductase involved in lincomycin biosynthesis in Streptomyces lincolnensis.
High‐level Production, Chemical Modification and Site‐directed Mutagenesis of a Cephalosporin C Acylase from Pseudomonas Strain N176
Authors: Ishii Yoshinori , Saito Yoshimasa , Fujimura Takao , Sasaki Hitoshi , Noguchi Yuji , Yamada Hisashi , Niwa Mineo , Shimomura Kyoichi
Published: March 2005

A cephalosporin acylase from Pseudomonas strain N176 hydrolyses both 7‐β‐(4‐carboxybutanamido)‐cephalosporanic acid (glutarylcephalosporanic acid) and cephalosporin C to 7‐amino‐cephalosporanic acid. However, its productivity in the original host was low and its activity against cephalosporin C was not sufficient for direct large‐scale production of 7‐amino‐cephalosporanic acid. In order to overcome these problems, we established a high‐level expression system for the acylase in Escherichia coli. Tyr270 in the acylase is reported to play an important role in the interaction with glutarylcephalosporanic acid, as determined from the reaction with an affinity‐label reagent, 7β‐(6‐bromohexanoylamido) cephalosporanic acid [Ishii, Y., Saito, Y., Sasaki, H., Uchiyama, F., Hayashi, M., Nakamura, S. & Niwa, M. (1994) J. Ferment. Bioeng. 77, 598–603] and modification with tetranitromethane [Nobbs, T. J., Ishii, Y., Fujimura, T., Saito, Y. & Niwa, M. (1994) J. Ferment. Bioeng. 77, 604–609]. From carbamoylation with potassium cyanate and site‐directed point mutagenesis of the cephalosporin C acylase, we have deduced that Tyr270 exists at a position where it can interact with a residue (possibly Ser239) corresponding to inactivation by carbamoylation. We mutated Met269 and Ala271 of the acylase and found that mutation of Met269 to Tyr or Phe caused a 1.6‐fold and 1.7‐fold increase, respectively, of specific activity against cephalosporin C as compared to that of the wild‐type enzyme. Kinetic studies of these mutants revealed that their kcat values increased, although their Km values against cephalosporin C were not changed. These data indicate that the mutation of Met269 near Tyr270 induces a minor conformational change to increase the stability of the activated complex with the enzyme and cephalosporin C. In particular, a mutant in which Met269 was replaced by Tyr was 2.5‐fold more efficient in converting cephalosporin C to 7‐amino‐cephalosporanic acid than the wild‐type enzyme under conditions similar to those in a bio‐reactor system.
Lactose‐Specific Enzyme II of the Phosphoenol Pyruvate‐Dependent Phosphotransferase System of Staphylococcus aureus
Authors: Peters Dirk , Frank Rainer , Hengstenberg Wolfgang
Published: March 2005

The lactose‐specific integral‐membrane‐protein enzyme II (IICBLac) of the bacterial phosphoenolpy‐ruvate‐dependent phosphotransferase system of Staphylococcus aureus catalyses the uptake and phosphorylation of lactose. It consists of an N‐terminal membrane‐spanning IIC domain and a C‐terminal hydrophilic IIB domain. IICBLac was fused with a C‐terminal tag of six histidine residues using recombinant DNA technology. The resulting protein, IICBLac‐His, was produced in Escherichia coli and purified under non‐denaturing conditions to homogenity. The purification procedure consits of a NaOH extraction step followed by solubilisation with Triton X‐100, and metal‐affinity chromatography using Ni2+‐nitrilotriacetic acid resin. The purified recombinant His‐tagged protein possessed subtrate specifity identical to that of the wild‐type protein. To investigate the hydrophilic IIB domain, the DNA sequence coding for IIB and the His tag were fused in‐frame to a DNA sequence specific for an initiation signal. The overproduced recombinant IIBLac‐His was obtained by metal‐affinity chromatography in pure form. Bacterial phospho‐transferase‐system‐dependent phosphorylation of IIB‐His was demonstrated in a photometric assay and by urea/polyacrylamide gel electrophoresis. The phosphorylation activity of the mutant protein [C476S]‐IICB1, containing the mutagenized phosphorylation site, was restored in the presence of IIBLac‐His in a phosphorylation assay.
The Structure of U17 Isolated from Streptomyces clavuligerus and its Properties as an Antioxidant Thiol
Authors: Newton Gerald L. , Bewley Carole A. , Dwyer Tammy J. , Horn Ronda , Aharonowitz Yair , Cohen Gerald , Davies Julian , Faulkner D. John , Fahey Robert C.
Published: March 2005

