The present work deals with a theoretical model of catalysis by enzymes entrapped in reverse mice... more The present work deals with a theoretical model of catalysis by enzymes entrapped in reverse micelles. Three aspects of the enzyme-reverse-micelle system have been considered: structure, dynamics and enzyme distribution and catalysis in reverse micelles. A proposed structural model of reverse micelles [El Seoud (1984) in Reverse Micelles (Luisi, P. L. & Straub, B. E., eds.), p. 81, Plenum Press, New York] consists of three domains: surfactant apolar tails, bound water and free water. Dynamics are based on a dynamic equilibrium of association-dissociation that lead one to consider the dispersed polar phase as a pseudo-continuous phase [Luisi, Giomini, Pileni & Robinson (1988) Biochim. Biophys. Acta 947, 207-246]. Enzyme is distributed among the reverse-micelle domains and it expresses a catalytic constant for each one of them. The overall activity is calculated taking into account the volume in which enzyme is solubilized, and expressed as a function of the whole volume (V). The char...
Tyrosinase usually catalyses the conversion of monophenols into o-diphenols and the oxidation of ... more Tyrosinase usually catalyses the conversion of monophenols into o-diphenols and the oxidation of diphenols to the corresponding o-quinones. Sugumaran [(1986) Biochemistry 25, 4489-4492] has previously proposed an unusual oxidative decarboxylation of 3,4-dihydroxymandelate catalysed by tyrosinase. Our determination of the intermediates involved in the reaction demonstrated that 3,4-dihydroxybenzaldehyde is not the first intermediate appearing in the medium during the enzymic reaction. Re-examination of this new activity of tyrosinase has demonstrated that the product of the enzyme action is the o-quinone, which, owing to its instability, evolves to the final product, 3,4-dihydroxybenzaldehyde, by a chemical reaction of oxidative decarboxylation.
Gamay cell cultures were treated with dimethyl-β-cyclodextrins in order to ascertain their effect... more Gamay cell cultures were treated with dimethyl-β-cyclodextrins in order to ascertain their effect on resveratrol metabolism before and after inoculation with Xylophilus ampelinus. The results showed that, in grapevine cell suspensions, dimethyl-β-cyclodextrins themselves do not need the co-cultivation with bacteria to act as elicitors of the cells producing trans-resveratrol, a phytoalexin of grapevines. Dimethyl-β-cyclodextrins protected cell suspensions against bacteria by
Journal of Chromatography B-Biomedical Applications, 1996
Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. T... more Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The phenols were reduced to 8% of the original content, avoiding pre-and post-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH and to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetically characterized, latent enzyme showed both monophenolase and diphenolase activities, the latter activity displaying an unexpected lag period before reaching the steady-state rate. This behaviour is characteristic of a hysteretic enzyme, and has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhibitor.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1997
The capability of cyclodextrins (CDs) to greatly enhance the solubility in water of poorly water-... more The capability of cyclodextrins (CDs) to greatly enhance the solubility in water of poorly water-soluble substances makes them an ideal alternative system for studying the expression of enzyme activity with such substrates in aqueous solution. In order to evaluate the behaviour of the enzymes in presence of CDs a study of the lipoxygenase (LOX)-catalyzed oxidation of polyunsaturated fatty acids (PUFA) as model reaction has been carried out. This was done by using LOX from two different sources (soybean and potato tuber), at two pH values (6.3 and 9.0), with two substrates (linoleic acid and arachidonic acid) and three types of CD (beta-CD, methyl-beta-CD and monoglucosyl-beta-CD). PUFA have been shown to form inclusion complexes of 1:2 stoichiometry which are in equilibrium with free PUFA and free CD, thus complexation is governed by two equilibrium constants, K1 and K2 (J.M. López-Nicolás et al., Biochem. J. 308 (1995) 151-154; R. Bru et al., Colloids Surf. 97 (1995) 263-269). For the oxidation of PUFA by LOX in the presence of beta-CD we propose a model in which free PUFA is the only effective substrate, thus the oxidation of the complexed substrate requires the previous dissociation of the complex. The equilibrium constants of complex formation are determined by both a physico-chemical and an enzymatic method. In spite of giving quite similar results, the second was proven to be more accurate so it was employed in further studies. CD was shown to slow down the reaction rate of LOX, specifically due to the increase of Km, Vmax remaining unchanged. That apparent inhibition is due to removal of effective (free) substrate in the form of inclusion complexes. This 'sequestered' substrate can, however, be converted since it is in equilibrium with the free. The feasibility of realizing a CD-mediated accurate control over the conversion rate is demonstrated in the experiment called 'cyclodextrin assay' in which the concentration of the free substrate is calculated by using the equilibrium constants of complex formation and setting the initial concentrations of total substrate and total CD. From the observation of the reaction progress curves in the conditions of the CD assay, we have studied some characteristic parameters of the oxidation of PUFA by LOX in this new medium, such as enzymatic activity, duration of linear product accumulation and the lag phase.
