Veronique Larreta-garde - Academia.edu (original) (raw)

Papers by Veronique Larreta-garde

Research paper thumbnail of Structural modifications of oxidized insulin B-chain induced by glycerol addition

Letters in Peptide Science, 1997

1H-NMR studies of the bovine insulin S-sulfonated B-chain are reported in H20/D2 O (9/1) and in g... more 1H-NMR studies of the bovine insulin S-sulfonated B-chain are reported in H20/D2 O (9/1) and in glycerol-d5 (5 M) using two-dimensional NMR spectroscopy. The first results show that the oxidized insulin B-chain secondary structure differs from that of native insulin by a loss of the a-helix between the two disulfide bridges and that the glycerol favours the structuring of the peptide.

Research paper thumbnail of Micro-environment effect on the structure of synthetic substrates of thermolysin

Letters in Peptide Science, 1997

The conformations of thermolysin synthetic substrates in H20/D20 (9/1) and glycerol-d5 (5 M) are ... more The conformations of thermolysin synthetic substrates in H20/D20 (9/1) and glycerol-d5 (5 M) are investigated using two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling. The structures obtained from molecular modeling and NMR studies are compared. Comparisons of these structures with bound inhibitor in the active site of thermolysin are also discussed.

Research paper thumbnail of Carbamylation differentially alters type I collagen sensitivity to various collagenases

Matrix Biology, 2007

Carbamylation is a post-translational modification due to nonenzymatic binding of cyanate, a by-p... more Carbamylation is a post-translational modification due to nonenzymatic binding of cyanate, a by-product of urea, on free amino groups of proteins. Post-translational modifications are known to induce alterations in structural and functional properties of proteins, thus disturbing protein-protein or cell-protein interactions. We report the impact of carbamylation on type I collagen sensitivity to enzymatic proteolysis. Type I collagen was extracted from rat tail tendons and carbamylated by incubation with 0.1 M potassium cyanate at 37°C for 2, 6 or 24 h. Degradation assays revealed that carbamylated collagen exhibited a greater resistance to collagenases (i.e. bacterial collagenase, matrix metalloproteinase (MMP)-1, MMP-8 and MMP-13), together with an increased sensitivity to MMP-2. Evaluation of collagen triple helix conformation by polarimetry indicated that local destabilizations of triple helix structure related to carbamylation could be responsible for the observed differences in sensitivity. These results confirm the crucial role of triple helix integrity in the degradation of type I collagen by MMPs, and support the deleterious impact of post-translational modifications in vivo by altering the balanced remodeling of collagen within connective tissue.

Research paper thumbnail of Structural studies of a small (linear, cyclic) peptide as a synthetic substrate for thermolysin

Journal of Molecular Structure, 1999

A synthetic peptide substrate, allowing a cleavage bond (GF) for the specific thermolysin and a s... more A synthetic peptide substrate, allowing a cleavage bond (GF) for the specific thermolysin and a switchable S-S bridge (reduced -linear-and oxidised -cyclic-form) was designed, synthesised, and enzymatically studied. Despite the fact that strong substrate structural constraints are supposed to appear with the S-S bridge presence, the catalytic behaviour of the thermolysin does not seem strongly affected on the cyclic form compared to the linear one. The two different forms of the peptide structure was studied using theoretical secondary structure prediction, optical spectroscopic methods (CD, FTIR, Raman) and molecular modelling studies. Our results are discussed and correlated in term of structure-function and/or structure activity relationships. ᭧

Research paper thumbnail of Raman spectroscopic study of a non-heme iron enzyme soybean lipoxygenase - 1 in low hydrated media

Journal of Molecular Structure, 1995

This work is a contribution to the experimental study of the structure-function relationship of e... more This work is a contribution to the experimental study of the structure-function relationship of enzymes in non conventional media. As it is known that bound water play a determining role on affinity and specificity, we have chosen aqueous media with restricted water content. Water activity of the reaction media was decreased by addition of specific hydrosoluble cosolvents.