The predominant low‐molecular‐mass thiol produced by streptomycetes is a cysteine derivative previously designated as U17 [Newton, G. L., Fahey, R. C., Cohen, G. & Aharonowitz, Y. (1993) J. Bacteriol. 175, 2734–2742]. In this study we report the elucidation of the structure of the monobromobimane derivative of U17, which establishes the structure of U17 as 2‐(N‐acetylcysteinyl)amido‐2‐deoxy‐α‐d‐glucopyranosyl‐myo‐inositol. The presence of the N‐acetylcysteine moiety was indicated by formation of N‐acetylcysteine‐monobromobimane during acid hydrolysis of the monobromobimane derivative of U17. Complete hydrolysis released 1 mol glucosamine/mol cysteine as determined by carbohydrate and amino acid analysts. High‐resolution mass spectral analysis gave a precise mass consistent with the molecular formula C27H40N4O14S. Analysis of 13C‐NMR, one‐dimensional 1H‐NMR and two‐dimensional NMR experiments identified the remaining C6H12O6 moiety as myo‐inositol, confirmed the presence of N‐acetyl‐cysteine and glucosamine, and established the connectivity of the components. Two chemical properties of this novel thiol make it suitable as an intracellular storage form of cysteine and as an antioxidant thiol. First, it undergoes heavy‐metal‐ion catalyzed autoxidation at a rate dramatically lower than that for cysteine and markedly lower than that for glutathione or N‐acetylcysteine. Secondly, the α‐(1→1) glycosidic link between glucosamine and myo‐inositol is resistant to acid hydrolysis, hydrolysing at a rate comparable to that of the two amide bonds in the molecule.
Tyrosine Kinase Activity of a Chimeric Insulin‐Like‐Growth‐Factor‐l Receptor Containing the Insulin Receptor C‐Terminal Domain
Authors: Mothe Isabelle , Tartare Sophie , Kowalski‐Chauvel Aline , Kaliman Perla , Van Obberghen Emmanuel , Ballotti Robert
Published: March 2005

In a previous study, we showed that a chimeric insulin‐like‐growth‐factor‐1 (IGF‐1) receptor, with the β subunit C‐terminal part of the insulin receptor was more efficient in stimulating glycogen synthesis and p44mapk activity compared to the wild‐type IFG‐1 receptor [Tartare, S., Mothe, I., Kowalski‐Chauvel, A., Breittmayer, J.‐F., Ballotti, R. & Van Obberghen, E. (1994) J. Biol. Chem. 269, 11449–11455]. These data indicate that the receptor C‐terminal domain plays an important role in the transmission of biological effects. To understand the molecular basis of the differences in receptor specificity, we studied the characteristics of insulin, IGF‐1 and chimeric receptor tyrosine kinase activities in a cell‐free system. We found that, compared to wild‐type insulin and IGF‐1 receptors, the chimeric receptor showed a decrease in (a) autophosphorylation, (b) tyrosine kinase activity towards insulin receptor substrate‐1 and the insulin re‐ceptor‐(1142–1158)‐peptide, and (c) the ability to activate phosphatidylinositol 3‐kinase. However, for all the effects measured in a cell‐free system, the chimeric receptor displayed an increased response to IGF‐1 compared to the native IGF‐1 receptor. Concerning the cation dependence of the tyrosine kinase activity, we showed that, at 10 mM Mg2+, the ligand‐stimulated phosphorylation of poly(Glu80Tyr20) by both insulin receptor and chimeric receptor was increased by Mn2+. Conversely at 50 mM Mg2+, the chimeric receptor behaved like the IGF‐1 receptor, since the presence of Mn2+ decreased the stimulatory effect of IGF‐1 on their kinase activity. Furthermore, the Km of the chimeric receptor for ATP was increased compared to the wild‐type receptors. These data demonstrate that the replacement of the C‐terminal tail of the IGF‐1 receptor by that of the insulin receptor has changed the receptor characteristics studied in a cell‐free system. Our findings indicate that the C‐terminal domain of the insulin receptor β subunit plays a key role in regulation of the tyrosine kinase activity. The fine‐tuning of the tyrosine kinase by the C‐terminal tail could participate in the receptor specificity.
Manganese Peroxidase from Phanerochaete chrysosporium
Authors: Selvaggini Carlo , Salmona Mario , De Gioia Luca
Published: March 2005

A detailed three‐dimensional model of manganese peroxidase was constructed using lignine peroxidase as the structural scaffold. This is the only protein in the peroxidase family except for cytochrome c peroxidase for which a resolved crystal structure is available.

