Vesna Hadži-Tašković Šukalović

Characterization of cell wall oxalate oxidase from maize roots

Oxalate oxidase activity was detected in the cell wall fraction isolated from maize roots (Zea mays L.). The enzyme was active at acidic pH with optimal activity at pH 3.2. It was thermally extremely stable and resistant to high salt concentration, SDS and pepsin. The enzyme activity was inhibited by sulphydryl reagents 2-mercaptoethanol (2-ME), N-ethyl maleimide (NEM) and dithiotreitol (DTT), but was insensitive to EDTA, KCN and metal ions. Measurements of enzyme activity were performed using colorimetric assay of H(2)O(2), as well as polarographic detection of O(2) consumption. Maximal activity was obtained with 5 mM oxalic acid for the colorimetric method, and 10 mM oxalic acid for the polarographic method. Both methods were applicable in oxalate oxidase characterization, the polarographic method being more suitable under conditions of H(2)O(2) interaction with some of the analyzed substances.

Cell wall-associated malate dehydrogenase activity from maize roots

Isolated cell walls from maize (Zea mays L.) roots exhibited ionically and covalently bound NAD-specific malate dehydrogenase activity. The enzyme catalyses a rapid reduction of oxaloacetate and much slower oxidation of malate. The kinetic and regulatory properties of the cell wall enzyme solubilized with 1M NaCl were different from those published for soluble, mitochondrial or plasma membrane malate dehydrogenase with respect to their ATP, Pi, and pH dependence. Isoelectric focusing of ionically-bound proteins and specific staining for malate dehydrogenase revealed characteristic isoforms present in cell wall isolate, different from those present in plasma membranes and crude homogenate. Much greater activity of cell wall-associated malate dehydrogenase was detected in the intensively growing lateral roots compared to primary root with decreased growth rates. Presence of Zn(2+) and Cu(2+) in the assay medium inhibited the activity of the wall-associated malate dehydrogenase. Exposure of maize plants to excess concentrations of Zn(2+) and Cu(2+) in the hydroponic solution inhibited lateral root growth, decreased malate dehydrogenase activity and changed isoform profiles. The results presented show that cell wall malate dehydrogenase is truly a wall-bound enzyme, and not an artefact of cytoplasmic contamination, involved in the developmental processes, and detoxification of heavy metals.

The characterization of peroxidases in mitochondria of maize roots

Abstract In the present study, we provide the evidence about the presence of peroxidases in mitochondria isolated from maize (Zea mays L.) roots with characteristics of both, peroxidase and oxidase activities. Oxidase activity, mediating reduction of oxygen to superoxide anion (O2− ) and H2O2 with NAD(P)H as a substrate, in the presence of MnCl2 and SHAM or p-coumaric acid, was demonstrated by using various known effectors and inhibitors of this reaction. Mitochondrial enzyme had some characteristics similar to MnCl2/SHAM-stimulated NAD(P)H oxidase from cell wall and plasma membrane. Peroxidatic activity was detected as NAD(P)H oxidation in the presence of exogenously added H2O2 and various phenolic compounds. This peroxidase dependent NAD(P)H oxidation via phenolics had similar characteristics with guaiacol peroxidase from the same mitochondrial preparation. Different effects of Cu2+, Fe2+ and phenolic compounds on oxidase and peroxidase reactions suggest that these two activities are catalyzed by two separate, well-coordinated isoenzymes. Their role in generation and removal of reactive oxygen species (ROS) in plant cell was proposed.

The effects of manganese and copper in vitro and in vivo on peroxidase catalytic cycles.

Here we present the results of in vitro and in vivo studies of the influence of Mn²+ and Cu²+ on the peroxidative and oxidative catalytic functions of class III peroxidase. Complex peroxidase catalysis by intermediates generated in the reaction was analyzed by utilizing the activating effect of Mn²+ and the inhibitory effect of Cu²+ on the oxidative reaction in vitro. p-Coumaric acid was used as an enzyme substrate in the peroxidative reaction and as a cofactor in the oxidative reaction. In order to correlate the observed in vitro effects with the in vivo situation, we exposed maize plants to excess concentrations of Mn²+ and Cu²+ in the hydroponic solutions. Copper severely arrested plant growth, while manganese exerted no significant effect. The effects on peroxidase activity and isoforms profile of root soluble and cell wall bound fractions were studied. Inhibition of the peroxidase oxidative function by copper was reversible, localized in the cell wall, and accompanied by disappearance of some and appearance of new cationic isoforms. Copper-mediated changes were suppressed by the presence of manganese, although Mn²+ treatment per se did not affect the activity of the peroxidase enzyme. The results on the peroxidase activity in maize roots grown with excess Mn²+ and Cu²+ point to the coupling between the oxidative cycle, root growth and different peroxidase isoforms.

