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C Balny

Publications and source records attributed to C Balny.

At least 37 records · Page 2Linked to original sources
PubMed1997

A stable partly denatured state of trypsin induced by high hydrostatic pressure.

The effect of hydrostatic pressure on the unfolding of trypsin was studied by fluorescence spectroscopy under pressure from 1 to 7000 bar. It was found that, at pH 3.0 or pH 7.3, a stable partly denatured state of trypsin was obtained when the applied pressure was about 6.5 kbar. This transient denatured state did not show any enzymatic activity and was different from that denatured by 8 M urea or high temperature in both intrinsic fluorescence spectrum and 8-anilino-1-naphtalene sulfonate (ANS) binding, having some obvious characteristics of 2 molten globule state of protein. It was also found that the formation of this partly denatured state of trypsin was temperature dependent. Energenic values of the process were also given.

Anilino Naphthalenesulfonates↗
PubMed1997

The effect of high pressure on thermolysin.

The effects of high pressure on thermolysin activity and spectroscopic properties were studied. Thermolysin showed distinct pressure-induced activation with a maximum observed at 200-250 MPa for a dipeptide amide substrate and at 100-120 MPa for a heptapeptide substrate. By examining the pressure dependence of the hydrolytic rate for the former substrate using a high pressure stopped-flow apparatus as a mixing device under elevated pressures, the activation volume of the reaction was -71 ml mol(-1) at 25 degrees C. Delta V++ was accompanied by a negative activation expansibility and a value of -95 ml mol(-1) was obtained at 45 degrees C. A prolonged incubation of thermolysin under high pressure, however, caused a time-dependent deactivation. These changes due to pressure were monitored by several spectroscopic methods. The fourth-derivative absorbance spectrum showed an irreversible change, mostly in the tyrosine and tryptophan regions, at a pressure higher than 300 MPa. Intrinsic fluorescence and circular dichroism measurements of thermolysin in solution also detected irreversible changes. All these measurements indicated that a change occurred at higher pressures and are explained by a simple two-state transition model accompanied by a large, negative change in the volume of reaction.

Biochemistry↗
PubMed1996

High pressure effects on protein structure and function.

Many biochemists would regard pressure as a physical parameter mainly of theoretical interest and of rather limited value in experimental biochemistry. The goal of this overview is to show that pressure is a powerful tool for the study of proteins and modulation of enzymatic activity.

Biophysics↗
PubMed1995

Kinetics of butyrylcholinesterase in reversed micelles under high pressure.

The combined effects of high pressure and reversed micelles have been studied to modulate the catalytic behaviour of butyrylcholinesterase. The purpose of this study was to determine whether the conformational plasticity of the enzyme is altered by entrapment in reversed micelles. The presence of soman, an irreversible inhibitor of cholinesterase was used to bring to the fore a possible modification of the enzyme behaviour in this system under pressure. Results show differences between enzyme in conventional medium and in reversed micelles regarding the mechanism of BuChE catalyzed hydrolysis of acetylthiocholine. In both systems, the enzyme displays a non-Michaelian behaviour with this substrate. In conventional medium the kinetics is multiphasic with an activation phase followed by an inhibition phase at high concentration. In reversed micelles there is inhibition by excess substrate but the activation phase is missing. This behaviour may be the result of a change of the enzyme conformational plasticity when is entrapped in reversed micelles.

Acetylthiocholine↗
PubMed1995

Reaction with cyanide of hydroxylamine oxidoreductase of Nitrosomonas europaea.