Proceedings of the Ninth International Symposium on Cyclodextrins, 1999
Lipoxygenases (LOX; linoleate: oxygen oxidoreductase, EC 1.13.11.12) are a group of enzymes that ... more Lipoxygenases (LOX; linoleate: oxygen oxidoreductase, EC 1.13.11.12) are a group of enzymes that catalyze the dioxygenation of polyunsaturated fatty acids (PUFAs) containing one or more 1,4-cis,cis-pentadiene systems to conjugated hydroperoxy fatty acids (1). In solution, PUFAs exhibit an aggregation beheviour that, depending on the pH, may lead to the formation of true micelles or merely to a dispersed oil phase. At high pH (above 9), the aggregates form true transparent micellar solutions whilst the preparations of PUFA at neutral pH are turbid suspensions. Most LOXs display optimum activity at pH values around neutrality (1) but in these conditions PUFAs are quite insoluble. For this reason the kinetic properties of LOX are well known at pH 9–10 (2) and practically unknown at neutral or slightly acid pH values. The ability of cyclodextrins (CDs) to enhance the aqueous solubility of many different compounds (3) makes them an ideal alternative system for performing enzyme-catalyzed conversions of poorly water-soluble substrates compared with organic media or two-phase system. In a previous study (4) the complexes between β-CD and PUFAs were shown to have a stoichiometry of 2:1 (β-CD: PUFAs). In this study the oxidation of linoleic acid entrapped in β-CD by lipoxygenase has been characterized and a model for enzyme catalysis in a CD medium is proposed
The use of grapevine cell cultures to increase trans-resveratrol and stilbene-related production ... more The use of grapevine cell cultures to increase trans-resveratrol and stilbene-related production under controlled conditions is viewed as a promising biotechnological alternative to their extraction from the whole plant or their chemical synthesis. At present, in vitro plant cultures are rarely used as a commercial method for the production of bioactive compounds. The major problems hindering the development of the large-scale culture of plant cells include low productivity, cell line instability, and the difficulties involved in scaling up. This chapter describes how the composition of the medium, culture conditions, elicitors, bioreactor design, and other critical parameters influence the behavior of grapevine cell cultures and how optimization of these factors could allow improvements in trans-resveratrol and stilbene-related production. For these reasons, this chapter describes how high-producing Vitis cell lines, nutrient and precursor feeding, elicitation with stress factors, and signaling molecules, as well as metabolic engineering can be used to increase the production of these secondary metabolites.
trans-Resveratrol (trans-R) has been reported to be a potential cancer chemopreventive agent. Alt... more trans-Resveratrol (trans-R) has been reported to be a potential cancer chemopreventive agent. Although its cytotoxic activity against different cancer cell lines has been tested, its effect on human acute leukemia cell lines has scarcely been investigated, and only a few in vitro studies were performed using human breast epithelial cell lines. Due to its potential value for human health, demand for trans-R has rapidly increased, and new biotechnological strategies to obtain it from natural edible sources have been developed. Thus, grapevine cell cultures represent a reliable system of trans-R production since they biosynthesize trans-R constitutively or in response to elicitation. In addition, there are no studies deepen on the inhibitory effect of trans-R, produced by elicited grapevine cell cultures, on growth of human tumor cell lines. In this work, the effect of trans-R extracted from the culture medium, after elicitation of grapevine cell cultures, was tested on two human acute lymphocytic and monocytic leukemia cell lines, and one human breast cancer cell line. The effect of trans-R on cell proliferation was not only dose-and time-dependent but also cell type-dependent, as seen from the different degrees of susceptibility of cancer cell lines tested. As regards the effect of trans-R on cell cycle distribution, low trans-R concentrations increased cells in the S phase whereas a high trans-R concentration increased G 0 /G 1 phase in all cell lines. Perturbation of the cell cycle at low trans-R concentrations did not correlate with the induction of cell death, whereas a high trans-R concentration, cell proliferation decreased as a result of increasing apoptosis in the three cell lines. In leukemia cells, trans-R up-regulated the expression of caspase-3 while trans-R-induced apoptosis in breast cells occur through a caspase-3-independent mechanism mediated by a downregulation of Bcl-2.
