Frederic CADET - Academia.edu (original) (raw)
Papers by Frederic CADET
Spectroscopy Letters, 1996
Collected Mid-IR Attenuated Total Reflectance (ATR) spectra of various sugars were assessed by mu... more Collected Mid-IR Attenuated Total Reflectance (ATR) spectra of various sugars were assessed by multidimensional statistical analysis. Through Principal Component Analysis (PCA) of collected spectra of various pure 10% sugar solutions and from the spectroscopic representation of the factorial axes, characteristic frequencies of monosaccharides and oligosaccharides were directly and automatically obtained within a few seconds. Monosugars are characterised by a hollow
Biochemical Education, 1997
Introduction In the process of photosynthesis complex enzymatic reactions are involved particular... more Introduction In the process of photosynthesis complex enzymatic reactions are involved particularly in the Benson-Calvin cycle which occurs in the chloroplasts. Some of these enzymes are inactive in darkness but are activated by light, hence, the cycle functions only during the day. Examples of such enzymes are fructose bisphosphatase (FBPase) and sedoheptulose bisphosphatase (SBPase) which are inactivated in darkness. Several mechanisms have been proposed that account for the activation and deactivation of the Benson-Calvin cycle. For example, upon illumination, the pH of the stroma increases from pH 7 to pH 8 owing to the active pumping of protons into the intrathylakoid space. This is accompanied by a transfer of Mg 2+ ions via the thylakoid membrane into the stroma. As the envelope of the chloroplast is impermeable to Mg 2+ ions, the concentration of Mg 2+ in the stroma increase from 1 to 2-5mm. Most of the enzymes of the Benson-Calvin cycle are sensitive to pH and to changes in Mg 2+ concentration but these changes, by themselves, do not wholly account for the activation of the cycle. Two other types of activation mechanisms have been shown: (1) the photoregulation of these enzymes in which light-regulated redox systems such as ferredoxins are involved, and (2) regulation via energy load and via the concentration of metabolites from the cycle.
Talanta, 1999
The advent of more and more powerful micro-computers has allowed the introduction of multidimensi... more The advent of more and more powerful micro-computers has allowed the introduction of multidimensional analysis in research laboratories. Complex mathematical treatments are now possible within a few seconds. Prediction equations that linked sucrose, fructose, glucose, total sugars and reducing sugars concentrations to the spectral data, were established by regression on the principal components. Very high correlation coefficient values between the first ten axes and the chemical values were obtained. The bias and standard deviation (S.D.) values obtained between reference and predicted values were good. From such aqueous biological samples containing a ternary mixture of sucrose, fructose and glucose it was possible to (i) identify the characteristic IR bands of these different sugars (and their combination: reducing sugars, total sugars)-using spectral pattern; and (ii) to specifically measure their concentrations with good accuracy.
Libraries of structural prototypes that abstract protein local structures are known as structural... more Libraries of structural prototypes that abstract protein local structures are known as structural alphabets and have proven to be very useful in various aspects of protein structure analyses and predictions. One such library, Protein Blocks (PBs), is composed of 16 standard 5-residues long structural prototypes. This form of analyzing proteins involves drafting its structure as a string of PBs. Thus, predicting the local structure of a protein in terms of protein blocks is a step towards the objective of predicting its 3-D structure. Here a new approach, kPred, is proposed towards this aim that is independent of the evolutionary information available. It involves (i) organizing the structural knowledge in the form of a database of pentapeptide fragments extracted from all protein structures in the PDB and (ii) apply a purely knowledge-based algorithm, not relying on secondary structure predictions or sequence alignment profiles, to scan this database and predict most probable backbo...
Biochemical Education, 1996
This issue of Biochemical Education has two offerings in the Problem-based Learning Page by a reg... more This issue of Biochemical Education has two offerings in the Problem-based Learning Page by a regular contributor, Dr Cadet 1"2 of the Universit6 de la R6union, France, and concerns the thermal denaturation and activity of the enzyme polyphenol oxidase (EC 1.14.18.1). The first problem is interesting in its own right and, I think, enzymes are fascinating in themselves, but this paper has several features of more general appeal. Polyphenol oxi- dase should gain the attention of students because its activity has commercial implications being a major cause of the enzyme-catalyzed browning in fruits and vegetables during storage. Also, this first problem nicely comple- ments an earlier one from Dr Jones of the University of Manchester (UK) on thermal denaturation and protein stability) These two problems together therefore could form an attractive package; perhaps one used as a worked/ tutorial example, the other set as a revision or test example. The Problem-based Learning Page is always pleased to receive letters, so do write to express your views on the issues and topics raised here. Do keep submitting articles for the Page, they are always gratefully received! References
Biochemical Education, 1996
Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie, 1994
An alternate method for enzyme study is proposed. This technique allows enzymatic reactions by a ... more An alternate method for enzyme study is proposed. This technique allows enzymatic reactions by a one step assay, and visualisation of variations in FTIR spectral data of substrate during the reaction. Hydrolysis of sucrose by beta-fructosidase is carried out as an example.