Research paper thumbnail of Solute-Solvent and Water Activity of Small Carbohydrates: Application to the Study of Enzyme Stability in Aqueous Sugar Solutions

Journal of Carbohydrate Chemistry, 1989

Viscometric constants of small carbohydrates are determined and interpreted in terms of specific ... more Viscometric constants of small carbohydrates are determined and interpreted in terms of specific hydration. Water activity of their saturated solutions is measured. Their effect on water structure is deduced from the deconvolution of the Raman bands in the OH region. The influence of water activity lowering of D-fructose, D-glucose and sucrose on initial lysozyme activity is studied. The effect of

Research paper thumbnail of Structural Studies by 1 H NMR and Molecular Modeling of Peptide Substrates of Thermolysin in Relation with its Proteasic Activity in Water and Glycerol

Journal of Biomolecular Structure and Dynamics, 1999

In an attempt to explain the relationship between conformations of peptide substrates of thermoly... more In an attempt to explain the relationship between conformations of peptide substrates of thermolysin in natural form and the experimental enzymatic cleavages, five peptides of various length were studied in two solvents H2O and glycerol, which may mimic the catalytic environmental conditions. As NMR failed to define sufficently rough constraints to ensure a convergence of a refinement process for such

Research paper thumbnail of Removal of surface by-products from sintered hydroxyapatite: Effect of a chelation treatment on fibronectin adsorption and cell adhesion

Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2006

It was observed that fibronectin precipitates when deposited on hydroxyapatite (HA) ceramics. Fib... more It was observed that fibronectin precipitates when deposited on hydroxyapatite (HA) ceramics. Fibronectin's known affinity for calcium and the composition of the ceramic itself suggested that calcium release could be the main cause of this aggregation effect. It was then decided to investigate the effect of a surface chelation treatment on fibronectin adsorption, and MG63 cell adhesion, onto porous ceramics of hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP), and HA/TCP biphasic material (BCP). Those ceramics were immersed in an EDTA solution and the effect of this treatment on the material composition was assayed. X-ray diffraction data showed the presence of alpha- and beta-TCP phases in HA and BCP materials, which were both completely removed by the chelation treatment in the case of HA. On BCP, alpha-TCP was removed and beta-TCP partially dissolved. The TCP material, which was pure beta-TCP, underwent a mass loss, but no change in composition was observed. Adhesion of MG63 cells was overall higher on the fibronectin-coated EDTA-treated HA material, but was especially enhanced on EDTA-treated HA. Changes in surface morphologies, as compared with the use of scanning electron microscopy, did not seem to be related to the effects observed. The EDTA treatment proved to be a very efficient way of removing by-products of HA sintered materials, and thus enhancing the biocompatibility of the material.

Research paper thumbnail of Effect of polyhydroxylic additives on the catalytic activity of thermolysin

Enzyme and Microbial Technology, 1998

The catalytic activity of thermolysin during the hydrolysis of N-(3-[2-furyl]acryloyl)-Gly-Leu am... more The catalytic activity of thermolysin during the hydrolysis of N-(3-[2-furyl]acryloyl)-Gly-Leu amide is noticeably enhanced in the presence of sugars and polyols. A series of polyhydroxylic additives were tested and the degree of activation was found to depend on both the concentration and nature of the additive. Sucrose and trehalose were found to yield the higher activation effect whereas glycerol was found to yield an inhibition of thermolysin in a large domain of concentration. A mechanism of activation based on the lowering of the energy barrier for the enzymatic reaction is proposed. This free energy barrier lowering is very likely due to water structure modification by the additives.

