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B Roux

Publications and source records attributed to B Roux.

At least 19 recordsLinked to original sources
PubMed1995

Thermal and pH stabilities of alkaline phosphatase from bovine intestinal mucosa: a FTIR study.

The inactivation of alkaline phosphatase (AP) from bovine intestinal mucosa caused by lowering the p2H from 10.4 to 5.4 or by increasing the temperature from 25 degrees C to 70 degrees C were not followed by significant FTIR changes, indicating that the native conformation of AP was preserved under these conditions. Further decrease of p2H from 5.4 to 3.4 leaded to small infrared spectral changes of AP in the amide I' and amide II regions that were similar to the infrared spectral changes of AP induced by raising the temperature from 70 degrees C to 80 degrees C. The increase of temperature from 70 degrees C to 80 degrees C promoted the formation of intermolecular beta-sheets at the expense of some alpha-helix structures as evidenced by the appearance of the 1684 cm-1 and 1620 cm-1 component bands and the disappearance of the 1651-1657 cm-1 component band. This conformational change was followed by a sharp increase of the 2H/H exchange rate. CD spectra confirmed the FTIR results and were very sensitive to the variation of alpha-helix content while FTIR spectra were more receptive to the changes of beta-sheet structures.

Alkaline Phosphatase↗
PubMed1995

Functional interactions in bacteriorhodopsin: a theoretical analysis of retinal hydrogen bonding with water.

The light-driven proton pump, bacteriorhodopsin (bR) contains a retinal molecule with a Schiff base moiety that can participate in hydrogen-bonding interactions in an internal, water-containing channel. Here we combine quantum chemistry and molecular mechanics techniques to determine the geometries and energetics of retinal Schiff base-water interactions. Ab initio molecular orbital calculations are used to determine potential surfaces for water-Schiff base hydrogen-bonding and to characterize the energetics of rotation of the C-C single bond distal and adjacent to the Schiff base NH group. The ab initio results are combined with semiempirical quantum chemistry calculations to produce a data set used for the parameterization of a molecular mechanics energy function for retinal. Using the molecular mechanics force field the hydrated retinal and associated bR protein environment are energy-minimized and the resulting geometries examined. Two distinct sites are found in which water molecules can have hydrogen-bonding interactions with the Schiff base: one near the NH group of the Schiff base in a polar region directed towards the extracellular side, and the other near a retinal CH group in a relatively nonpolar region, directed towards the cytoplasmic side.

Bacteriorhodopsins↗
PubMed1995

Ion transport in the gramicidin channel: molecular dynamics study of single and double occupancy.

The structural and thermodynamic factors responsible for the singly and doubly occupied saturation states of the gramicidin channel are investigated with molecular dynamics simulations and free energy perturbation methods. The relative free energy of binding of all of the five common cations Li+, Na+, K+, Rb+, and Cs+ is calculated in the singly and doubly occupied channel and in bulk water. The atomic system, which includes the gramicidin channel, a model membrane made of neutral Lennard-Jones particles and 190 explicit water molecules to form the bulk region, is similar to the one used in previous work to calculate the free energy profile of a Na+ ion along the axis of the channel. In all of the calculations, the ions are positioned in the main binding sites located near the entrances of the channel. The calculations reveal that the doubly occupied state is relatively more favorable for the larger ions. Thermodynamic decomposition is used to show that the origin of the trend observed in the calculations is due to the loss of favorable interactions between the ion and the single file water molecules inside the channel. Small ions are better solvated by the internal water molecules in the singly occupied state than in the doubly occupied state; bigger ions are solvated almost as well in both occupation states. Water-channel interactions play a role in the channel response. The observed trends are related to general thermodynamical properties of electrolyte solutions.

Binding Sites↗
PubMed1994

Molecular dynamics simulation of the gramicidin channel in a phospholipid bilayer.