Biochemical Education, 1999
A problem for students on the mechanisms of leaf senescence is presented as a series of questions... more A problem for students on the mechanisms of leaf senescence is presented as a series of questions requiring the interpretation of experimental data.
Biophysical Reviews, 2010
Prot ines de la membrane rythrocytaire et homologues non-rythroides 4 é é é
TAG Theoretical and Applied Genetics, 2002
Spectroscopy Letters, 1996
Complex-formation between carbohydrates and cations could have important biological implications.... more Complex-formation between carbohydrates and cations could have important biological implications.In this work, Mid-Infrared spectra of pure sucrose solutions and of biological solutions containing sucrose and potassium ions (K+) were investigated by Principal Component Analysis (PGA).By direct examination of the Mid-Infrared spectra of the biological solutions containing K ions, no interactions between the cations and sucrose molecules could be observed. However, when the spectral pattern obtained by PCA and which is associated with sucrose, was examined, splitting and shifts in the characteristic absorption bands were observed owing to interactions between sucrose molecules and K ions. The 997 cm-1 peak which had a visible shoulder at 991 cm-1 and that is observed in pure solutions, was decomposed in the biological solutions into 3 distinct peaks at 1004, 996 and 990 cm-1. The two peaks centered at 1053 cm-1 were split into 3 peaks: 1060, 1051, 1045 cm-1. Hence by PCA, shoulders were characterized in biological solutions and more distinct peaks could be observed. These split and shift phenomena are similar to those obtained when crystalline sugar salts were investigated. This type of interaction, involving potassium ions and sucrose molecules, would be responsible for the storage of this cation which role is essential in plant metabolism.
Spectroscopy Letters, 1996
... Sciences, Universit6 de la %union, 15 avenue, Ren6 Cassin, BP 7151, 97715 Saint-Denis Messag ... more ... Sciences, Universit6 de la %union, 15 avenue, Ren6 Cassin, BP 7151, 97715 Saint-Denis Messag Cedex 9, Rkunion, France-Dom. ... of physical quantitative analysis methods such as Mid infrared spectroscopy have considerably expanded (Depecker et al., 1985; Crocombe et al ...
Spectroscopy Letters, 1998
ABSTRACT In a previous paper we have shown that it was possible to quantify protein solutions at ... more ABSTRACT In a previous paper we have shown that it was possible to quantify protein solutions at very weak concentrations directly by UV-visible spectroscopy. Nevertheless the protein quantification could not be possible if there is any trace of interferents left in the solution. So it is necessary to eliminate all the interferents to make the measure at 190 and/or 277 nm possible. We have developped a method based on the use of Microcon membranes and centrifugation. Interferents could be eliminated from protein solutions after four centrifugations at 13000 g during 5 min. This procedure allowed the recovering of proteins, with 80 to 99 % yield, and thus making microquantification possible.This method is particularly interesting for enzymatic solutions after a purification procedure by HPLC where very tiny quantities of protein are recovered. This protocol has been tested on three enzymes; enzymatic activity recovered after four centrifugations was quite high for PEPcase and malic enzyme (71 and 64 % respectively).