The behavior of potato 5-lipoxygenase acting on both monomeric and aggregated linoleic acid has b... more The behavior of potato 5-lipoxygenase acting on both monomeric and aggregated linoleic acid has been studied. While the substrate preparation was transparent, lipoxygenase activity was determined by means of a spectrophotometric method, which was not useful to determine the activity in turbid samples. In the latter case a polarographic method was used. Cyclodextrins were used in order to increase the
Blossom-end rot (BER) is a physiopathy that affects tomato fruits causing disorganisation, cell b... more Blossom-end rot (BER) is a physiopathy that affects tomato fruits causing disorganisation, cell breakage and darkening of the tissues. In this study we describe a tomato fruit protein extraction protocol that includes polyvinyl polypyrrolidone, ascorbic acid and protease inhibitors to promote depletion of phenolics and to avoid protein degradation. The temperature-induced phase separation of plant extracts with nonionic detergent Triton X-114 favours the solubilisation of partially-hydrophobic species in the low-detergent upper phase, making them suitable for further analysis using two-dimensional gel electrophoresis. The analysis of two-dimensional images revealed differences in number and expression levels of several proteins from the control and BER-affected tomato fruits. Although the appearance of BER in tomato is primarily attributed to a lack of calcium supply to fruits, very little is known about the molecular and biochemical mechanisms involved. The identification of differential proteins from affected fruits with matrix-assisted laser desorption/ionisation-time of flight and peptide mass fingerprinting analysis revealed the induction of proteins participating in antioxidant processes (ascorbate-glutathione cycle) and the pentose phosphate pathway. We suggest that these two biochemical pathways, acting as reactive oxygen species scavengers in BER-affected fruits, restrain the spread of the blackening to the whole fruit.
Tobacco mosaic virus (TMV) is a widespread plant virus from the genus Tobamovirus that affects to... more Tobacco mosaic virus (TMV) is a widespread plant virus from the genus Tobamovirus that affects tobacco and tomato plants causing a pathology characterised by cell breakage and disorganisation in plant leaves and fruits. In this study we undertook a proteomic approach to investigate the molecular and biochemical mechanisms potentially involved in tomato fruit defence against the viral infection. The comparison of 2-D gels from control and TMV-infected but asymptomatic tomato fruits revealed changes in several proteins. The differential expression of peptidases, endoglucanase, chitinase and proteins participating in the ascorbate-glutathione cycle in infected fruits suggests that pathogenesis-related proteins and antioxidant enzymes may play a role in the protection against TMV infection. TMV coat protein appeared as a prominent spot in 2-D gels from TMV-infected asymptomatic fruits. A Triton X-114 phase-partitioning step of tomato protein extracts favoured the solubilisation of TMV coat protein and the enrichment of two aminopeptidases not present in control fruits. PMF and MS/MS data of the 2-D gel-isolated TMV coat protein is proposed as a powerful analysis method for the simultaneous tobamovirus detection, species determination and strain differentiation in virus-infected fruit commodities.
We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmon... more We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmonate (MJ) and cyclodextrins (CDs) in tobacco (Nicotiana tabacum) cell cultures, paying particular attention to changes in cytosolic free Ca 2þ concentration ([Ca 2þ ] cyt), the production of hydrogen peroxide (H 2 O 2) and nitric oxide (NO), and late events like the induction of capsidiol. Our data indicate that MJ and CDs trigger a [Ca 2þ ] cyt rise promoted by Ca 2þ influx through Ca 2þ-permeable channels. The joint presence of MJ and CDs provokes a first increase in [Ca 2þ ] cyt similar to that observed in MJ-treated cells, followed by a second peak similar to that found in the presence of CDs alone. Moreover, oxidative burst induced by MJ is more pronounced when tobacco cells are incubated with CDs alone or in combination with MJ. The presence of both elicitors provokes H 2 O 2 production similar to that found in CD-treated cells, and a sustained response similar to that found in MJ-treated cells. In all treatments, H 2 O 2 production is dependent on Ca 2þ influx and protein phosphorylation events. Similarly, the joint action of both elicitors provokes NO accumulation, although to a lesser extent that in MJ-treated cells because CDs alone do not trigger this accumulation. This NO production is dependent on Ca 2þ influx but independent of both H 2 O 2 production and staurosporine-sensitive phosphorylation events. Taken as a whole, these results suggest the existence of different intracellular signaling pathways for both elicitors. Likewise, CDs might act by regulating the signaling pathway triggered by MJ since, in the presence of both compounds, CDs neutralize the strong oxidative and nitrosative bursts triggered by MJ and therefore, they regulate both H 2 O 2 and NO levels.