A molecular dynamics simulation of the gramicidin A channel in an explicit dimyristoyl phosphatidylcholine bilayer was generated to study the details of lipid-protein interactions at the microscopic level. Solid-state NMR properties of the channel averaged over the 500-psec trajectory are in excellent agreement with available experimental data. In contrast with the assumptions of macroscopic models, the membrane/solution interface region is found to be at least 12 A thick. The tryptophan side chains, located within the interface, are found to form hydrogen bonds with the ester carbonyl groups of the lipids and with water, suggesting their important contribution to the stability of membrane proteins. Individual lipid-protein interactions are seen to vary from near 0 to -50 kcal/mol. The most strongly interacting conformations are short-lived and have a nearly equal contribution from both van der Waals and electrostatic energies. This approach for performing molecular dynamics simulations of membrane proteins in explicit phospholipid bilayers should help in studying the structure, dynamics, and energetics of lipid-protein interactions.

Gramicidin↗
PubMed1994

Nuclear proteins interacting with DNA and tubulin. Study of the interaction of the High Mobility Group protein 1 with tubulin.

Fractionation of a 0.2 M NaCl nuclear extract from rat liver cells by both tubulin and DNA affinity chromatography steps allowed us to find three polypeptides interacting in vitro with both DNA and tubulin. A 22 kDa polypeptide was identified as a proteolytic fragment of High Mobility Group proteins 1 or 2 (HMG 1 or 2). Purified rat liver HMG 1 immobilized on nitrocellulose was found to bind radioiodinated dimeric tubulin through its central B domain. The C domain of HMG 1 appeared to play a negative role in this association process. Soluble HMG 1 depleted of its C-terminal domain interacted with tubulin immobilized on an agarose gel and with microtubules formed from purified tubulin. In contrast, undigested HMG 1 did not interact with tubulin in these conditions. The modification of HMG 1 with amine by 1-ethyl-3-(dimethylaminopropyl)carbodiimide which caused the neutralization of the C domain carboxyl groups restored the ability of HMG 1 to interact with microtubules. These results show that: (a) HMG 1, through its central B domain, binds to both assembled and non-assembled tubulin in vitro and (b) the C-terminal domain of HMG 1 exerts a negative regulatory action on the interaction.

Animals↗
PubMed1994

Identification of a new transposon Tn5403 in a Klebsiella pneumoniae strain isolated from a polluted aquatic environment.

A Klebsiella pneumoniae strain having mobilization "helper" potential has been isolated from the river Rhine. Analysis of the transconjugants resulting from the mobilization of non-conjugative pBR-type plasmids and RSF1010 derivatives showed that the transfer-helper capacity of the K. pneumoniae strain is related to the presence of a Tn3-like transposable element, Tn5403. This element has been identified and localized in a plasmid.

Base Sequence↗
PubMed1994

A molecular dynamics study of gating in dioxolane-linked gramicidin A channels.

The gating transition of the RR and SS dioxolane ring-linked gramicidin A channels were studied with molecular dynamics simulations using a detailed atomic model. It was found that the probable reaction path, describing the transition of the ring from the exterior to the interior of the channel where it blocked the permeation pathway, involved several steps including the isomerization of the transpeptide plane dihedral angle of Val1. Reaction coordinates along this pathway were defined, and the transition rates between the stable conformers were calculated. It was found, in good accord with experimental observations, that the calculated blocking rate for the RR-linked channel was 280/s with a mean blocking time of 0.04 ms, whereas such blocking did not occur in the case of the SS-linked channel. An important observation is that the resulting lifetime for the blocked state of the RR-linked channel was in good accord with the experimental observations only when the calculations were performed in the presence of a potassium ion inside the channel.

Dioxolanes↗
PubMed1993

p-Aminobenzoate synthesis in Escherichia coli: mutational analysis of three conserved amino acid residues of the amidotransferase PabA.

p-Aminobenzoate synthesis in Escherichia coli requires three enzymes, PabA, PabB, and PabC, acting respectively as glutaminase, chorismate aminase, and 4-amino-4-deoxychorismate aromatase. PabA requires stoichiometric amounts of PabB to display glutaminase activity. PabA has conserved cysteine (C79), histidine (H168), and glutamate (E170) residues that have been suggested in the analogous anthranilate synthase to form a type of catalytic triad in an acylenzyme mechanism. Mutations at each of these residues of PabA lead to the following observations. C79S PabA has 40-fold lower kcat and 10(4) lower kcat/Km with no detectable acylenzyme accumulation in steady-state turnover (vs wild-type PabA at 0.56 mol fraction of gamma-glutamyl-enzyme). H168Q has no catalytic activity and does not compete with wild-type PabA for PabB (this may indicate a folding defect). Four E170 mutants give three outcomes. E170D and E170A yield active PabA species, down 4-fold and 150-fold, respectively, in kcat/Km ratios from wild-type PabA. E170Q has no detectable glutaminase activity but does bind to PabB in competition with wild-typoe PabA while E170K has neither detectable catalytic activity nor the ability to be recognized by PabB.