Nucleic Acids Research, 2006
Encoding protein 3D structures into 1D string using short structural prototypes or structural alp... more Encoding protein 3D structures into 1D string using short structural prototypes or structural alphabets opens a new front for structure comparison and analysis. Using the well-documented 16 motifs of Protein Blocks (PBs) as structural alphabet, we have developed a methodology to compare protein structures that are encoded as sequences of PBs by aligning them using dynamic programming which uses a substitution matrix for PBs. This methodology is implemented in the applications available in Protein Block Expert (PBE) server. PBE addresses common issues in the field of protein structure analysis such as comparison of proteins structures and identification of protein structures in structural databanks that resemble a given structure. PBE-T provides facility to transform any PDB file into sequences of PBs. PBE-ALIGNc performs comparison of two protein structures based on the alignment of their corresponding PB sequences. PBE-ALIGNm is a facility for mining SCOP database for similar structures based on the alignment of PBs. Besides, PBE provides an interface to a database (PBE-SAdb) of preprocessed PB sequences from SCOP culled at 95% and of all-against-all pairwise PB alignments at family and superfamily levels.
Journal of Enzyme Inhibition and Medicinal Chemistry, 1998
The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halid... more The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halide salts have the same inhibitory effect on the two forms of palmito PPO separated by hydrophobic chromatography. Fluoride and chloride ions showed a non-competitive, mixed type inhibition while bromide and iodide ions were found to be non-competitive inhibitors. A study of the Ki for the different halide salts showed that the smaller Fion is a stronger inhibitor than Iand Brand that CIhas the highest K, value. This suggests that the active site of the palmito PPO is not easily accessible. The inhibition by chloride and fluoride ion was found to be pH-dependent. The inhibitory effects of these ions increased with a decrease in pH. It is suggested that halide ions (X) could bind to either the protonated enzyme (EH) or the protonated substrate-enzyme complex (EHS) to yield inactive forms EHX and EHSX, respectively.
Journal of Agricultural and Food Chemistry, 1995
ABSTRACT The optimal temperature of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is 30... more ABSTRACT The optimal temperature of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is 30 degrees C. The Arrhenius activation energy was calculated to be 5.41 kJ mol(-1), Delta H degrees of the reaction is -60.99 kJ mol(-1). At 25 degrees C, Delta G degrees and Delta S degrees were, respectively, 16.75 kJ mol(-1) and -260.87 J mol(-1) K-1. The enzyme heated at temperatures above 30 degrees C loses its activity. Fifty percent inhibition is reached in 18 min at 70 degrees C, in 8 min at 75 degrees C, and in 2.5 min at 80 degrees C. The kinetics of the thermal irreversible denaturation of this enzyme is characterized by two steps: N --> X(T-d) --> D, where N represents the native form, X represents an intermediate form, the structure of which depends on the deactivation temperature T-d, and D is the completely denatured form of the enzyme. Our experimental results rule out a two-isoenzyme (with varying heat sensitivity) model. The thermodynamic parameters of the irreversible denaturation of the intermediate form were 102.70 and 97.10 kJ mol(-1) for Delta H and Delta G, respectively, and 16.85 J mol(-1) K-1 for Delta S at 60 degrees C. Furthermore, this paper describes the effect of pH on the activity of the PPO. Studies were carried out with. 4-methylcatechol and pyrogallol as substrates. The pH profile was not a function of the nature of the substrate assayed. The pH optimum was 5.2. The plot of logV(max app) vs pH indicates that the oxidation of the substrates depended of the ionization of two groups in the enzyme-substrate complex with apparent pK values of 3.06 and 7.29 and 3.44 and 7.12, respectively, for 4-methylcatechol and pyrogallol. The very slight differences between the values suggest the existence of only one site on the molecule for both substrates.
Journal of Agricultural and Food Chemistry, 1996
Polyphenol oxidases (PPO) are responsible for the oxidation of phenols into quinones that give br... more Polyphenol oxidases (PPO) are responsible for the oxidation of phenols into quinones that give brown or black pigments. The aim of the work presented in this paper was to study the mechanism of inhibition of palmito (Acanthophoenix rubra) PPO by metabisulfite. When monitored spectrophotometrically and with an oxymeter, a decrease in enzymatic activity was observed. When measured with the oxymeter, there was a decrease in oxygen comsumption, indicating enzyme inhibition. When monitored with the spectrophotometer, the existence of a lag phase was noted, indicating that the quinones formed either are reduced into phenols or react with metabisulfite to give a colorless complex. The oxidation of phenol took place during the lag phase, and when metabisulfite was depleted, the amount of oxygen was not enough to allow the oxidation of the substrate in the optimal conditions. The plot of inhibition versus metabisulfite showed an allosteric behavior of PPO with positive cooperativity. The incubation of PPO with metabisulfite resulted in inhibition of enzymatic activity. This inhibition was as strong as the concentration of metabisulfite was high. The plot of log(residual activity) versus incubation time showed a diphasic behavior. All of these results indicate that PPO inhibition by metabisulfite does not obey a first-order reaction and that the action of metabisulfite is accompanied by a conformational change of the enzyme. Accordingly, bisulfite would react with the enzyme and form an intermediate complex that is more stable than the native form. The decomposition of this complex would give an inactive enzymatic form (E i) through a slower second step. Fitting experimental values to the equation describing a two-step denaturation model confirmed the structural changes observed by electrophoresis by other authors.