Research paper thumbnail of A new possibility for the direct use of soybean lipoxygenase on concentrated triglycerides

Enzyme and Microbial Technology, 1993

... Acta 1972, 284, 54-62 8 Vertrue, WM and Francke, A. Biochim. Biophys. Acta 1972 284,43-53 9 F... more ... Acta 1972, 284, 54-62 8 Vertrue, WM and Francke, A. Biochim. Biophys. Acta 1972 284,43-53 9 Funk, MO, Whitney, MA, Hausknecht, EC and O'Brien, EM Anal. Biochem. ... Biophys. Acta 1986, 870, 367-371 14 Datcheva, VK, Kiss, K., Solomon, L. and Kyler, KSJ Am. Chem. Soc. ...

Research paper thumbnail of Enzyme-Catalyzed Gel Proteolysis: An Anomalous Diffusion-Controlled Mechanism

Biophysical Journal, 2003

Enzyme-catalyzed proteolysis of gelatin gels has been studied. We report a gel degradation rate v... more Enzyme-catalyzed proteolysis of gelatin gels has been studied. We report a gel degradation rate varying as the square of the enzyme concentration. The diffusion motion of enzymes in the gel has been measured by two-photon fluorescence correlation spectroscopy and identified as being anomalously slow. These experimental results are discussed from a theoretical point of view and interpreted in terms of a diffusion-controlled mechanism for the gel degradation. These results make a step toward the understanding of enzyme-catalyzed gel degradation and give new insight on biological processes such as the action of metalloproteinases in the extracellular matrix involved in cellular invasion.

Research paper thumbnail of Enzyme-Catalyzed Phase Transition of Alginate Gels and Gelatin−Alginate Interpenetrated Networks

Biomacromolecules, 2007

The enzyme-catalyzed gel-sol transition of calcium-alginate obtained by internal gelling strategy... more The enzyme-catalyzed gel-sol transition of calcium-alginate obtained by internal gelling strategy with the help of an entrapped alginate lyase is described. We show that alginate molecules and enzyme-produced oligoalginates shorten the gel time of physical gelatin gels (5% and 1.5%), probably due to local protein concentration increase. Interpenetrated networks composed of calcium-alginate and of gelatin were obtained only if elongation of gelatin helices inside a pre-existing calcium-alginate network could occur and only for low gelatin concentration (1.5%). The physical gelatin network is almost reversible inside the alginate one. Both networks can be obtained in the presence of alginate lyase, but gel-sol transition of calcium-alginate cannot be obtained in the presence of gelatin.

Research paper thumbnail of Influence of Weak and Covalent Bonds on Formation and Hydrolysis of Gelatin Networks

Biomacromolecules, 2004

The relative influence of physical and chemical bonds to overall gel properties are explored in g... more The relative influence of physical and chemical bonds to overall gel properties are explored in gelatin gels. Physical, chemical, chemical-physical, and physical-chemical gels are obtained by cooling the protein solution and/or by transglutaminase reaction. Each type of network is characterized by rheology and polarimetry. It is shown that the overall properties as well as the dynamics inside the gels are dependent upon the order of formation and on the relative amount of triple helices and covalent bonds. Enzyme hydrolysis of covalent gels is slower than that of physical gels, as confirmed by the kinetics of helix release and degradation. A scheme is proposed to explain the results at both the physicochemical and the molecular levels.

Research paper thumbnail of Lysozyme kinetics in low water activity media a possible hydration memory

Biochemical and Biophysical Research Communications, 1988

The influence of water activity on initial lysozyme kinetics is studied. Enzyme catalytic capacit... more The influence of water activity on initial lysozyme kinetics is studied. Enzyme catalytic capacity exponentially increases with thermodynamical water activity of the reactant medium ; this relation seems independent of the chemical structure of the water activity depressors but dependent on the structuration of water molecules they induce.This influence of water organization on initial enzyme activity is immediate and may be preserved even after a large dilution, thus lysozyme presents a "hydration memory" phenomenon, This effect is in accordance with sorption / desorption isotherms, an hysteresis loop being observed.