4-Aminobenzoic Acid↗
PubMed1993

Fine mapping of neutralization epitopes on duck hepatitis B virus (DHBV) pre-S protein using monoclonal antibodies and overlapping peptides.

To define the residues involved in duck hepatitis B virus (DHBV) neutralization at the amino acid level, we have used a procedure combining monoclonal antibodies (MAbs) and overlapping octapeptides (Pepscan). Two neutralizing MAbs (SD20 and 900), specific for the pre-S protein were shown to reduce DHBV infectivity in vivo by 75 and 90%, respectively, while complete protection of ducklings was achieved with a polyclonal antiserum raised against the bacterially expressed first 131 amino acids of the DHBV pre-S region (DHBpre-S). Using fusion polypeptides, the binding sites of these MAbs were localized between aa 77 and 100 on pre-S protein. We have used octapeptides spanning the pre-S sequence from aa 64 to 115 for fine mapping of these epitopes. Within the sequence scanned, the polyclonal anti-DHBpre-S antiserum recognized a region exclusively limited to the residues E82-K95, suggesting immunodominance of this region in the sequence aa 64-115. The epitope recognized by Mab 900 was mapped within the same region, whereas the epitope recognized by Mab SD20 was localized downstream from this region. To define the amino acids essential for binding to the highly neutralizing Mab 900, we have used single amino acid replacement and demonstrated that two residues Q87 and W88 were important for antibody recognition.

Amino Acid Sequence↗
PubMed1992

p-aminobenzoate synthesis in Escherichia coli: kinetic and mechanistic characterization of the amidotransferase PabA.

p-Aminobenzoic acid (PABA) is an important precursor in the bacterial biosynthetic pathway for folate enzymes. This biosynthesis requires three separate proteins: PabA, PabB, and PabC. Together PabA and PabB convert glutamine and chorismate to glutamate and 4-amino-4-deoxychorismate. This aminochorismate is subsequently transformed to PABA by PabC. In this study, PabA from Escherichia coli has been purified to homogeneity from an overproducing construct and found to have no detectable glutaminase activity until addition of the E. coli PabB subunit. PabB forms a 1:1 complex with PabA to yield a glutaminase k(cat) of 17 min-1. The addition of chorismate, the substrate of PabB, induces a 2-fold increase of k(cat) as well as a 3-fold increase of Km for glutamine. The PabA/PabB complex has Kd less than 10(-8) M but does not form a stable complex isolable by gel filtration. Studies with the glutamine affinity label diazooxonorleucine (DON) reveal it is an inactivator of the glutaminase activity of the PabA/PabB complex, but DON does not alkylate and inactivate PabA alone. Similarly, while isolated PabA shows no tendency to form a glutamyl-enzyme intermediate, the PabA/PabB complex forms a covalent intermediate with [14C]glutamine on PabA that accumulates to 0.56 mol/mol in hydrolytic turnover. PabA is thus a conditional glutaminase, activated by 1:1 complexation with PabB.

4-Aminobenzoic Acid↗
PubMed1992

Histone H1a subtype presents structural differences compared to other histone H1 subtypes. Evidence for a specific motif in the C-terminal domain.