Immunogenetics, 1999
Regulation of HLA-DQ gene transcription is a complex phenomenon because the allelic polymorphism ... more Regulation of HLA-DQ gene transcription is a complex phenomenon because the allelic polymorphism associated with these genes and their promoters is a putative source of differential allele expression. Both transcriptional and post-transcriptional regulation could account for the density of the molecules expressed at the cell surface and then for the specificity of the immune response. Different methods have been developed to evaluate the functional consequences of this polymorphism, but at present no universal method allows measurement of either the steady-state level or the half-life time of mRNA species of both DQA1 and DQB1 polymorphic genes in heterozygous cell lines. Here, we propose a potent method, based on relative quantification of reverse transcriptase-polymerase chain reaction products, which analyzes the differential expression of all DQA1 or DQB1 allele combinations. This method is used to analyze the differential expression of HLA-DQB1*0201/0402 alleles in the human heterozygous lymphoblastoid B-cell line. Nucleotidic sequences of the proximal upstream regulatory region of these alleles exhibit significant differences. We show that the DQB1*0402 promoter is able to mediate a transcription strength twice as efficiently as *0201. In addition, the *0402 mRNA steady-state level is also governed by a remarkable post-transcriptional regulation. Indeed, an important part (20%) of the *0402 primary transcript is derived by alternative splicing in a short mRNA translated into a nonfunctional protein. Despite their variable sequence and length, no difference in the half-life of DQB1*0201 and both DQB*0402 mRNAs was observed in B-lymphoblastoid cells. The implications of these findings are discussed.
Spectroscopy Letters, 1996
Collected Mid-IR Attenuated Total Reflectance (ATR) spectra of various sugars were assessed by mu... more Collected Mid-IR Attenuated Total Reflectance (ATR) spectra of various sugars were assessed by multidimensional statistical analysis. Through Principal Component Analysis (PCA) of collected spectra of various pure 10% sugar solutions and from the spectroscopic representation of the factorial axes, characteristic frequencies of monosaccharides and oligosaccharides were directly and automatically obtained within a few seconds. Monosugars are characterised by a hollow
Biochemical Education, 1997
Introduction In the process of photosynthesis complex enzymatic reactions are involved particular... more Introduction In the process of photosynthesis complex enzymatic reactions are involved particularly in the Benson-Calvin cycle which occurs in the chloroplasts. Some of these enzymes are inactive in darkness but are activated by light, hence, the cycle functions only during the day. Examples of such enzymes are fructose bisphosphatase (FBPase) and sedoheptulose bisphosphatase (SBPase) which are inactivated in darkness. Several mechanisms have been proposed that account for the activation and deactivation of the Benson-Calvin cycle. For example, upon illumination, the pH of the stroma increases from pH 7 to pH 8 owing to the active pumping of protons into the intrathylakoid space. This is accompanied by a transfer of Mg 2+ ions via the thylakoid membrane into the stroma. As the envelope of the chloroplast is impermeable to Mg 2+ ions, the concentration of Mg 2+ in the stroma increase from 1 to 2-5mm. Most of the enzymes of the Benson-Calvin cycle are sensitive to pH and to changes in Mg 2+ concentration but these changes, by themselves, do not wholly account for the activation of the cycle. Two other types of activation mechanisms have been shown: (1) the photoregulation of these enzymes in which light-regulated redox systems such as ferredoxins are involved, and (2) regulation via energy load and via the concentration of metabolites from the cycle.