Research paper thumbnail of Comparison of Macromolecular and Molecular Effects of Cosolvents on the Catalytic Behaviour of Soybean Lipoxygenase-1

Biocatalysis and Biotransformation, 1997

... CHRISTINE LAMBERT, CHRISTINE POURPLANCHE and VERONIQUE LARRETA-GARDE ... subsequent enzyme ac... more ... CHRISTINE LAMBERT, CHRISTINE POURPLANCHE and VERONIQUE LARRETA-GARDE ... subsequent enzyme activities (Gavish and Yedgar, 1995) but conflicting effects have been observed de-pending on the range of viscosites studied (de Almeida Cunha and Vitolo, 1984 ...

Research paper thumbnail of Glycerol’s influence on the oxidized insulin B-chain conformation in relation to the selectivity variation of subtilisin: an nuclear magnetic resonance and simulated annealing study

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1999

Glycerol, employed to mimic biological media with restricted water activity, has been shown to mo... more Glycerol, employed to mimic biological media with restricted water activity, has been shown to modify the activity of subtilisin BPNP, an endopeptidase, towards the oxidized insulin B-chain, a well-studied substrate (FEBS Lett., 279 (1991) 123^131). Without minimizing the role of the microenvironment on the enzyme, we have studied the effect of glycerol addition on the structure of the enzyme substrate by homonuclear NMR spectroscopy and simulated annealing. Our results show that, in water, the oxidized insulin B-chain tertiary structure loses its central helix (residues B9^B19) and presents a folded structure with a flexible turn (residues B18^B24) in the L-turn region of the insulin B-chain; whereas, in glycerol, the peptide is more rigid and is not folded. Moreover, in our experimental conditions, glycerol favors L-strand secondary structure formation. Following these results, hypotheses about the differences observed in enzymatic activity on this substrate in glycerol have been postulated. ß

Research paper thumbnail of Additional data about thermolysin specificity in buffer- and glycerol-containing media

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1997

Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recogn... more Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recognition and elucidation of several new criteria affecting enzyme specificity. Nature and position of the recognized residue, role of adjacent amino acids, lateral chain hydrophobicity, and volume and length of peptides were all considered. Hydrolysis reactions were also carried out in the presence of glycerol; the effect of microenvironment modifications was quantitative, for example, in inducing variations in catalytic reaction rates, and also qualitative, such as in influencing affinity.

Research paper thumbnail of Gel–sol transition can describe the proteolysis of extracellular matrix gels

Biochimica et Biophysica Acta (BBA) - General Subjects, 2000

We monitored the cell-free solubilization of extracellular matrix and fibronectin gels, catalyzed... more We monitored the cell-free solubilization of extracellular matrix and fibronectin gels, catalyzed by exogenous proteinases. The corresponding measurements could not be interpreted according to usual proteinase kinetics. The observation that this experimental system did not consist in surface but in bulk degradation and appeared specific to gel substrates, incited us to use gelation-related approaches to describe these kinetics. We show that the gel^sol transition theory adequately describes the enzyme reactions. This allowed formulation and experimental confirmation of a power law relating macroscopic changes of the gel to enzyme kinetics. This approach could also be used for other power laws predicted by the gel^sol transition theory, allowing a better understanding of macroscopic modification of the extracellular matrix during proteolysis, which is implied in many biological situations, especially tumor dissemination. ß

Research paper thumbnail of Kinetic and Structural Observations of a Nonheme Iron Enzyme Contribution of Conformational Changes to the Modification of Lipoxygenase Catalysis

Annals of the New York Academy of Sciences, 1995

Research paper thumbnail of Proteolysis of Aggregated Fibronectin A Model for In Vivo Matrix Degradation

Annals of the New York Academy of Sciences, 1998

Research paper thumbnail of Structural modifications of oxidized insulin B-chain induced by glycerol addition

Letters in Peptide Science, 1997

1H-NMR studies of the bovine insulin S-sulfonated B-chain are reported in H20/D2 O (9/1) and in g... more 1H-NMR studies of the bovine insulin S-sulfonated B-chain are reported in H20/D2 O (9/1) and in glycerol-d5 (5 M) using two-dimensional NMR spectroscopy. The first results show that the oxidized insulin B-chain secondary structure differs from that of native insulin by a loss of the a-helix between the two disulfide bridges and that the glycerol favours the structuring of the peptide.