Hydroxylamine oxidoreductase (HAO) catalyzes the reaction NH2OH+H2O-->HNO2+4e- + 4H+, a step in the energy-generating oxidation of ammonia to nitrite by the bacterium Nitrosomonas europaea. Each subunit of HAO contains 7 c-hemes and 1 heme P460. The latter, c-heme cross-linked from a methylene carbon to the ring of a protein tyrosine, forms part of the active site. The iron of heme P460 is probably linked by a bridging ligand to the iron of a c-heme. Here, the reaction of cyanide with ferric HAO was studied by optical, transient, and steady state kinetic techniques. The molecules, F-, Cl-, Br-, N3-, SCN-, and OCN- did not react with HAO. A single molecule of cyanide bound with high affinity to heme P460 of HAO. The optical and kinetic characteristics of formation of the monocyano complex of HAO resembled those of cyanide derivatives of other heme proteins. Cyanide, in the monocyano complex, was a noncompetitive inhibitor and remained bound during turnover. HAO was found in two forms. The most common form, HAO-A, formed only the monocyano derivative of heme P460, whereas the other, HAO-B, formed a mono- and dicyano complex. The optical properties and kinetics of formation of the mono- and dicyano complexes were different enough to easily allow independent analysis. The optical and kinetic characteristics of formation of the monocyano complex of heme P460 of HAO A and B were very similar. The dicyano complex of HAO-B appeared to result from the addition of a second molecule of cyanide to heme P460. The rate of conversion of the monocyano to the dicyano complex was stimulated 100-fold by the binding of substrate. Formation of the monoheme complex inhibited enzyme activity. The kinetic constants for the first-order formation of the monocyano derivative and the inhibition of substrate oxidation (under either transient or steady-state conditions) were different. The apparent discrepancy could be resolved by the hypothesis that HAO is functionally a dimer in which electrons rapidly equilibrate between the c-hemes of each subunit but not between oligomers. The results form the basis for the use of cyanide as a probe of the active site of HAO.

Binding Sites↗
PubMed1995

Substrate dependence of amiloride- and soman-induced conformation changes of butyrylcholinesterase as evidenced by high-pressure perturbation.

Previous results on butyrylcholinesterase-catalyzed hydrolysis of o-nitrophenylbutyrate in the presence of soman, an irreversible inhibitor of cholinesterases, suggested that reversible binding of soman preceding enzyme phophonylation induced a new enzyme conformational state (E'). The purpose of the present study was to determine whether this effect depends on soman itself or is dependent on the presence and nature of substrate or ligand. First, we examined the effect of amiloride, a reversible cholinesterase effector, upon the butyrylcholinesterase-catalyzed hydrolysis of nitrophenyl esters. The effect of amiloride was found to be dependent on the position ortho or para of the substrate nitro group: amiloride acts as a non-linear reversible activator of p-nitrophenyl ester hydrolysis and as a non-linear reversible inhibitor of o-nitrophenyl ester hydrolysis. Second, the effect of amiloride upon hydrolysis of o/p-nitrophenylbutyrate was also studied under perturbing conditions, i.e., as a function of pressure (1-1600 bar) in the presence and absence of soman. Results show that the effect of reversible soman binding on butyrylcholinesterase activity in the presence of amiloride depends on the position of the substrate nitro group and amiloride concentration. Molecular modelling suggests that the presence of amiloride determines the orientation of ortho- and para-nitrophenyl esters in the active-site. gorge. The nitro group of o-nitrophenylbutyrate interacts with the oxyanion hole via hydrogen bonds and its phenyl ring interacts with amiloride whose heterocycle faces Trp-82. The nitro group of p-nitrophenylbutyrate does not interact with the oxyanion hole but points towards Tyr-332; the phenyl ring of p-nitrophenylbutyrate interacts with amiloride but there is no steric constraint on the acyl chain. Thus, the network of interactions in ternary complexes is tighter with o-nitrophenylbutryate as the substrate. There is no evidence for the existence of amiloride and/or soman-induced E' state when p-nitrophenylbutyrate is the substrate. On the other hand, reversible binding of amiloride and/or soman induces new active conformational states that may be either binary (or ternary) enzyme-ligand complex or new free enzyme conformation resulting from long-lived ligand-induced enzyme conformational change when o-nitrophenylbutyrate is the substrate. These ligand-induced states are stabilized by high pressure.

Amiloride↗
PubMed1995

High-pressure stabilization of alpha-chymotrypsin entrapped in reversed micelles of aerosol OT in octane against thermal inactivation.

alpha-Chymotrypsin (CT) solubilized in reversed micelles of sodium bis-(2-ethylhexyl)-sulfosuccinate (AOT) undergoes thermal inactivation and the enzyme stability decreases significantly when temperature increases (25-40 degrees C). The half-life of CT in micelles shows a bell-shaped dependence on the degree of hydration of AOT (wo) analogous to the previously obtained dependence on wo for the enzyme activity. The optima of catalytic activity and thermal stability have been observed under conditions where the diameter of the inner aqueous cavity of the micelle is close to the size of the enzyme molecule (wo = 10). Application of high hydrostatic pressure in the range of 1-1500 atm (bar) stabilizes CT against thermal inactivation at all hydration degrees (wo) from 7 to 20; the stabilization effect is most pronounced under the experimental conditions being far from the optimum for catalytic activity.