Following a previous isolation by reverse-phase HPLC of five histone H1 subtypes from adult rat liver, purity of three of them, H1a, H1b and H1d (according to Lennox's nomenclature), was achieved. Structural features of these three subtypes were investigated. Partial cleavage of these subtypes by endoproteinase Glu-C showed a different behavior of the H1a subtype when compared to the H1b and H1d subtypes. Under the conditions used in this work, the H1b and H1d subtypes present three major sites accessible to the endoproteinase Glu-C, while the H1a subtype presents only one major site accessible to the proteinase. Partial N-terminal sequence of the different fragments obtained after proteolysis indicated that the two H1b and H1d subtypes were cleaved inside the globular domain (Glu-54,-75) and between the globular domain and the C-terminal one (Glu-116). The H1a subtype was only cleaved between the globular domain and the C-terminal tail (Glu-116), though Glu-54 and Glu-75 sites were present. These results would suggest some differences in the conformation of these proteins. Furthermore, the partial determined sequences of H1b and H1d showed 85% similarity to each other (the main differences were threonine residues instead of alanine residues in the C-terminal domain) while H1a was only 60% similar to H1b and H1d, for the sequences which aligned. The strongest differences between the H1a subtype and the two other subtypes were observed in the first amino acid residues of the C-terminal domain. The 117-126 amino acid residues (SKASTTKVTV) of H1a were quite different from those of H1b and H1d. This sequence, which showed a number of serine and threonine residues, was not found in any other histone sequence, after consultation with data bases. This H1a subtype was a minor component in adult liver (2.4%). As it was described in testis as a major component, testis histone H1 proteins were fractionated onto reverse-phase HPLC under the same conditions as those used for histone H1 proteins from liver. The pure testis H1a fraction was submitted to the endoproteinase Glu-C digestion. The pattern digestion was the same as that observed for liver H1a. The two 44-76 and 117-126 determined amino acid residues of H1a from testis were strictly identical to those of liver H1a. We demonstrate that H1a is the same protein in liver and testis and we give evidence for a specific motif SKASTTKVTV (117-126 residues) in the sequence of the C-terminal domain.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Differential nucleotide binding to catalytic and noncatalytic sites and related conformational changes involving alpha/beta-subunit interactions as monitored by sensitive intrinsic fluorescence in Schizosaccharomyces pombe mitochondrial F1.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe exhibits an intrinsic tryptophan fluorescence sensitive to adenine nucleotides and inorganic phosphate [Divita, G., Di Pietro, A., Deléage, G., Roux, B., & Gautheron, D.C. (1991) Biochemistry 30, 3256-3262]. The present results indicate that the intrinsic fluorescence is differentially modified by nucleotide binding to either catalytic or noncatalytic sites. Guanine or hypoxanthine nucleotides, which selectively bind to the catalytic site, produce a hyperbolic saturation monitored by fluorescence quenching at 332 nm, the maximal emission wavelength. On the contrary, adenine nucleotides, which bind to both catalytic and noncatalytic sites, exhibit a biphasic saturation. High-affinity ATP binding produces a marked quenching as opposed to the lower-affinity one. In contrast, ADP exhibits a sigmoidal saturation, with high-affinity binding producing no quenching but responsible for positive cooperativity of binding to the lower-affinity site. The catalytic-site affinity for GDP is almost 20-fold higher at pH 5.0 as compared to pH 9.0, and the high sensitivity of the method allows detection of the 10-fold lower-affinity GMP binding. In contrast, high-affinity binding of ADP, or AMP, is not pH-dependent. The selective catalytic-site saturation induces a F1 conformational change decreasing the Stern-Volmer constant for acrylamide and the tryptophan fraction accessible to iodide. ATP saturation of both catalytic and noncatalytic sites produces an additional reduction of the accessible fraction to acrylamide.

Adenine Nucleotides↗
PubMed1992

Sequence of the phosphoenolpyruvate carboxykinase-encoding cDNA from the rumen anaerobic fungus Neocallimastix frontalis: comparison of the amino acid sequence with animals and yeast.

The nucleotide sequence of the cDNA of the phosphoenolpyruvate carboxykinase-encoding gene from the fungus Neocallimastix frontalis, was determined. The deduced amino acid sequence (608 residues) and the predicted protein structure were compared to their counterparts in animals and yeast. Catalytic regions (substrate-binding site and nucleotide-binding domains) are highly conserved among fungal and animal organisms. The yeast sequence showed no similarity to the fungal sequence.

Amino Acid Sequence↗
PubMed1992

[Can solid state Holter monitoring replace endomyocardial biopsy in patients with heart transplantation?].