Research paper thumbnail of Micro-environment effect on the structure of synthetic substrates of thermolysin

Letters in Peptide Science, 1997

The conformations of thermolysin synthetic substrates in H20/D20 (9/1) and glycerol-d5 (5 M) are ... more The conformations of thermolysin synthetic substrates in H20/D20 (9/1) and glycerol-d5 (5 M) are investigated using two-dimensional nuclear magnetic resonance spectroscopy and molecular modeling. The structures obtained from molecular modeling and NMR studies are compared. Comparisons of these structures with bound inhibitor in the active site of thermolysin are also discussed.

Research paper thumbnail of Carbamylation differentially alters type I collagen sensitivity to various collagenases

Matrix Biology, 2007

Carbamylation is a post-translational modification due to nonenzymatic binding of cyanate, a by-p... more Carbamylation is a post-translational modification due to nonenzymatic binding of cyanate, a by-product of urea, on free amino groups of proteins. Post-translational modifications are known to induce alterations in structural and functional properties of proteins, thus disturbing protein-protein or cell-protein interactions. We report the impact of carbamylation on type I collagen sensitivity to enzymatic proteolysis. Type I collagen was extracted from rat tail tendons and carbamylated by incubation with 0.1 M potassium cyanate at 37°C for 2, 6 or 24 h. Degradation assays revealed that carbamylated collagen exhibited a greater resistance to collagenases (i.e. bacterial collagenase, matrix metalloproteinase (MMP)-1, MMP-8 and MMP-13), together with an increased sensitivity to MMP-2. Evaluation of collagen triple helix conformation by polarimetry indicated that local destabilizations of triple helix structure related to carbamylation could be responsible for the observed differences in sensitivity. These results confirm the crucial role of triple helix integrity in the degradation of type I collagen by MMPs, and support the deleterious impact of post-translational modifications in vivo by altering the balanced remodeling of collagen within connective tissue.

Research paper thumbnail of Structural studies of a small (linear, cyclic) peptide as a synthetic substrate for thermolysin

Journal of Molecular Structure, 1999

A synthetic peptide substrate, allowing a cleavage bond (GF) for the specific thermolysin and a s... more A synthetic peptide substrate, allowing a cleavage bond (GF) for the specific thermolysin and a switchable S-S bridge (reduced -linear-and oxidised -cyclic-form) was designed, synthesised, and enzymatically studied. Despite the fact that strong substrate structural constraints are supposed to appear with the S-S bridge presence, the catalytic behaviour of the thermolysin does not seem strongly affected on the cyclic form compared to the linear one. The two different forms of the peptide structure was studied using theoretical secondary structure prediction, optical spectroscopic methods (CD, FTIR, Raman) and molecular modelling studies. Our results are discussed and correlated in term of structure-function and/or structure activity relationships. ᭧

Research paper thumbnail of Raman spectroscopic study of a non-heme iron enzyme soybean lipoxygenase - 1 in low hydrated media

Journal of Molecular Structure, 1995

This work is a contribution to the experimental study of the structure-function relationship of e... more This work is a contribution to the experimental study of the structure-function relationship of enzymes in non conventional media. As it is known that bound water play a determining role on affinity and specificity, we have chosen aqueous media with restricted water content. Water activity of the reaction media was decreased by addition of specific hydrosoluble cosolvents.