The 4-plex iTRAQ platform was utilized to analyze the protein profiles in four stages of grapevin... more The 4-plex iTRAQ platform was utilized to analyze the protein profiles in four stages of grapevine berry skin ripening, from pre-veraison to fully ripening. Mass spectrometric data were acquired from three replicated analyses using a parallel acquisition method in an Orbitrap instrument by combining collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) peptide ion fragmentations. As a result, the number of spectra suitable for peptide identification (either from CID or HCD) increased 5-fold in relation to those suitable for quantification (from HCD). Spectra were searched against an NCBInr protein database subset containing all the Vitis sequences, including those derived from whole genome sequencing. In general, 695 unique proteins were identified with more than one single peptide, and 513 of them were quantified. The sequence annotation and GO term enrichment analysis assisted by the automatic annotation tool Blast2GO permitted a pathway analysis which resulted in finding that biological processes and metabolic pathways de-regulated throughout ripening. A detailed analysis of the function-related proteins profiles helped discover a set of proteins of known Vitis gene origin as the potential candidates to play key roles in grapevine berry quality, growth regulation and disease resistance.
The present work deals with a theoretical model of catalysis by enzymes entrapped in reverse mice... more The present work deals with a theoretical model of catalysis by enzymes entrapped in reverse micelles. Three aspects of the enzyme-reverse-micelle system have been considered: structure, dynamics and enzyme distribution and catalysis in reverse micelles. A proposed structural model of reverse micelles [El Seoud (1984) in Reverse Micelles (Luisi, P. L. & Straub, B. E., eds.), p. 81, Plenum Press, New York] consists of three domains: surfactant apolar tails, bound water and free water. Dynamics are based on a dynamic equilibrium of association-dissociation that lead one to consider the dispersed polar phase as a pseudo-continuous phase [Luisi, Giomini, Pileni & Robinson (1988) Biochim. Biophys. Acta 947, 207-246]. Enzyme is distributed among the reverse-micelle domains and it expresses a catalytic constant for each one of them. The overall activity is calculated taking into account the volume in which enzyme is solubilized, and expressed as a function of the whole volume (V). The char...
Tyrosinase usually catalyses the conversion of monophenols into o-diphenols and the oxidation of ... more Tyrosinase usually catalyses the conversion of monophenols into o-diphenols and the oxidation of diphenols to the corresponding o-quinones. Sugumaran [(1986) Biochemistry 25, 4489-4492] has previously proposed an unusual oxidative decarboxylation of 3,4-dihydroxymandelate catalysed by tyrosinase. Our determination of the intermediates involved in the reaction demonstrated that 3,4-dihydroxybenzaldehyde is not the first intermediate appearing in the medium during the enzymic reaction. Re-examination of this new activity of tyrosinase has demonstrated that the product of the enzyme action is the o-quinone, which, owing to its instability, evolves to the final product, 3,4-dihydroxybenzaldehyde, by a chemical reaction of oxidative decarboxylation.