The Coexistence of the Oxidative and Reductive Systems in Roots: The Role of Plasma Membranes

Abstract: Different components of the plasma membrane bound and associated redox system, which participate in the energy transfer from the predominantly reducing intercellular environment to the extracellular oxidizing environment, are reviewed. Special attention is given to plant root cells. An analysis of the plasma membrane‐associated redox components, such as the cytochromes, quinones, and different types of oxidoreductases (dehydrogenases, oxidases, peroxidases, and superoxide dismutases), is made, as well as their coupling with naturally occurring extracellular substrates, such as oxygen and its reactive forms, phenols, ascorbate, nitrate, ferric ion, and organic acids. The participation of different free radical species in most of the plasma membrane‐bound redox reactions is documented.

Properties of Maize Root Mitochondria from Plants Grown on Different Nitrogen Sources

Summary The role of maize ( Zea mays L. inbred line VA35) root mitochondria in nitrogen assimilation was investigated. Maize plants were grown for 2 weeks on a modified Knopp solution containing different forms of nitrogen: 10.9 mM nitrate or 10.9 mM nitrate + 7.2 mM ammonium. Mitochondria were isolated from root tissue and purified on a Percoll gradient. Glutamate dehydrogenase (GDH; EC 1.4.1.2), alanine aminotransferase (GPT; EC 2.6.1.2), NAD + -isocitrate dehydrogenase (NAD + -ICDH; EC 1.1.1.41), succinate dehydrogenase (SDH; EC 1.3.99.1) and malate dehydrogenase (MDH; EC 1.1.1.37) all showed increased specific activities in the case of mitochondria isolated from plants grown in the presence of ammonia. These results indicate the involvement of such mitochondria in ammonia assimilation by synthesizing glutamate in a reductive animation reaction catalyzed by glutamate dehydrogenase and enhanced TCA metabolism. Increased oxygen consumption rates of mitochondria isolated from ammonia grown plants was demonstrated in our experiments, supporting the occurrence of such a metabolic shift. The possibility that higher oxygen consumption rates of both respiratory pathways (phosphorylative and non-phosphorylative) with malate as substrate for oxidation result from stimulation of biosynthetic and catabolic functions of mitochondria from plants grown in the presence of ammonia is discussed.

Effectiveness of phenoxyl radicals generated by peroxidase/H2O2-catalyzed oxidation of caffeate, ferulate, and p-coumarate in cooxidation of ascorbate and NADH

The rate of ascorbate and nicotinamide adenine dinucleotide plus hydrogen (NADH) cooxidation (i.e., their nonenzymic oxidation by peroxidase/H2O2-generated phenoxyl radicals of three hydroxycinnamates: caffeate, ferulate and p-coumarate) was studied in vitro. The reactions initiated by different sources of peroxidase (EC 1.11.1.7) [isolates from soybean (Glycine max L.) seed coat, maize (Zea mays L.) root-cell wall, and commercial horseradish peroxidase] were monitored. Native electrophoresis of samples and specific staining for peroxidase activity revealed various isoforms in each of the three enzyme sources. The peroxidase sources differed both in the rate of H2O2-dependent hydroxycinnamate oxidation and in the order of affinity for the phenolic substrates. The three hydroxycinnamates did not differ in their ability to cooxidize ascorbate, whereas NADH cooxidation was affected by substitution of the phenolic ring. Thus, p-coumarate was more efficient than caffeate in NADH cooxidation, with ferulate not being effective at all. Metal ions (Zn2+ and Al3+) inhibited the reaction of peroxidase with p-coumarate and affected the cooxidation rate of ascorbate and the peroxidase reaction in the same manner with all substrates used. However, inhibition of p-coumarate oxidation by metal ions did not affect NADH cooxidation rate. We propose that both the ascorbate and NADH cooxidation systems can function as mechanisms to scavenge H2O2 and regenerate phenolics in different cellular compartments, thus contributing to protection from oxidative damage.