The model was built using the following procedure: (a) structurally preserved regions were derived from similar regions in the sequence alignment of the two proteins; (b) non‐similar regions were modelled by searching a set of resolved protein structures for fragments which fitted in geometrically and choosing the best fitting fragment. Side chains were constructed by calculating rotamer‐rotamer interaction energies and minimizing intramolecular energy. Model refinement was performed by molecular mechanics calculation.

The quality of the model was assessed on the basis of the propensity of the amino acids to be inserted into regular secondary‐structure elements and to be exposed to solvent.

All the lignine peroxidase regions not used for model construction because of the lack of similarity, except the helix fragment Leu261‐Phe269, correspond to external loops, suggesting reliable modelling.

The manganese peroxidase model structure was analyzed in detail and several functionally relevant structural features were predicted, the most important being: (a) the very close structural similarity between lignine and manganese peroxidase active sites, suggesting a similar mode of hydrogen peroxide activation; (b) the substitution of polar residues for the hydrophobic amino acids exposed at the edge of the channel involved in substrate recognition in lignine peroxidase, suggesting that manganese peroxidase does not directly bind aromatic substrates; (c) the location of residues potentially able to bind Mn2+, spatially positioned on the side of the 3‐CH3 heme edge.
Myc Inhibits CCAAT/Enhancer‐Binding Protein α‐Gene Expression in HIB‐1B Hibernoma Cells Through Interactions with the Core Promoter Region
Authors: Antonson Per , Pray Marilyn G. , Jacobsson Anders , Xanthopoulos Kleanthis G.
Published: March 2005

The product of the c‐myc proto‐oncogene, Myc, has been implicated in the transcriptional regulation of several genes, acting either as an activator or represser of gene expression. To determine whether Myc is involved in the modulation of the expression of the CCAAT/enhancer‐binding protein α (C/EBPα) gene, we used both stable cell lines overexpressing Myc and transient co‐transfection assays. We show that the endogenous C/EBPα protein level is repressed in stable cell lines overexpressing Myc. We also show that enforced expression of Myc in mouse hibernoma HIB‐1B cells dramatically repressed the expression of C/EBPα‐promoter‐reporter fusion genes. This effect of Myc was mediated through the core promoter region. Mutation of the initiator site could not abolish this affect, indicating that Myc may interact with some component(s) of the basal transcription machinery.
Involvement of Retinoic Acid Receptor α in the Stimulation of Tissue‐Type Plasminogen‐Activator Gene Expression in Human Endothelial Cells
Authors: Kooistra Teake , Lansink Mirian , Arts Janine , Sitter Thomas , Toet Karin
Published: March 2005

Retinoids stimulate tissue‐type plasminogen‐activator (t‐PA) gene expression in human endothelial cells, and are likely to do so by binding to one or more nuclear retinoid receptors. The present study was initiated to identify the retinoid receptor(s) involved in this process. Expression and regulation of retinoic acid receptors (RARs) and retinoid X receptors (RXRs) were analyzed by Northern‐blot analysis of total or poly(A)‐rich RNA prepared from cultured human umbilical vein endothelial cells (HUVEC). Prior to any exposure to retinoids, HUVEC express two transcripts for RAR‐α (3.6 kb and 2.8 kb), and low levels of transcripts for RAR‐β (3.4 kb and 3.2 kb) and RAR‐γ (3.3 kb and 3.1 kb). Two RXR subtypes were identified, RXR‐α (4.8kb) and, at a much lower concentration, RXR‐β (2.4kb); no evidence for the presence of RXR‐γ was found. Furthermore, HUVEC express cellular retinol‐binding protein I (CRBP‐I) and cellular retinoic‐acid‐binding protein I (CRABP‐I) mRNA. Exposure of HUVEC to 1 μM retinoic acid or the retinobenzoic acid, Ch55, led to the induction of the two RAR‐β mRNAs, RXR‐α mRNA and CRBP‐I mRNA, whereas the expression of the other receptor and CRABP‐I transcripts did not change appreciably. Using retinoid analogues that bind preferentially to one of the RAR or RXR subtypes, we found evidence that RAR‐α is involved in the retinoid‐induced t‐PA expression in HUVEC. This conclusion was strengthened by experiments in which blocking of RAR‐α with a specific RAR‐α antagonist, Ro 41‐5253, was demonstrated to suppress the induction of t‐PA by retinoids.
NMR Studies of the Major Coat Protein of Bacteriophage M13
Authors: Papavoine Christina H. M. , Aelen Jan M. A. , Konings Ruud N. H. , Hilbers Cornelis W. , Van De Ven Frank J. M.
Published: March 2005