Gamay cell cultures were treated with dimethyl-β-cyclodextrins in order to ascertain their effect... more Gamay cell cultures were treated with dimethyl-β-cyclodextrins in order to ascertain their effect on resveratrol metabolism before and after inoculation with Xylophilus ampelinus. The results showed that, in grapevine cell suspensions, dimethyl-β-cyclodextrins themselves do not need the co-cultivation with bacteria to act as elicitors of the cells producing trans-resveratrol, a phytoalexin of grapevines. Dimethyl-β-cyclodextrins protected cell suspensions against bacteria by
Journal of Chromatography B-Biomedical Applications, 1996
Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. T... more Mushroom tyrosinase was partially purified using an aqueous two-phase system with Triton X-114. The purification achieved was 5.5-fold from a crude extract of mushroom pileus, with a high recovery of 84%. The phenols were reduced to 8% of the original content, avoiding pre-and post-purification tanning of the enzyme. The enzyme obtained was latent and was activated 3-fold by trypsin, 2.7-fold by changes in the pH and to different extents by cationic and anionic detergents, the latter being the more effective. There was also a synergistic effect between trypsin and detergent, at low detergent concentrations. When kinetically characterized, latent enzyme showed both monophenolase and diphenolase activities, the latter activity displaying an unexpected lag period before reaching the steady-state rate. This behaviour is characteristic of a hysteretic enzyme, and has not been previously described for this enzyme. In addition, inhibition studies with substrate analogues were carried out, tropolone being found to be the most effective inhibitor.
Biochimica et Biophysica Acta (BBA) - Lipids and Lipid Metabolism, 1997
The capability of cyclodextrins (CDs) to greatly enhance the solubility in water of poorly water-... more The capability of cyclodextrins (CDs) to greatly enhance the solubility in water of poorly water-soluble substances makes them an ideal alternative system for studying the expression of enzyme activity with such substrates in aqueous solution. In order to evaluate the behaviour of the enzymes in presence of CDs a study of the lipoxygenase (LOX)-catalyzed oxidation of polyunsaturated fatty acids (PUFA) as model reaction has been carried out. This was done by using LOX from two different sources (soybean and potato tuber), at two pH values (6.3 and 9.0), with two substrates (linoleic acid and arachidonic acid) and three types of CD (beta-CD, methyl-beta-CD and monoglucosyl-beta-CD). PUFA have been shown to form inclusion complexes of 1:2 stoichiometry which are in equilibrium with free PUFA and free CD, thus complexation is governed by two equilibrium constants, K1 and K2 (J.M. López-Nicolás et al., Biochem. J. 308 (1995) 151-154; R. Bru et al., Colloids Surf. 97 (1995) 263-269). For the oxidation of PUFA by LOX in the presence of beta-CD we propose a model in which free PUFA is the only effective substrate, thus the oxidation of the complexed substrate requires the previous dissociation of the complex. The equilibrium constants of complex formation are determined by both a physico-chemical and an enzymatic method. In spite of giving quite similar results, the second was proven to be more accurate so it was employed in further studies. CD was shown to slow down the reaction rate of LOX, specifically due to the increase of Km, Vmax remaining unchanged. That apparent inhibition is due to removal of effective (free) substrate in the form of inclusion complexes. This 'sequestered' substrate can, however, be converted since it is in equilibrium with the free. The feasibility of realizing a CD-mediated accurate control over the conversion rate is demonstrated in the experiment called 'cyclodextrin assay' in which the concentration of the free substrate is calculated by using the equilibrium constants of complex formation and setting the initial concentrations of total substrate and total CD. From the observation of the reaction progress curves in the conditions of the CD assay, we have studied some characteristic parameters of the oxidation of PUFA by LOX in this new medium, such as enzymatic activity, duration of linear product accumulation and the lag phase.
Proceedings of the Ninth International Symposium on Cyclodextrins, 1999
Lipoxygenases (LOX; linoleate: oxygen oxidoreductase, EC 1.13.11.12) are a group of enzymes that ... more Lipoxygenases (LOX; linoleate: oxygen oxidoreductase, EC 1.13.11.12) are a group of enzymes that catalyze the dioxygenation of polyunsaturated fatty acids (PUFAs) containing one or more 1,4-cis,cis-pentadiene systems to conjugated hydroperoxy fatty acids (1). In solution, PUFAs exhibit an aggregation beheviour that, depending on the pH, may lead to the formation of true micelles or merely to a dispersed oil phase. At high pH (above 9), the aggregates form true transparent micellar solutions whilst the preparations of PUFA at neutral pH are turbid suspensions. Most LOXs display optimum activity at pH values around neutrality (1) but in these conditions PUFAs are quite insoluble. For this reason the kinetic properties of LOX are well known at pH 9–10 (2) and practically unknown at neutral or slightly acid pH values. The ability of cyclodextrins (CDs) to enhance the aqueous solubility of many different compounds (3) makes them an ideal alternative system for performing enzyme-catalyzed conversions of poorly water-soluble substrates compared with organic media or two-phase system. In a previous study (4) the complexes between β-CD and PUFAs were shown to have a stoichiometry of 2:1 (β-CD: PUFAs). In this study the oxidation of linoleic acid entrapped in β-CD by lipoxygenase has been characterized and a model for enzyme catalysis in a CD medium is proposed
The use of grapevine cell cultures to increase trans-resveratrol and stilbene-related production ... more The use of grapevine cell cultures to increase trans-resveratrol and stilbene-related production under controlled conditions is viewed as a promising biotechnological alternative to their extraction from the whole plant or their chemical synthesis. At present, in vitro plant cultures are rarely used as a commercial method for the production of bioactive compounds. The major problems hindering the development of the large-scale culture of plant cells include low productivity, cell line instability, and the difficulties involved in scaling up. This chapter describes how the composition of the medium, culture conditions, elicitors, bioreactor design, and other critical parameters influence the behavior of grapevine cell cultures and how optimization of these factors could allow improvements in trans-resveratrol and stilbene-related production. For these reasons, this chapter describes how high-producing Vitis cell lines, nutrient and precursor feeding, elicitation with stress factors, and signaling molecules, as well as metabolic engineering can be used to increase the production of these secondary metabolites.