Talanta, 1999
The advent of more and more powerful micro-computers has allowed the introduction of multidimensi... more The advent of more and more powerful micro-computers has allowed the introduction of multidimensional analysis in research laboratories. Complex mathematical treatments are now possible within a few seconds. Prediction equations that linked sucrose, fructose, glucose, total sugars and reducing sugars concentrations to the spectral data, were established by regression on the principal components. Very high correlation coefficient values between the first ten axes and the chemical values were obtained. The bias and standard deviation (S.D.) values obtained between reference and predicted values were good. From such aqueous biological samples containing a ternary mixture of sucrose, fructose and glucose it was possible to (i) identify the characteristic IR bands of these different sugars (and their combination: reducing sugars, total sugars)-using spectral pattern; and (ii) to specifically measure their concentrations with good accuracy.
Libraries of structural prototypes that abstract protein local structures are known as structural... more Libraries of structural prototypes that abstract protein local structures are known as structural alphabets and have proven to be very useful in various aspects of protein structure analyses and predictions. One such library, Protein Blocks (PBs), is composed of 16 standard 5-residues long structural prototypes. This form of analyzing proteins involves drafting its structure as a string of PBs. Thus, predicting the local structure of a protein in terms of protein blocks is a step towards the objective of predicting its 3-D structure. Here a new approach, kPred, is proposed towards this aim that is independent of the evolutionary information available. It involves (i) organizing the structural knowledge in the form of a database of pentapeptide fragments extracted from all protein structures in the PDB and (ii) apply a purely knowledge-based algorithm, not relying on secondary structure predictions or sequence alignment profiles, to scan this database and predict most probable backbo...
Biochemical Education, 1996
This issue of Biochemical Education has two offerings in the Problem-based Learning Page by a reg... more This issue of Biochemical Education has two offerings in the Problem-based Learning Page by a regular contributor, Dr Cadet 1"2 of the Universit6 de la R6union, France, and concerns the thermal denaturation and activity of the enzyme polyphenol oxidase (EC 1.14.18.1). The first problem is interesting in its own right and, I think, enzymes are fascinating in themselves, but this paper has several features of more general appeal. Polyphenol oxi- dase should gain the attention of students because its activity has commercial implications being a major cause of the enzyme-catalyzed browning in fruits and vegetables during storage. Also, this first problem nicely comple- ments an earlier one from Dr Jones of the University of Manchester (UK) on thermal denaturation and protein stability) These two problems together therefore could form an attractive package; perhaps one used as a worked/ tutorial example, the other set as a revision or test example. The Problem-based Learning Page is always pleased to receive letters, so do write to express your views on the issues and topics raised here. Do keep submitting articles for the Page, they are always gratefully received! References
Biochemical Education, 1996
Comptes rendus de l'Académie des sciences. Série III, Sciences de la vie, 1994
An alternate method for enzyme study is proposed. This technique allows enzymatic reactions by a ... more An alternate method for enzyme study is proposed. This technique allows enzymatic reactions by a one step assay, and visualisation of variations in FTIR spectral data of substrate during the reaction. Hydrolysis of sucrose by beta-fructosidase is carried out as an example.
Biochemical Education, 1999
A problem for students on the mechanisms of leaf senescence is presented as a series of questions... more A problem for students on the mechanisms of leaf senescence is presented as a series of questions requiring the interpretation of experimental data.
Biophysical Reviews, 2010
Prot ines de la membrane rythrocytaire et homologues non-rythroides 4 é é é
TAG Theoretical and Applied Genetics, 2002
Spectroscopy Letters, 1996
Complex-formation between carbohydrates and cations could have important biological implications.... more Complex-formation between carbohydrates and cations could have important biological implications.In this work, Mid-Infrared spectra of pure sucrose solutions and of biological solutions containing sucrose and potassium ions (K+) were investigated by Principal Component Analysis (PGA).By direct examination of the Mid-Infrared spectra of the biological solutions containing K ions, no interactions between the cations and sucrose molecules could be observed. However, when the spectral pattern obtained by PCA and which is associated with sucrose, was examined, splitting and shifts in the characteristic absorption bands were observed owing to interactions between sucrose molecules and K ions. The 997 cm-1 peak which had a visible shoulder at 991 cm-1 and that is observed in pure solutions, was decomposed in the biological solutions into 3 distinct peaks at 1004, 996 and 990 cm-1. The two peaks centered at 1053 cm-1 were split into 3 peaks: 1060, 1051, 1045 cm-1. Hence by PCA, shoulders were characterized in biological solutions and more distinct peaks could be observed. These split and shift phenomena are similar to those obtained when crystalline sugar salts were investigated. This type of interaction, involving potassium ions and sucrose molecules, would be responsible for the storage of this cation which role is essential in plant metabolism.