The membrane‐bound form of the major coat protein (gVIIIp) of bacteriophage M13 has been studied using nuclear magnetic resonance spectroscopy. As membrane mimetics, we used dodecylphosphocholine (DodPCho) detergent micelles to solubilize the protein. We were able to nearly completely assign all resonances of the protein solubilized in DodPCho micelles by using both homonuclear and heteronuclear multidimensional experiments. Based on the patterns of the nuclear Overhauser enhancements and the chemical shifts of the resonances, we deduced the secondary structure of the protein. Additional structural information was obtained from amide proton exchange data and J‐coupling constants. The protein consists of two α‐helices which are connected by a hinge region around residue 21. From spin‐label experiments, the location of the protein relative to the DodPCho micelles was determined. One, hydrophobic, helix spans the micelle, and another, amphipathic, helix, is located beneath the surface of the micelle. Comparison of the data of gVIIIp in DodPCho micelles with those of gVIIIp in sodium dodecyl sulfate (SDS) micelles [Van de Ven, F. J. M., van Os, J. W. M., Aelen, J. M. A., Wymenga, S. S., Remerowski, M. L., Konings, R. N. H. & Hilbers, C. W. (1993) Biochemistry 32, 8322–8328; Papavoine, C. H. M., Konings, R. N. H., Hilbers, C. W. & Van de Ven, F. J. M. (1994) Biochemistry 33, 12990–12997] reveals that the structures of the protein in the two detergent micelles are very similar. They differ only in the arrangement of the detergent molecules around the protein. For gVIIIp in SDS micelles, we found a micellar structure which is distorted near the C‐terminus of the protein; whereas for DodPCho micelles, both distorted and regular elliptical micelles occur. This distortion is probably due to the interaction of the positively charged lysine side chains with the negatively charged head group of the detergent molecules.
Determination of Haem Electronic Structure in Cytochrome b5 and Metcyanomyoglobin
Authors: Banci Lucia , Pierattelli Roberta , Turner David L.
Published: March 2005

The paramagnetic shifts of 13C nuclei positioned α to the haems in the A and B forms of rat cytochrome b5 and in metcyanomyoglobin have been analysed in terms of molecular orbitals based on D4h symmetry with a rhombic perturbation. The contribution to the 13C shifts from pseudocontact interactions is calculated from parameters obtained for a metal‐centred dipolar shift tensor by fitting 1H shifts. The effect of electron delocalisation onto the vinyl groups of these haems b is separated with reference to the shifts of the vinyl β carbons. In each case, it was found that the orientation of the magnetic axes in the plane of the haem is rotated away from the iron‐nitrogen vectors in the opposite sense to the rotation of the rhombic perturbation and the molecular orbitals. The orientation of the orbitals is closely aligned with the normal to the single His ligand in metcyanomyoglobin, and with the average of the two normals in the bis‐His cytochrome b5. It is concluded that the in‐plane anisotropy of haems b is dominated by the orientation of the axial ligands in a similar manner to that in haems c and that the approximations used are weakened, but not invalidated, by the presence of partially conjugated vinyl groups.
Anti‐La Monoclonal Antibodies Recognizing Epitopes Within the RNA‐Binding Domain of the La Protein Show Differential Capacities to Immunoprecipitate RNA‐Associated La Protein
Authors: Pruijn Ger J. M. , Thijssen José P. H. , Smith Paul R. , Williams David G. , Van Venrooij Walther J.
Published: March 2005