trans-Resveratrol (trans-R) has been reported to be a potential cancer chemopreventive agent. Alt... more trans-Resveratrol (trans-R) has been reported to be a potential cancer chemopreventive agent. Although its cytotoxic activity against different cancer cell lines has been tested, its effect on human acute leukemia cell lines has scarcely been investigated, and only a few in vitro studies were performed using human breast epithelial cell lines. Due to its potential value for human health, demand for trans-R has rapidly increased, and new biotechnological strategies to obtain it from natural edible sources have been developed. Thus, grapevine cell cultures represent a reliable system of trans-R production since they biosynthesize trans-R constitutively or in response to elicitation. In addition, there are no studies deepen on the inhibitory effect of trans-R, produced by elicited grapevine cell cultures, on growth of human tumor cell lines. In this work, the effect of trans-R extracted from the culture medium, after elicitation of grapevine cell cultures, was tested on two human acute lymphocytic and monocytic leukemia cell lines, and one human breast cancer cell line. The effect of trans-R on cell proliferation was not only dose-and time-dependent but also cell type-dependent, as seen from the different degrees of susceptibility of cancer cell lines tested. As regards the effect of trans-R on cell cycle distribution, low trans-R concentrations increased cells in the S phase whereas a high trans-R concentration increased G 0 /G 1 phase in all cell lines. Perturbation of the cell cycle at low trans-R concentrations did not correlate with the induction of cell death, whereas a high trans-R concentration, cell proliferation decreased as a result of increasing apoptosis in the three cell lines. In leukemia cells, trans-R up-regulated the expression of caspase-3 while trans-R-induced apoptosis in breast cells occur through a caspase-3-independent mechanism mediated by a downregulation of Bcl-2.
The behavior of potato 5-lipoxygenase acting on both monomeric and aggregated linoleic acid has b... more The behavior of potato 5-lipoxygenase acting on both monomeric and aggregated linoleic acid has been studied. While the substrate preparation was transparent, lipoxygenase activity was determined by means of a spectrophotometric method, which was not useful to determine the activity in turbid samples. In the latter case a polarographic method was used. Cyclodextrins were used in order to increase the
Blossom-end rot (BER) is a physiopathy that affects tomato fruits causing disorganisation, cell b... more Blossom-end rot (BER) is a physiopathy that affects tomato fruits causing disorganisation, cell breakage and darkening of the tissues. In this study we describe a tomato fruit protein extraction protocol that includes polyvinyl polypyrrolidone, ascorbic acid and protease inhibitors to promote depletion of phenolics and to avoid protein degradation. The temperature-induced phase separation of plant extracts with nonionic detergent Triton X-114 favours the solubilisation of partially-hydrophobic species in the low-detergent upper phase, making them suitable for further analysis using two-dimensional gel electrophoresis. The analysis of two-dimensional images revealed differences in number and expression levels of several proteins from the control and BER-affected tomato fruits. Although the appearance of BER in tomato is primarily attributed to a lack of calcium supply to fruits, very little is known about the molecular and biochemical mechanisms involved. The identification of differential proteins from affected fruits with matrix-assisted laser desorption/ionisation-time of flight and peptide mass fingerprinting analysis revealed the induction of proteins participating in antioxidant processes (ascorbate-glutathione cycle) and the pentose phosphate pathway. We suggest that these two biochemical pathways, acting as reactive oxygen species scavengers in BER-affected fruits, restrain the spread of the blackening to the whole fruit.