Acute cardiac graft rejection after transplantation, the diagnosis of which is based on the findings of endomyocardial biopsy, is associated with a reduction in coronary reserve due to abnormalities of the microcirculation. But this reduction in coronary reserve cause silent myocardial ischaemia (SMI)? In order to assess the frequency of SMI and ventricular arrhythmias during rejection, 53 consecutive Holter recordings were performed in 32 patients (28 men, 4 women, average age 47 +/- 11 years) 11 months after transplantation and within 24 hours of endomyocardial biopsy. The recorder which was used (Monitor One TC) analysed the ST segment in 2 leads in real time: ST segment depression of more than 1 mm lasting over 40 ms, 0.08 s after the J point were considered to be diagnostic of myocardial ischaemia. Although the frequency of SMI is low and not specific for cardiac rejection, its duration was twice as long (80 mn vs 38 mn) in this condition. On the other hand, ventricular arrhythmias are common in cardiac rejection and correlated with its severity according to Billingham's classification (VES p = 0.045; doublets p = 0.035; non-sustained VT p = 0.006).

Adolescent↗
PubMed1992

Interactions of bioactive lipopeptides, iturin A and surfactin from Bacillus subtilis.

The antifungal activity of iturin A and its interaction with erythrocyte membranes were enhanced in the presence of surfactin. The modification of the properties of iturin A was explained by the formation of mixed iturin A-surfactin micelles. Such mixed micelles were easily generated when both lipopeptides were in aqueous solutions in the absence of mineral salts but the formation of these micelles did not occur when the solutions contained a high molarity of mineral cations.

Amino Acid Sequence↗
PubMed1991

Intrinsic tryptophan fluorescence of Schizosaccharomyces pombe mitochondrial F1-ATPase. A powerful probe for phosphate and nucleotide interactions.

Mitochondrial F1 from the yeast Schizosaccharomyces pombe, in contrast to the mammalian enzyme, exhibits a characteristic intrinsic tryptophan fluorescence with a maximal excitation at 291 nm and a maximal emission at 332 nm. Low values of Stern-Volmer quenching constants, 4.0 M-1 or 1.8 M-1, respectively, in the presence of either acrylamide or iodide, indicate that tryptophans are mainly buried inside the native enzyme. Upon subunit dissociation and unfolding by 6 M guanidine hydrochloride (Gdn.HCl), the maximal emission is shifted to 354 nm, a value very similar to that obtained with N-acetyltryptophanamide, a solute-tryptophan model compound. The tryptophan content of each isolated subunit has been estimated by fluorescence titration in the presence of Gdn.HCl with free tryptophan as a standard. Two tryptophans and one tryptophan are found respectively in the alpha and epsilon subunits, whereas none is detected in the beta, gamma, and delta subunits. These subunit contents are consistent with the total of seven tryptophans estimated for native F1 with alpha 3 beta 3 gamma 1 delta 1 epsilon 1 stoichiometry. The maximal emission of the isolated epsilon subunit is markedly blue-shifted to 310-312 nm by interaction with the isolated delta subunit, which suggests that the epsilon subunit tryptophan might be a very minor contributor to the native F1 fluorescence measured at 332 nm. This fluorescence is very sensitive to phosphate, which produces a marked blue shift indicative of tryptophans in a more hydrophobic environment. On the other hand, ADP and ATP quench the maximal emission at 332 nm, lower tryptophan accessibility to acrylamide, and reveal tryptophan heterogeneity.

Acrylamide↗
PubMed1991

ANTHEPROT: an interactive graphics software for analyzing protein structures from sequences.

ANTHEPROT is a fully interactive program devoted to the analysis of protein structures using a graphics workstation. It presents four options: The first option can predict secondary structures using five methods, and hydrophobicity, solvent accessibility, flexibility and antigenicity profiles using eighteen scales. The user may introduce his own scales. The results displayed on the screen can be easily analyzed. The second option is for representing results concerning up to eight proteins by one method. To compare these proteins, it is possible to align the profiles or the predicted secondary structure according to various motifs. The secondary structure deduced from crystallographic data may also be introduced. The third option is designed to compare the primary structure of two proteins and to visualize on the screen regions that exhibit similarity. Six different comparison matrices may be used, but the user can also introduce his own matrices. The last option is for studying the proteolytic peptides resulting from a chemical or enzymatic digestion of a given protein. It is possible to analyze the protein cleavage using eleven chemical reagents or enzymes. The results are displayed on the screen as RP-HPLC chromatogram.

Amino Acid Sequence↗