The La (SS‐B) autoimmune antigen is an RNA‐binding protein that is present in both the nucleus and cytoplasm of eukaryotic cells, where it is found associated with RNA polymerase III transcripts. We have investigated the capacity of anti‐La monoclonal antibodies SW1, SW3, and SW5 to immunoprecipitate human La ribonucleoprotein particles. Distinct differences were observed for SW3 in comparison with SW1 and SW5. While SW1 and SW5 precipitated ribonucleoproteins containing pre‐tRNA, pre‐5S rRNA, hY RNAs, pre‐U6 snRNA or the viral EBER1 and VA RNAs, SW3 precipitated only ribonucleoproteins containing VA RNAs or (the precursor of) 7‐2 RNA.

Mapping of the epitopes recognized by SW1, SW3, and SW5 revealed that all three monoclonal antibodies recognize an epitope within the domain of the protein formed by the ribonucleoprotein motif. Cross‐competition studies suggested that the epitope recognized by SW1 and SW5 are identical but distinct from the epitope recognized by SW3. Further analyses of the recognition of La from other species by these monoclonal antibodies revealed that they all reacted with bovine La and were not reactive with La from rodents and Xenopus laevis. Replacement of a single amino acid in the human protein by its murine counterpart abolished recognition by SW1 and SW5, but had no effect on recognition by SW3.

Taken together, our results indicate that SW1 and SW5 recognize the same epitope and that SW3 recognizes a distinct epitope, both of which are located in the RNA‐binding domain of La, and that the accessibility of these epitopes is differentially influenced by the association of La with various RNA polymerase III transcripts.
Characterization of a cAMP‐Binding Protein from the Bivalve Mollusc Mytilus galloprovincialis
Authors: Cao Jesús , Ramos‐Martinez J. Ignacio , Villamarín J. Antonio
Published: March 2005

Three cAMP‐binding proteins have been identified by photoaffinity labeling with 8‐azido[32P]cAMP and purified from the mantle tissue of the sea mussel Mytilus galloprovincialis. Their molecular masses, determined by SDS/PAGE, were 54, 42 and 37 kDa. The purified 54‐kDa protein, which had two cAMP‐binding sites/monomer, was judged to be a regulatory (R) subunit of cAMP‐dependent protein kinase since it re‐associated with and inhibited purified catalytic (C) subunit of this enzyme from mussel, in the absence but not in the presence of cAMP. The molecular mass of the complex between Mytilus cAMP‐binding protein and C subunit, estimated by analytical gel‐filtration, was 220 kDa, a value which agrees with a R2C2 stoichiometry for the mussel cAMP‐dependent protein kinase holoenzyme. On the basis of the elution pattern from DEAE‐cellulose chromatography and its ability to be phosphorylated by purified C subunit of cAMP‐dependent protein kinase, the 54‐kDa protein could be classified as a type II regulatory subunit. Furthermore, no mobility shift on SDS/PAGE upon phosphorylation/dephosphorylation of Mytilus protein was observed, a similar behaviour to that shown by the mammalian RIIβ isoform. The 42‐kDa and 37‐kDa proteins, which were recognized by a specific antiserum against the 54‐kDa protein and fail to be phosphorylated by Mytilus C subunit, are probably products generated by proteolysis of the 54‐kDa protein, although they were shown even when inhibitors of the major types of proteases were used.
A Single Point Mutation in the Splice Donor Site of the Low‐Density‐Lipoprotein‐Receptor Gene Produces Intron Read‐Through, Exon‐Skipped and Cryptic‐Site‐Utilized Transcripts
Authors: Maruyama Takao , Miyake Yasuko , Tajima Shoji , Funahashi Tohru , Matsuzawa Yuji , Yamamoto Akira
Published: March 2005