Animals↗
PubMed1994

Interaction of tryptophan residues of cytochrome P450scc with a highly specific fluorescence quencher, a substrate analogue, compared to acrylamide and iodide.

The cytochrome P450scc tryptophan fluorescence was studied by the use of the three quenchers acrylamide, 25-doxyl-27-nor-cholesterol (CNO) and potassium iodide (KI). All the nine tryptophan residues were accessible to acrylamide. Whereas a strong interaction (static quenching) between acrylamide and tryptophan in the active site had been found previously for cytochrome P450c21 [Narasimhulu, S. (1988) Biochemistry 27, 1147-1153], in the case of P450scc the temperature dependence of the slope of the linear Stern-Volmer plots indicated a dynamic quenching mechanism. This mechanism was confirmed by fluorescence lifetime measurements. Of the three observed life-times tau 1 = 3.1 +/- 0.5 ns, tau 2 = 0.7 +/- 0.25 ns and tau 3 = 20 +/- 10 ps, tau 1 decreased noticeably as a function of the acrylamide concentration. CNO, a spin-labeled substrate which is known to bind tightly to the substrate-binding site of P450scc, quenched 15.5% of the total fluorescence. The Lehrer plot of this compound indicated a static quenching process with a reciprocal quenching constant of 1/Ks = 4 microM, a value which is in accord with the dissociation constant. Our data indicate that CNO quenches selectively one or two tryptophan residue(s) in the active site. The fluorescence spectrum of the residue(s) accessible to CNO was characterized by a red-shifted emission maximum (from 332 nm to 336 nm). The same residue(s) appeared to be quenched by potassium iodide, although much less effectively (1/Ks = 0.12 M). The most probable candidate for a complex formation with CNO is Trp417, which is rather close to Cys422 (the fifth heme ligand). Four arginine residues (Arg411, Arg420, Arg425 and Arg426) in the heme peptide may constitute the iodide-binding site.

Acrylamide↗
PubMed1994

Activation volume and energetic properties of the binding of CO to hemoproteins.

We have investigated the CO binding to various reduced hemoproteins by stopped-flow rapid mixing as a function of pressure (from 0.1 to 200 MPa) and temperature (from 4 to 35 degrees C). In particular, we studied several varieties of cytochrome P-450: CYP11A1 (scc), CYP2B4 (LM2), CYP3A6 (LM3c), and Cyp2a (7 alpha), as well as chloroperoxidase and lactoperoxidase, and compared the results to data reported for other hemoproteins. Whereas the CO binding activation enthalpy delta H++ and entropy delta S++ (correlated through a compensation effect) varied greatly between the hemoproteins, with no apparent relation to structural features, the pressure effect depended on the nature of the proximal axial heme ligand: the activation volume was very small for cysteine (S-) ligand hemoproteins (delta V++ = +1 to +6 ml mol-1), and markedly negative for histidine (N) ligand hemoproteins (delta V++ = -3 to -36 ml mol-1). Furthermore, the transition state volume of the histidine ligand class enzymes, but not that of the cysteine ligand enzymes, depended on the solvent composition. These results suggest that the CO-binding transition state of the S-ligand class has a molecular conformation similar to the ground state. In the histidine class, however, the transition state appears to involve protein conformational changes and/or solvation processes.

Adrenal Cortex↗
PubMed1994

Pressure effects on enzyme reactions in mainly organic media: alpha-chymotrypsin in reversed micelles of Aerosol OT in octane.

Biocatalytic transformations in reversed micelles formed by anionic surfactant Aerosol OT in octane have been studied at high pressures by an example of alpha-chymotrypsin-catalyzed hydrolysis of N-carbobenzoxy-L-tyrosine p-nitrophenyl ester and N-succinyl-L-phenylalanine p-nitroanilide. For the first time it has been found that the enzyme retains high activity in these water-in-oil microemulsions up to a pressure of 2 kbar. The value of the activation volume (delta V*) for the enzyme reactions shows a dependence on the water content in the system. When the size of the micellar aqueous inner cavity (as evaluated at 1 atm) approaches the molecular size of alpha-chymotrypsin, delta V* becomes significantly different from the value in aqueous solution and in the micelles with a larger size. Possibilities of regulating the enzyme activity by pressure in systems with a low content of water are discussed.

