Changes in antioxidant and lignifying enzyme activities in sunflower roots (Helianthus annuus L.) stressed with copper excess

Treatment with 50 microM CuSO4 for five days caused significant decrease in dry-matter production and protein level of ten-day-old sunflower seedling roots. An increase of lipoperoxidation product rate was also observed. The involvement of some enzyme activities in the sunflower root defence against Cu-induced oxidative stress was studied. Copper treatment induced several changes in antioxidant enzymes. SOD (superoxide dismutase, EC 1.15.1.1) activity was reduced but CAT (catalase, EC 1.11.1.6) and GPX (guaiacol peroxidase, EC 1.11.1.7) activities were significantly enhanced. The lignifying peroxidase activities, assayed using coniferyl alcohol and syringaldazine, were also stimulated. Analysis by native gel electrophoresis of syringaldazine peroxidase activity showed the stimulation of an isoform (A2) and the induction of another one (A1) under cupric stress conditions. On the other hand, the activity of PAL (phenylalanine ammonia lyase, EC 4.3.1.5), which plays an important role in plant defence, was also activated. The possible mechanisms by which Cu-induced growth delay and changes in enzymatic activities involved in plant defence processes are discussed.     

The relationship between the activity of various iron-containing and iron-free enzymes and the presence of nicotianamine in tomato seedlings

The influence of nicotianamine (NA) and iron on the activities of 4 iron-containing and two iron-free enzymes in leaves and roots of the NA-free tomato mutant chloronerva and its NA-containing wild-type ( Lycopersicon esculentum Mill. cv. Bonner Beste) was investigated. Aconitase (EC 4.2.1.3) activity in both leaves and roots was much higher in the mutant under normal iron supply (10 μ M FeEDTA) and in wild-type under iron deficiency than in wild-type supplied with 10 μ M FeEDTA. Application of NA to chloronerva leaves led to a decrease of aconitase activity in leaves and roots. NA had no effect on the enzyme activity when added to the assay medium.
Similar results were obtained for the iron-containing enzymes catalase (EC 1.11.1.6), ascorbate-dependent peroxidase (EC 1.11.1.11) and guaiacol-dependent peroxidase (EC 1.11.1.7) in roots. NA treatment of the mutant leaves decreased enzyme activities in roots down to wild-type values. In vivo NA application had no effect on enzyme activities in leaf extracts.
The activities of the iron-free enzymes NAD⁺-malate dehydrogenase (EC 1.1.1.37) and phosphofructokinase (EC 2.7.1.11) in root and leaf extracts were not influenced by the iron supply to the plants.     

Plant peroxidases—an organismic perspective

Peroxidases are ubiquitous enzymes found in virtually all green plants, many fungi and aerobic bacteria. The isozymic heterogeneity of peroxidases appears to result from de novo synthesis, as well as an array of physiological/ecological determinants including hormones, light, gravity, and infection. Homologies among isoperoxidases from the same species are largely distinguished by the isoelectric point and as well as by protein sequence data. The basic and acidic peroxidases from a number of angiosperms show a greater functional and structural relationship within rather than between these groups. Peroxidases have phylogenetically-correlated similarities based on the chemical nature and redox potentials of the substrates which they can oxidize. Peroxidases often increase as a response to stress, and one of the principle roles of peroxidase appears to be cellular protection from oxidative reactions imposed on all photosynthetic plants. The relationships among the peroxidases, IAA and lignification emerge as a particular adaptation of vascular plants to the land environment. The great catalytic versatility of peroxidase as its predominant character and, therefore, no single major role need necessarily exist for this multifaceted enzyme.     

Gallic acid oxidation by turnip peroxidase

Both ellagic and gallic acids non competitively inhibited guaiacol oxidation by turnip peroxidase. The K_i values were 3 and 26 μm for ellagic and gallic acid respectively. Enzymatic oxidation of gallic acid by the isolated major turnip peroxidase was characterized with respect to spectral behaviour, affinity constant and pH effect. The K_m for H₂O₂ and gallic acid are 2.5 and 8.0 mM for turnip peroxidase. The pH optimum for gallic acid oxidation is about 6.5 and the rate constant k₄ decreased with the increase of pH in presence of both guaiacol and Gallic acid. When the gallic acid oxidation products were subjected to chromatographic analysis, it was found to be converted mainly to ellagic and an unknown quinone.     