Thermal Inactivation of Soybean Bioactive Proteins

Abstract Inactivation of trypsin inhibitor, lipoxygenase 1, and urease during the processes of dry extrusion, wet extrusion, micronisation, microwave roasting and autoclaving were studied. Cultivars Bosa, ZPS 015, Goyou Kurakake and L93-7290 were used in the experiment. Depending on a technological procedure of processing, kernels were exposed to temperatures from 57 to 150 oC for different time: from 25-30 sec in the process of dry and wet extrusion, to 30 min in autoclaving. The process of dry extrusion had the greatest influence on reduction of the trypsin inhibitor content and inactivation of urease and lipoxygenase 1. During the dry extrusion at 100 oC trypsin inhibitor declined by 74.2 % and the residual activity of a lipoxygenase 1 and urease was only 0.76 i.e. 0.55 %, respectively. Lipoxygenase 1 was the most heat labile enzyme, followed by urease and than trypsin inhibitor.

Comparative biochemical characterization of peroxidases (class III) tightly bound to the maize root cell walls and modulation of the enzyme properties as a result of covalent binding

Comparative biochemical characterization of class III peroxidase activity tightly bound to the cell walls of maize roots was performed. Ionically bound proteins were solubilized from isolated walls by salt washing, and the remaining covalently bound peroxidases were released, either by enzymatic digestion or by a novel alkaline extraction procedure that released covalently bound alkali-resistant peroxidase enzyme. Solubilized fractions, as well as the salt-washed cell wall fragments containing covalently bound proteins, were analyzed for peroxidase activity. Peroxidative and oxidative activities indicated that peroxidase enzymes were predominately associated with walls by ionic interactions, and this fraction differs from the covalently bound one according to molecular weight, isozyme patterns, and biochemical parameters. The effect of covalent binding was evaluated by comparison of the catalytic properties of the enzyme bound to the salt-washed cell wall fragments with the corresponding solubilized and released enzyme. Higher thermal stability, improved resistance to KCN, increased susceptibility to H2O2, stimulated capacity of wall-bound enzyme to oxidize indole-3-acetic acid (IAA) as well as the difference in kinetic parameters between free and bound enzymes point to conformational changes due to covalent binding. Differences in biochemical properties of ionically and covalently bound peroxidases, as well as the modulation of the enzyme properties as a result of covalent binding to the walls, indicate that these two fractions of apoplastic peroxidases play different roles.

Effects of enzyme activities during steeping and sprouting on the solubility and composition of proteins, their bioactivity and relationship with the bread making quality of wheat flour

The aim was to determine the effect of steeping and sprouting on wheat grain proteins and the functional consequences in this regard. The solubility of proteins and the polypeptide composition of albumins, globulins, gliadins and glutenins were determined, as well as the content of non-protein nitrogen and free sulfhydryl groups (-SH), and the activity of peroxidase (POD) and lipoxygenase (LOX). In addition, the pasting viscosity of flour and protein bioactivity such as antioxidant capacity and immunoreactivity were evaluated. The increase of non-protein nitrogen and free -SH groups by about 62.09 and 96.7%, respectively, as well as the decrease of albumin + globulin polypeptides with a molecular weight over 85.94 kDa and between 85.94-48.00 kDa by about 34 and 8.7%, respectively, were the most notable changes observed in the flour from whole sprouted wheat that clearly point to the intensive protein hydrolysis. The reduction of disulfide bonds and increased concentrations of free -SH groups significantly modify the visco-elastic properties of gliadins and glutenins causing pasting viscosity reduction. However, sprouted wheat flour could be considered as a potential food ingredient because of its improved antioxidant capacity that is a result of protein hydrolysis inter alia. As protein modification induced by steeping may have beneficial effects on the antigenicity of the glutenin fraction, this kind of wheat pretreatment can represent a putative strategy in the dietary modulation of diseases related to glutenin immunoreactivity, e.g. dermatitis herpetiformis.