Spectroscopy Letters, 1996
... Sciences, Universit6 de la %union, 15 avenue, Ren6 Cassin, BP 7151, 97715 Saint-Denis Messag ... more ... Sciences, Universit6 de la %union, 15 avenue, Ren6 Cassin, BP 7151, 97715 Saint-Denis Messag Cedex 9, Rkunion, France-Dom. ... of physical quantitative analysis methods such as Mid infrared spectroscopy have considerably expanded (Depecker et al., 1985; Crocombe et al ...
Spectroscopy Letters, 1998
ABSTRACT In a previous paper we have shown that it was possible to quantify protein solutions at ... more ABSTRACT In a previous paper we have shown that it was possible to quantify protein solutions at very weak concentrations directly by UV-visible spectroscopy. Nevertheless the protein quantification could not be possible if there is any trace of interferents left in the solution. So it is necessary to eliminate all the interferents to make the measure at 190 and/or 277 nm possible. We have developped a method based on the use of Microcon membranes and centrifugation. Interferents could be eliminated from protein solutions after four centrifugations at 13000 g during 5 min. This procedure allowed the recovering of proteins, with 80 to 99 % yield, and thus making microquantification possible.This method is particularly interesting for enzymatic solutions after a purification procedure by HPLC where very tiny quantities of protein are recovered. This protocol has been tested on three enzymes; enzymatic activity recovered after four centrifugations was quite high for PEPcase and malic enzyme (71 and 64 % respectively).
Nucleic Acids Research, 2006
Encoding protein 3D structures into 1D string using short structural prototypes or structural alp... more Encoding protein 3D structures into 1D string using short structural prototypes or structural alphabets opens a new front for structure comparison and analysis. Using the well-documented 16 motifs of Protein Blocks (PBs) as structural alphabet, we have developed a methodology to compare protein structures that are encoded as sequences of PBs by aligning them using dynamic programming which uses a substitution matrix for PBs. This methodology is implemented in the applications available in Protein Block Expert (PBE) server. PBE addresses common issues in the field of protein structure analysis such as comparison of proteins structures and identification of protein structures in structural databanks that resemble a given structure. PBE-T provides facility to transform any PDB file into sequences of PBs. PBE-ALIGNc performs comparison of two protein structures based on the alignment of their corresponding PB sequences. PBE-ALIGNm is a facility for mining SCOP database for similar structures based on the alignment of PBs. Besides, PBE provides an interface to a database (PBE-SAdb) of preprocessed PB sequences from SCOP culled at 95% and of all-against-all pairwise PB alignments at family and superfamily levels.
Journal of Enzyme Inhibition and Medicinal Chemistry, 1998
The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halid... more The inhibitory properties of halide salts on palmito polyphenoloxidase (PPO) are described. Halide salts have the same inhibitory effect on the two forms of palmito PPO separated by hydrophobic chromatography. Fluoride and chloride ions showed a non-competitive, mixed type inhibition while bromide and iodide ions were found to be non-competitive inhibitors. A study of the Ki for the different halide salts showed that the smaller Fion is a stronger inhibitor than Iand Brand that CIhas the highest K, value. This suggests that the active site of the palmito PPO is not easily accessible. The inhibition by chloride and fluoride ion was found to be pH-dependent. The inhibitory effects of these ions increased with a decrease in pH. It is suggested that halide ions (X) could bind to either the protonated enzyme (EH) or the protonated substrate-enzyme complex (EHS) to yield inactive forms EHX and EHSX, respectively.