New species ofRelbunium (Rubiaceae) from Brazil,with notes on flavonoid and peroxidase patterns

Three new species ofRelbunium, R. humilioides, R. catarinense, andR. longipedunculatum (Rubiaceae), endemic in the southern states of Brazil, are described. Flavonoid patterns from two-dimensional thin layer chromatography and peroxidase patterns from gel electrophoresis are presented for two of them. The relationships of the new species, their taxonomic position, and their evolutionary significance within the genus are discussed.The Systematics and Evolution of the GenusRelbunium (Rubiaceae), I.     

Effect of calcium on subcellular distribution of peroxidases

About 40% of the peroxidase activity extracted from hypocotyl hooks of etiolated Cucurbita pepo was pelletable at 20000 g. The activity in the pellet was partially solubilized by the addition of 5 mM EGTA. This effect of EGTA was reversed by Ca²⁺, but not by Mg²⁺. Reassociation of EGTA-solubilized peroxidases to 4 cellular fractions obtained by centrifugation on discontinuous sucrose gradients was assayed. It appeared that the enzymes could be linked to ribosomes or RNP particles through Ca²⁺. Two fractions, identified as smooth and rough endoplasmic reticulum, also bound peroxidases and, in this case, the Ca²⁺-mediated binding involved a loss of the enzyme activity. The fraction containing mitochondria and plasmalemma exhibited a slight binding capacity. Isoelectric focusing in thin layer polyacrylamide gel showed that only 5 out of the 10 isoperoxidases present in hypocotyl hooks had their pelletability level changed by Ca²⁺.     

Adenine recognition: a motif present in ATP-, CoA-, NAD-, NADP-, and FAD-dependent proteins.

Adenosine triphosphate (ATP) plays an essential role in energy transfer within the cell. In the form of NAD, adenine participates in multiple redox reactions. Phosphorylation and ATP-hydrolysis reactions have key roles in signal transduction and regulation of many proteins, especially enzymes. In each cell, proteins with many different functions use adenine and its derivatives as ligands; adenine, of course, is present in DNA and RNA. We show that an adenine binding motif, which differs according to the backbone chain direction of a loop that binds adenine (and in one variant by the participation of an aspartate side-chain), is common to many proteins; it was found from an analysis of all adenylate-containing protein structures from the Protein Data Bank. Indeed, 224 protein-ligand complexes (86 different proteins) from a total of 645 protein structure files bind ATP, CoA, NAD, NADP, FAD, or other adenine-containing ligands, and use the same structural elements to recognize adenine, regardless of whether the ligand is a coenzyme, cofactor, substrate, or an allosteric effector. The common adenine-binding motif shown in this study is simple to construct. It uses only (1) backbone polar interactions that are not dependent on the protein sequence or particular properties of amino acid side-chains, and (2) nonspecific hydrophobic interactions. This is probably why so many different proteins with different functions use this motif to bind an adenylate-containing ligand. The adenylate-binding motif reported is present in "ancient proteins" common to all living organisms, suggesting that adenine-containing ligands and the common motif for binding them were exploited very early in evolution. The geometry of adenine binding by this motif mimics almost exactly the geometry of adenine base-pairing seen in DNA and RNA.     

Electrostatic modulation of electron transfer in the active site of heme peroxidases

 The heme enyzmes cytochrome c peroxidase (CCP) and pea cytosolic ascorbate peroxidase (APX) show a high level of sequence identity. The main difference near the active sites is the presence of a cation binding site in APX located about 1 nm from the Trp-179 side chain, which is hydrogen-bonded to Asp-208. It is possible that this difference in electrostatics provided by the protein environment is an essential determinant of the stabilization of the ion-pair or neutral form of the Trp...Asp couple in APX and CCP. Semiempirical molecular orbital calculations support the hypothesis that the position of the moving proton inside the couple influences the location of the free electron, leading to radical formation either on the heme or on the Trp side chain of these enzymes. Received, accepted: 26 November 1996