Research paper thumbnail of Solute-Solvent and Water Activity of Small Carbohydrates: Application to the Study of Enzyme Stability in Aqueous Sugar Solutions

Journal of Carbohydrate Chemistry, 1989

Viscometric constants of small carbohydrates are determined and interpreted in terms of specific ... more Viscometric constants of small carbohydrates are determined and interpreted in terms of specific hydration. Water activity of their saturated solutions is measured. Their effect on water structure is deduced from the deconvolution of the Raman bands in the OH region. The influence of water activity lowering of D-fructose, D-glucose and sucrose on initial lysozyme activity is studied. The effect of

Research paper thumbnail of Structural Studies by 1 H NMR and Molecular Modeling of Peptide Substrates of Thermolysin in Relation with its Proteasic Activity in Water and Glycerol

Journal of Biomolecular Structure and Dynamics, 1999

In an attempt to explain the relationship between conformations of peptide substrates of thermoly... more In an attempt to explain the relationship between conformations of peptide substrates of thermolysin in natural form and the experimental enzymatic cleavages, five peptides of various length were studied in two solvents H2O and glycerol, which may mimic the catalytic environmental conditions. As NMR failed to define sufficently rough constraints to ensure a convergence of a refinement process for such

Research paper thumbnail of Removal of surface by-products from sintered hydroxyapatite: Effect of a chelation treatment on fibronectin adsorption and cell adhesion

Journal of Biomedical Materials Research Part B: Applied Biomaterials, 2006

It was observed that fibronectin precipitates when deposited on hydroxyapatite (HA) ceramics. Fib... more It was observed that fibronectin precipitates when deposited on hydroxyapatite (HA) ceramics. Fibronectin's known affinity for calcium and the composition of the ceramic itself suggested that calcium release could be the main cause of this aggregation effect. It was then decided to investigate the effect of a surface chelation treatment on fibronectin adsorption, and MG63 cell adhesion, onto porous ceramics of hydroxyapatite (HA), beta-tricalcium phosphate (beta-TCP), and HA/TCP biphasic material (BCP). Those ceramics were immersed in an EDTA solution and the effect of this treatment on the material composition was assayed. X-ray diffraction data showed the presence of alpha- and beta-TCP phases in HA and BCP materials, which were both completely removed by the chelation treatment in the case of HA. On BCP, alpha-TCP was removed and beta-TCP partially dissolved. The TCP material, which was pure beta-TCP, underwent a mass loss, but no change in composition was observed. Adhesion of MG63 cells was overall higher on the fibronectin-coated EDTA-treated HA material, but was especially enhanced on EDTA-treated HA. Changes in surface morphologies, as compared with the use of scanning electron microscopy, did not seem to be related to the effects observed. The EDTA treatment proved to be a very efficient way of removing by-products of HA sintered materials, and thus enhancing the biocompatibility of the material.

Research paper thumbnail of Effect of polyhydroxylic additives on the catalytic activity of thermolysin

Enzyme and Microbial Technology, 1998

The catalytic activity of thermolysin during the hydrolysis of N-(3-[2-furyl]acryloyl)-Gly-Leu am... more The catalytic activity of thermolysin during the hydrolysis of N-(3-[2-furyl]acryloyl)-Gly-Leu amide is noticeably enhanced in the presence of sugars and polyols. A series of polyhydroxylic additives were tested and the degree of activation was found to depend on both the concentration and nature of the additive. Sucrose and trehalose were found to yield the higher activation effect whereas glycerol was found to yield an inhibition of thermolysin in a large domain of concentration. A mechanism of activation based on the lowering of the energy barrier for the enzymatic reaction is proposed. This free energy barrier lowering is very likely due to water structure modification by the additives.