Animals↗
PubMed1992

Soman inhibition of butyrylcholinesterase in the presence of substrate: pressure and temperature perturbations.

Irreversible inhibition of butyrylcholinesterase by soman was studied in the presence of the substrate (o-nitrophenyl butyrate). Inhibition was found of the competitive complexing type. Study at different temperatures and pressures showed that the behavior of the enzyme differs from that of the inhibitor-free enzyme. In the absence of inhibitor, enzyme kinetics displayed a non-linear temperature dependence with a break at 21 degrees C. In the presence of a non-inhibitor structural analog of soman (pinacolyl dimethylphosphinate and methyl dimethylphosphinate), the Arrhenius plot break is slightly shifted (18 degrees C). On the other hand, in the presence of soman this break is abolished. The pressure-dependence of the substrate hydrolysis revealed also differences between the native enzyme and the enzyme in the presence of soman: the sign and magnitude of the apparent activation volume (delta V not equal to) were different for the two reactions. Beyond 300 bar, in the presence of soman, a plateau (delta V not equal to approx. 0) was observed over a large pressure range depending on temperature. Such a behavior with respect to temperature and pressure can reflect a soman-induced enzyme conformational state. Thus, temperature and pressure perturbations of the kinetics allow to complete the inhibition scheme of butyrylcholinesterase by soman. Our data suggest that upon soman binding, the enzyme undergoes a long-lived soman-induced-fit conformational change preceding the phosphonylation step. However, an alternative hypothesis according to which the enzyme processes a secondary soman-binding site cannot be ruled out.

Binding Sites↗
PubMed1992

Pressure modulation of cytochrome-to-cytochrome electron-transfer. Models and enzyme reactions.

The kinetics of electron-transfer involved in reactions of reduction of 2,6-dichlorophenol indophenol and Fe(CN)3-(6) by L-ascorbic acid and reduction of ferric cytochrome c by both L-ascorbic acid and reduced hydroxylamine oxidoreductase were studied as a function of three parameters: ionic strength, pressure (1-2000 bar) and temperature (4-20 degrees C) using the high-pressure stopped-flow method. From measurements, the thermodynamic parameters of activation volume (delta V++), and, when possible, activation enthalpy and entropy (delta H++ and delta S++) have been calculated. We found, for these four systems, that the pressure has revealed solvation effects involved in electron-transfer. For the reduction of ferric cytochrome c by reduced hydroxylamine oxidoreductase (a cytochrome-to-cytochrome electron-transfer), we have not obtained evidence for a conformational change.

2,6-Dichloroindophenol↗
PubMed1991

Spectroscopic and rapid kinetic studies of reduction of cytochrome c554 by hydroxylamine oxidoreductase from Nitrosomonas europaea.

During oxidation of hydroxylamine, hydroxylamine oxidoreductase (HAO) transfers two electrons to tetraheme cytochrome c554 at rates sufficient to account for physiological rates of oxidation of ammonia to nitrite in Nitrosomonas europaea. Spectroscopic changes indicate that the two electrons are taken up by a high-potential pair of hemes (E degrees' = +47 mV) (one apparently high spin and one low spin). During single-turnover experiments, in which the reduction of oxidized cytochrome c554 by NH2OH-reduced HAO is monitored, one electron is taken up by the high-spin heme at a rate too fast to monitor directly (greater than 100 s-1) but which is inferred either by a loss of amplitude (relative to that observed under multiple-turnover conditions) or is slowed down by increasing ionic strength (greater than or equal to 300 mM KCl). The second electron is taken up by the low-spin heme at a 10-30-fold slower rate. The latter kinetics appear multiphasic and may be complicated by a transient oxidation of HAO due to the rapid transfer of the first electron into the high-spin heme of cytochrome c554. Under multiple-turnover conditions, a "slower" rate of reduction is observed for the high-spin heme of cytochrome c554 with a maximum rate constant of approximately 30 s-1, a value also obtained for the reduction, by NH2OH, of the cytochrome c554 high-spin heme within an oxidized HAO/c554 complex. Under these conditions, the maximum rate of reduction of the low-spin heme was approximately 11.0 s-1. Both rates decreased as the concentration of cytochrome c554 was increased above the concentration of HAO.(ABSTRACT TRUNCATED AT 250 WORDS)

Ammonium Hydroxide↗
PubMed1991

The effect of hydrostatic pressure on the interaction of actomyosin subfragment 1 with nucleotides.