Tobacco mosaic virus (TMV) is a widespread plant virus from the genus Tobamovirus that affects to... more Tobacco mosaic virus (TMV) is a widespread plant virus from the genus Tobamovirus that affects tobacco and tomato plants causing a pathology characterised by cell breakage and disorganisation in plant leaves and fruits. In this study we undertook a proteomic approach to investigate the molecular and biochemical mechanisms potentially involved in tomato fruit defence against the viral infection. The comparison of 2-D gels from control and TMV-infected but asymptomatic tomato fruits revealed changes in several proteins. The differential expression of peptidases, endoglucanase, chitinase and proteins participating in the ascorbate-glutathione cycle in infected fruits suggests that pathogenesis-related proteins and antioxidant enzymes may play a role in the protection against TMV infection. TMV coat protein appeared as a prominent spot in 2-D gels from TMV-infected asymptomatic fruits. A Triton X-114 phase-partitioning step of tomato protein extracts favoured the solubilisation of TMV coat protein and the enrichment of two aminopeptidases not present in control fruits. PMF and MS/MS data of the 2-D gel-isolated TMV coat protein is proposed as a powerful analysis method for the simultaneous tobamovirus detection, species determination and strain differentiation in virus-infected fruit commodities.
We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmon... more We analyze, for the first time, the early signal transduction pathways triggered by methyl jasmonate (MJ) and cyclodextrins (CDs) in tobacco (Nicotiana tabacum) cell cultures, paying particular attention to changes in cytosolic free Ca 2þ concentration ([Ca 2þ ] cyt), the production of hydrogen peroxide (H 2 O 2) and nitric oxide (NO), and late events like the induction of capsidiol. Our data indicate that MJ and CDs trigger a [Ca 2þ ] cyt rise promoted by Ca 2þ influx through Ca 2þ-permeable channels. The joint presence of MJ and CDs provokes a first increase in [Ca 2þ ] cyt similar to that observed in MJ-treated cells, followed by a second peak similar to that found in the presence of CDs alone. Moreover, oxidative burst induced by MJ is more pronounced when tobacco cells are incubated with CDs alone or in combination with MJ. The presence of both elicitors provokes H 2 O 2 production similar to that found in CD-treated cells, and a sustained response similar to that found in MJ-treated cells. In all treatments, H 2 O 2 production is dependent on Ca 2þ influx and protein phosphorylation events. Similarly, the joint action of both elicitors provokes NO accumulation, although to a lesser extent that in MJ-treated cells because CDs alone do not trigger this accumulation. This NO production is dependent on Ca 2þ influx but independent of both H 2 O 2 production and staurosporine-sensitive phosphorylation events. Taken as a whole, these results suggest the existence of different intracellular signaling pathways for both elicitors. Likewise, CDs might act by regulating the signaling pathway triggered by MJ since, in the presence of both compounds, CDs neutralize the strong oxidative and nitrosative bursts triggered by MJ and therefore, they regulate both H 2 O 2 and NO levels.
The 4-plex iTRAQ platform was utilized to analyze the protein profiles in four stages of grapevin... more The 4-plex iTRAQ platform was utilized to analyze the protein profiles in four stages of grapevine berry skin ripening, from pre-veraison to fully ripening. Mass spectrometric data were acquired from three replicated analyses using a parallel acquisition method in an Orbitrap instrument by combining collision-induced dissociation (CID) and higher energy collision-induced dissociation (HCD) peptide ion fragmentations. As a result, the number of spectra suitable for peptide identification (either from CID or HCD) increased 5-fold in relation to those suitable for quantification (from HCD). Spectra were searched against an NCBInr protein database subset containing all the Vitis sequences, including those derived from whole genome sequencing. In general, 695 unique proteins were identified with more than one single peptide, and 513 of them were quantified. The sequence annotation and GO term enrichment analysis assisted by the automatic annotation tool Blast2GO permitted a pathway analysis which resulted in finding that biological processes and metabolic pathways de-regulated throughout ripening. A detailed analysis of the function-related proteins profiles helped discover a set of proteins of known Vitis gene origin as the potential candidates to play key roles in grapevine berry quality, growth regulation and disease resistance.
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