Research paper thumbnail of A new possibility for the direct use of soybean lipoxygenase on concentrated triglycerides

Enzyme and Microbial Technology, 1993

... Acta 1972, 284, 54-62 8 Vertrue, WM and Francke, A. Biochim. Biophys. Acta 1972 284,43-53 9 F... more ... Acta 1972, 284, 54-62 8 Vertrue, WM and Francke, A. Biochim. Biophys. Acta 1972 284,43-53 9 Funk, MO, Whitney, MA, Hausknecht, EC and O'Brien, EM Anal. Biochem. ... Biophys. Acta 1986, 870, 367-371 14 Datcheva, VK, Kiss, K., Solomon, L. and Kyler, KSJ Am. Chem. Soc. ...

Research paper thumbnail of Enzyme-Catalyzed Gel Proteolysis: An Anomalous Diffusion-Controlled Mechanism

Biophysical Journal, 2003

Enzyme-catalyzed proteolysis of gelatin gels has been studied. We report a gel degradation rate v... more Enzyme-catalyzed proteolysis of gelatin gels has been studied. We report a gel degradation rate varying as the square of the enzyme concentration. The diffusion motion of enzymes in the gel has been measured by two-photon fluorescence correlation spectroscopy and identified as being anomalously slow. These experimental results are discussed from a theoretical point of view and interpreted in terms of a diffusion-controlled mechanism for the gel degradation. These results make a step toward the understanding of enzyme-catalyzed gel degradation and give new insight on biological processes such as the action of metalloproteinases in the extracellular matrix involved in cellular invasion.

Research paper thumbnail of Enzyme-Catalyzed Phase Transition of Alginate Gels and Gelatin−Alginate Interpenetrated Networks

Biomacromolecules, 2007

The enzyme-catalyzed gel-sol transition of calcium-alginate obtained by internal gelling strategy... more The enzyme-catalyzed gel-sol transition of calcium-alginate obtained by internal gelling strategy with the help of an entrapped alginate lyase is described. We show that alginate molecules and enzyme-produced oligoalginates shorten the gel time of physical gelatin gels (5% and 1.5%), probably due to local protein concentration increase. Interpenetrated networks composed of calcium-alginate and of gelatin were obtained only if elongation of gelatin helices inside a pre-existing calcium-alginate network could occur and only for low gelatin concentration (1.5%). The physical gelatin network is almost reversible inside the alginate one. Both networks can be obtained in the presence of alginate lyase, but gel-sol transition of calcium-alginate cannot be obtained in the presence of gelatin.

Research paper thumbnail of Influence of Weak and Covalent Bonds on Formation and Hydrolysis of Gelatin Networks

Biomacromolecules, 2004

The relative influence of physical and chemical bonds to overall gel properties are explored in g... more The relative influence of physical and chemical bonds to overall gel properties are explored in gelatin gels. Physical, chemical, chemical-physical, and physical-chemical gels are obtained by cooling the protein solution and/or by transglutaminase reaction. Each type of network is characterized by rheology and polarimetry. It is shown that the overall properties as well as the dynamics inside the gels are dependent upon the order of formation and on the relative amount of triple helices and covalent bonds. Enzyme hydrolysis of covalent gels is slower than that of physical gels, as confirmed by the kinetics of helix release and degradation. A scheme is proposed to explain the results at both the physicochemical and the molecular levels.

Research paper thumbnail of Lysozyme kinetics in low water activity media a possible hydration memory

Biochemical and Biophysical Research Communications, 1988

The influence of water activity on initial lysozyme kinetics is studied. Enzyme catalytic capacit... more The influence of water activity on initial lysozyme kinetics is studied. Enzyme catalytic capacity exponentially increases with thermodynamical water activity of the reactant medium ; this relation seems independent of the chemical structure of the water activity depressors but dependent on the structuration of water molecules they induce.This influence of water organization on initial enzyme activity is immediate and may be preserved even after a large dilution, thus lysozyme presents a "hydration memory" phenomenon, This effect is in accordance with sorption / desorption isotherms, an hysteresis loop being observed.