Journal of Agricultural and Food Chemistry, 1995
ABSTRACT The optimal temperature of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is 30... more ABSTRACT The optimal temperature of palmito (Acanthophoenix rubra) polyphenol oxidase (PPO) is 30 degrees C. The Arrhenius activation energy was calculated to be 5.41 kJ mol(-1), Delta H degrees of the reaction is -60.99 kJ mol(-1). At 25 degrees C, Delta G degrees and Delta S degrees were, respectively, 16.75 kJ mol(-1) and -260.87 J mol(-1) K-1. The enzyme heated at temperatures above 30 degrees C loses its activity. Fifty percent inhibition is reached in 18 min at 70 degrees C, in 8 min at 75 degrees C, and in 2.5 min at 80 degrees C. The kinetics of the thermal irreversible denaturation of this enzyme is characterized by two steps: N --> X(T-d) --> D, where N represents the native form, X represents an intermediate form, the structure of which depends on the deactivation temperature T-d, and D is the completely denatured form of the enzyme. Our experimental results rule out a two-isoenzyme (with varying heat sensitivity) model. The thermodynamic parameters of the irreversible denaturation of the intermediate form were 102.70 and 97.10 kJ mol(-1) for Delta H and Delta G, respectively, and 16.85 J mol(-1) K-1 for Delta S at 60 degrees C. Furthermore, this paper describes the effect of pH on the activity of the PPO. Studies were carried out with. 4-methylcatechol and pyrogallol as substrates. The pH profile was not a function of the nature of the substrate assayed. The pH optimum was 5.2. The plot of logV(max app) vs pH indicates that the oxidation of the substrates depended of the ionization of two groups in the enzyme-substrate complex with apparent pK values of 3.06 and 7.29 and 3.44 and 7.12, respectively, for 4-methylcatechol and pyrogallol. The very slight differences between the values suggest the existence of only one site on the molecule for both substrates.
Journal of Agricultural and Food Chemistry, 1996
Polyphenol oxidases (PPO) are responsible for the oxidation of phenols into quinones that give br... more Polyphenol oxidases (PPO) are responsible for the oxidation of phenols into quinones that give brown or black pigments. The aim of the work presented in this paper was to study the mechanism of inhibition of palmito (Acanthophoenix rubra) PPO by metabisulfite. When monitored spectrophotometrically and with an oxymeter, a decrease in enzymatic activity was observed. When measured with the oxymeter, there was a decrease in oxygen comsumption, indicating enzyme inhibition. When monitored with the spectrophotometer, the existence of a lag phase was noted, indicating that the quinones formed either are reduced into phenols or react with metabisulfite to give a colorless complex. The oxidation of phenol took place during the lag phase, and when metabisulfite was depleted, the amount of oxygen was not enough to allow the oxidation of the substrate in the optimal conditions. The plot of inhibition versus metabisulfite showed an allosteric behavior of PPO with positive cooperativity. The incubation of PPO with metabisulfite resulted in inhibition of enzymatic activity. This inhibition was as strong as the concentration of metabisulfite was high. The plot of log(residual activity) versus incubation time showed a diphasic behavior. All of these results indicate that PPO inhibition by metabisulfite does not obey a first-order reaction and that the action of metabisulfite is accompanied by a conformational change of the enzyme. Accordingly, bisulfite would react with the enzyme and form an intermediate complex that is more stable than the native form. The decomposition of this complex would give an inactive enzymatic form (E i) through a slower second step. Fitting experimental values to the equation describing a two-step denaturation model confirmed the structural changes observed by electrophoresis by other authors.
Immunogenetics, 1999
Regulation of HLA-DQ gene transcription is a complex phenomenon because the allelic polymorphism ... more Regulation of HLA-DQ gene transcription is a complex phenomenon because the allelic polymorphism associated with these genes and their promoters is a putative source of differential allele expression. Both transcriptional and post-transcriptional regulation could account for the density of the molecules expressed at the cell surface and then for the specificity of the immune response. Different methods have been developed to evaluate the functional consequences of this polymorphism, but at present no universal method allows measurement of either the steady-state level or the half-life time of mRNA species of both DQA1 and DQB1 polymorphic genes in heterozygous cell lines. Here, we propose a potent method, based on relative quantification of reverse transcriptase-polymerase chain reaction products, which analyzes the differential expression of all DQA1 or DQB1 allele combinations. This method is used to analyze the differential expression of HLA-DQB1*0201/0402 alleles in the human heterozygous lymphoblastoid B-cell line. Nucleotidic sequences of the proximal upstream regulatory region of these alleles exhibit significant differences. We show that the DQB1*0402 promoter is able to mediate a transcription strength twice as efficiently as *0201. In addition, the *0402 mRNA steady-state level is also governed by a remarkable post-transcriptional regulation. Indeed, an important part (20%) of the *0402 primary transcript is derived by alternative splicing in a short mRNA translated into a nonfunctional protein. Despite their variable sequence and length, no difference in the half-life of DQB1*0201 and both DQB*0402 mRNAs was observed in B-lymphoblastoid cells. The implications of these findings are discussed.