Increased hydrostatic pressure has previously been shown to reduce the tension of isometrically contracting skinned muscle fibres. An isomerization of the actomyosin complex is known to be pressure sensitive, but the pressure sensitivity of other steps in the ATPase pathway has not been characterised. We report here the effect of pressure on the ATP hydrolysis step of the myosin subfragment 1 ATPase, ADP binding to actomyosin subfragment 1 and the rate of ATP induced dissociation of actomyosin subfragment 1. We discuss the relationship of these changes to the observed effect of pressure on skinned muscle fibres.

Actomyosin↗
PubMed1991

Preliminary studies on quinoprotein glucose dehydrogenase under extreme conditions of temperature and pressure.

The kinetics of the reduction of the quinoprotein glucose dehydrogenase by substrate were studied as a function of 3 parameters: pressure (1-1000 bar), temperature (down to -25 degrees C) and solvent (water and 40% dimethyl sulfoxide, DMSO) using a high-pressure low-temperature stopped-flow apparatus. A 2-step formation of the reduced enzyme by its substrate (xylose), was observed. A rapid equilibrium described by the constant K1 was followed by a slower process described by the constants k2 and k-2. By using the transition state theory, the thermodynamic quantities delta V (activation volumes) were determined for these various kinetics constants under different experimental conditions. The results are discussed in terms of conformational change and solvation effect on the protein shell, and compared with results obtained for other systems as the 2-step formation of horseradish peroxidase compound I.

Glucose Dehydrogenases↗
PubMed1990

Conformational plasticity of butyrylcholinesterase as revealed by high pressure experiments.

The ligand binding and kinetic behaviour of butyrylcholinesterase (EC 3.1.1.8, acylcholine acylhydrolase) from human plasma was studied at 35 degrees C under high hydrostatic pressure. The binding of phenyltrimethylammonium was studied by affinity electrophoresis at various pressures ranging from 10(-3) to 2 kbar. The kinetics of enzyme carbamylation with N-methyl(7-dimethylcarbamoxy)quinolinium iodide was studied in single-turnover conditions up to 1.2 kbar using a high-pressure stopped-flow fluorimeter. Experiments were carried out in different media: 1 mM Tris-HCl (pH 8) with water, water containing 0.1 M lithium chloride and deuterium oxide as solvents. The volume changes (delta V and delta V++) associated with each process were determined from the pressure-dependence of the binding and kinetic constants. Kinetic data show that the binding of substrate to the enzyme leads to a pressure-sensitive enzyme conformational state which cannot accomplish the catalytic act. The pressure-induced inhibitory effect is highly cooperative; it depends on both the nature (charged or neutral) and the concentration of the substrate. Also, large solvent effects indicate that enzyme sensitivity to pressure depends on the solvent structure. This findings suggests that the substrate-dependent pressure effect is modulated by the solvation state of the enzyme.

Butyrylcholinesterase↗
PubMed1990

Electrophoresis at elevated hydrostatic pressure of the multiheme hydroxylamine oxidoreductase.

The behavior of the multiheme protein hydroxylamine oxidoreductase (HAO) in polyacrylamide gel electrophoresis was studied at hydrostatic pressures up to 3 kbar at 25 degrees C. Due to the limited working volume of the high pressure vessel, the electrophoresis cells were miniaturized. A microcell which accommodates 6 capillary gel tubes is described. Between 1 bar and 1.5 kbar the enzyme did not undergo structural changes detectable in the gel system. At approximately 2 kbar the active form of the enzyme was partially dissociated. At higher pressures, the enzyme was converted to forms which were irreversibly inactive and had a higher apparent molecular mass, suggesting aggregation or denaturation.

Atmospheric Pressure↗