Research paper thumbnail of Comparison of Macromolecular and Molecular Effects of Cosolvents on the Catalytic Behaviour of Soybean Lipoxygenase-1

Biocatalysis and Biotransformation, 1997

... CHRISTINE LAMBERT, CHRISTINE POURPLANCHE and VERONIQUE LARRETA-GARDE ... subsequent enzyme ac... more ... CHRISTINE LAMBERT, CHRISTINE POURPLANCHE and VERONIQUE LARRETA-GARDE ... subsequent enzyme activities (Gavish and Yedgar, 1995) but conflicting effects have been observed de-pending on the range of viscosites studied (de Almeida Cunha and Vitolo, 1984 ...

Research paper thumbnail of Glycerol’s influence on the oxidized insulin B-chain conformation in relation to the selectivity variation of subtilisin: an nuclear magnetic resonance and simulated annealing study

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1999

Glycerol, employed to mimic biological media with restricted water activity, has been shown to mo... more Glycerol, employed to mimic biological media with restricted water activity, has been shown to modify the activity of subtilisin BPNP, an endopeptidase, towards the oxidized insulin B-chain, a well-studied substrate (FEBS Lett., 279 (1991) 123^131). Without minimizing the role of the microenvironment on the enzyme, we have studied the effect of glycerol addition on the structure of the enzyme substrate by homonuclear NMR spectroscopy and simulated annealing. Our results show that, in water, the oxidized insulin B-chain tertiary structure loses its central helix (residues B9^B19) and presents a folded structure with a flexible turn (residues B18^B24) in the L-turn region of the insulin B-chain; whereas, in glycerol, the peptide is more rigid and is not folded. Moreover, in our experimental conditions, glycerol favors L-strand secondary structure formation. Following these results, hypotheses about the differences observed in enzymatic activity on this substrate in glycerol have been postulated. ß

Research paper thumbnail of Additional data about thermolysin specificity in buffer- and glycerol-containing media

Biochimica et Biophysica Acta (BBA) - Protein Structure and Molecular Enzymology, 1997

Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recogn... more Synthesis and use of various substrates permit an improved approach to thermolysin-peptide recognition and elucidation of several new criteria affecting enzyme specificity. Nature and position of the recognized residue, role of adjacent amino acids, lateral chain hydrophobicity, and volume and length of peptides were all considered. Hydrolysis reactions were also carried out in the presence of glycerol; the effect of microenvironment modifications was quantitative, for example, in inducing variations in catalytic reaction rates, and also qualitative, such as in influencing affinity.

Research paper thumbnail of Gel–sol transition can describe the proteolysis of extracellular matrix gels

Biochimica et Biophysica Acta (BBA) - General Subjects, 2000

We monitored the cell-free solubilization of extracellular matrix and fibronectin gels, catalyzed... more We monitored the cell-free solubilization of extracellular matrix and fibronectin gels, catalyzed by exogenous proteinases. The corresponding measurements could not be interpreted according to usual proteinase kinetics. The observation that this experimental system did not consist in surface but in bulk degradation and appeared specific to gel substrates, incited us to use gelation-related approaches to describe these kinetics. We show that the gel^sol transition theory adequately describes the enzyme reactions. This allowed formulation and experimental confirmation of a power law relating macroscopic changes of the gel to enzyme kinetics. This approach could also be used for other power laws predicted by the gel^sol transition theory, allowing a better understanding of macroscopic modification of the extracellular matrix during proteolysis, which is implied in many biological situations, especially tumor dissemination. ß

Research paper thumbnail of Kinetic and Structural Observations of a Nonheme Iron Enzyme Contribution of Conformational Changes to the Modification of Lipoxygenase Catalysis

Annals of the New York Academy of Sciences, 1995

Research paper thumbnail of Proteolysis of Aggregated Fibronectin A Model for In Vivo Matrix Degradation

Annals of the New York Academy of Sciences, 1998