Familial hypercholesterolemia is a genetic disorder caused by mutations of the low‐density‐lipoprotein (LDL) receptor gene. We characterized the structures of LDL receptor mRNA transcripts in the fibroblasts of a homozygous patient carrying a single base substitution (T→C) at the 5′ splice donor site of intron 12 of the LDL receptor gene. We identified three aberrant transcripts as a consequence of intron‐12 read‐through, exon‐12 skipping and utilization of a cryptic splice donor site. Only a point mutation at the 5′ splice donor site caused the production of three alternatively spliced products. None of these transcripts produced a functional LDL receptor protein in this patient.
Construction of Stable BHK‐21 Cells Coexpressing Human Secretory Glycoproteins and Human Gal(β1‐4)GlcNAc‐R α2,6‐Sialyltransferase
Authors: Grabenhorst Eckart , Hoffman Andrea , Nimtz Manfred , Zettlmeissl Gerd , Conradt Harald S.
Published: March 2005

The human β‐trace protein has been cloned and has been expressed for the first time in a mammalian host cell line. Stable BHK‐21 cell lines exhibiting altered terminal sialylation properties were constructed by cotransfection of cells with the plasmids pMT‐βTP or pAB3‐1 which contain the cDNAs encoding the human secretory glycoproteins β‐trace protein or antithrombin III and pABSial containing the human Golgi enzyme CMP‐NeuAc:Gal(β1‐4)GlcNAc‐R α2,6‐sialyltransferase (ST6N) gene. The β‐trace protein was purified by immunoaffinity chromatography and N‐linked oligosaccharides were subjected to carbohydrate structural analysis. The enzymically liberated oligosaccharides were found to consist of 90% of diantennary chains as is the case for natural β‐trace protein from human cerebrospinal fluid. About 90% of the total oligosaccharides were recovered in the monosialo and disialo fractions in a ratio of 1:5. The monosialylated oligosaccharides of β‐trace protein coexpressed with human ST6N were found to contain NeuAc in α2,6‐or α2,3‐linkage in the same ratio. From 1H‐NMR analysis as well as calculations of peak areas obtained by HPLC, 60% of the molecules of the disialo fraction were found to contain NeuAc in both α2,3‐and α2,6‐linkage to Galβ(1‐4)GlcNAc‐R, whereas 40% of the molecules of this fraction contained NeuAc in only α2,3‐linkage to Gal(β1‐4)GlcNAc‐R. The α2,6‐linked NeuAc was shown to be attached preferentially to the Gal(β1‐4)GlcNAc(β1‐2)Man(α1‐3) branch of the diantennary structure. Therefore the in vivo specificity of the newly introduced recombinant human ST6N observed in this study supports the previously reported in vitro branch specificity of the bovine colostrum ST6N activity. Furthermore, these studies demonstrate the suitability of genetically engineered mammalian host cell lines with novel glycosylation properties for the production of human‐type glycosylated secretory recombinant polypeptides.
A Cytochrome‐b5‐Containing Fusion Protein Similar to Plant Acyl Lipid Desaturases
Authors: Sperling Petra , Schmidt Hermann , Heinz Ernst
Published: March 2005

The similarity between oleate and linoleate desaturase sequences from several plants was used lo construct degenerate oligonucleotide primers for PCR experiments with cDNA transcribed from mRNA of ripening sunflower embryos. A DNA fragment was amplified and sequenced. Specific primers derived from this partial sequence were used for rapid amplification of the 3′‐ and 5′‐ends of this cDNA. With appropriate primers derived from these sequences, a full‐length clone of 1377 bp was amplified by PCR which, after sequencing, showed an open reading frame of 458 amino acids corresponding to a putative protein of about 52 kDa.

Comparison with other desaturases showed the conserved three histidine boxes and the characteristic hydropathy profile of membrane‐bound desaturases, but the amino acid identity was restricted to 18 % and the N‐terminal region was about 100 amino acids longer. This N‐terminal extension showed high similarity with cytochrome b5, and, accordingly, the whole sequence can be considered as coding for a fusion protein between cytochrome b5 and a desaturase‐like enzyme. Furthermore, we detected a similar cytochrome b5 fold in the previously sequenced Δ9 acyl‐CoA desaturase from yeast, but in this enzyme it was located at the C‐terminus. An alignment of these fusion proteins with other heme‐binding proteins revealed desaturases to be novel members of the cytochrome b5, superfamily. A truncated DNA representing 366 bp of the 5′‐end was amplified from the cDNA clone and expressed in Escherichia coli. The truncated cDNA coded for a soluble protein of about 12 kDa as shown by SDS/PAGE and N‐terminal sequencing. The enriched recombinant protein exhibited redox absorbance spectra characteristic of plant microsomal cytochrome b5