Comparative studies on kinetic behavior of horseradish peroxidase isoenzymes

Kinetic parameters for each reaction step of the peroxidase-catalyzed reaction were determined by the stopped-flow technique on three distinct isoenzymes: acidic A2, neutral C1, and basic E5. The pH dependence of the reaction for the formation of compound I with hydrogen peroxide was examined. The three isoenzymes had a common ionizing group at about pK 4 which affects the rate constant for the formation of compound I. The heat of ionization determined from the temperature dependence of the dissociation constant of the group strongly suggested that it is of carboxyl nature. The rate constant for isoenzyme A2 was a tenth of those for the other two isoenzymes over the whole range of pH. Furthermore, the thermodynamic parameters of isoenzyme A2 were found to be different from those for the other two isoenzymes. These difference as well as the different behavior in alkaline transition of the isoenzymes are discussed in relation to the sixth ligand of the heme. The rate constant of the reduction of compound I and compound II by ferrocyanide were also determined. In both reduction steps, the pH profiles of the apparent rate constant for isoenzyme A2 and E5 were similar, but they were different from that of C1. The ionization with pK 5.29, which was detected only in isoenzyme C1, may be attributed to a group near the porphyrin ring as a stabilizer for the pi-cation radical.     

Persistence of differences between peroxidase isoenzymes of flax genotrophs in tissue culture

Anionic peroxidase isoenzymes from seedling root, hypocotyl and cotyledon regions of the large (L) and small (S) flax genotrophs were separated on acrylamide gels. Tissue cultures were initiated from each of these regions of the seedlings, and maintained for a 200-day period with six transfers. The differences in electrophoretic mobility of the peroxidase isoenzymes between L and S noted in seedlings, and also in main stems of adult plants, were still present in the tissue cultures.     

Leaf peroxidase activities in tomato mutants affecting plant morphology

The leaf peroxidase activity and the electrophoretic banding pattern of 69 tomato mutants affecting plant morphology have been studied. The zymograms of 63 mutants were normal, and their total peroxidase activities were not correlated with a particular plant or leaf trait. However, six mutations, lyrate x-1521, mottled, olivacea, monstrosa, extreme dwarf, and Curl, are characterized by one or two isozymic bands more intensely stained than in the normal electrophoretic pattern; concomitantly, their total peroxidase activities increase significantly, reaching, in the mutant olivacea, a value forty fold higher than in nonmutant stocks. These abnormal levels of total peroxidase activity are achieved by a promotion of the constitutive bands A5 or C2, singularly or simultaneously. It may be inferred that the six abovementioned mutations influence directly or indirectly the peroxidase activity level without affecting the genes which code for the peroxidase isozymes themselves. Parallels between the mutant effects on specific peroxidase bands and hormonally mediated control of the peroxidase isozymes are discussed.     

Kinetics of fenugreek peroxidase isoenzymes

Peroxidase from fenugreek seedlings was separated into 6 isoenzymes; 4 on CM-cellulose and 2 on DEAE-cellulose. The kinetics of these peroxidase isoenzymes with regard to o-dianisidine and catechol are described.     

Purification of two peroxidase isoenzymes of Aloe barbadensis which oxidize p-coumaric acid

Using a combination of hydrophobicity and ion-exchange chromatography methods, one cationic (pI 9.0) and one anionic (pI 4.5) peroxidase (donor: hydrogen-peroxide oxidoreductase; EC 1.11.1.7) isoenzymes of Aloe barbadensis have been purified (the cationic peroxidase to homogeneity as judged by SDS-PAGE analysis and microsequencing). This allowed us to initiate the investigation of individual catalytic properties to be related to their respective functions in vivo. The two peroxidases have an optimal activity at pH 6.0. Apparent affinities for H₂O₂ range between 0.01 and 0.14 mM depending on the phenolic substrate and the isoenzyme. The apparent K_m values for the phenolics (p-coumaric acid and hydroquinone) are some 25-fold lower in the anionic (around 0.02 mM) than in the cationic (around 0.77 and 0.34 mM, respectively) isoenzyme. The possible functions of the activities are discussed.     

Regulation and function of ascorbate peroxidase isoenzymes

Even under optimal conditions, many metabolic processes, including the chloroplastic, mitochondrial, and plasma membrane-linked electron transport systems of higher plants, produce active oxygen species (AOS). Furthermore, the imposition of biotic and abiotic stress conditions can give rise to excess concentrations of AOS, resulting in oxidative damage at the cellular level. Therefore, antioxidants and antioxidant enzymes function to interrupt the cascades of uncontrolled oxidation in each organelle. Ascorbate peroxidase (APX) exists as isoenzymes and plays an important role in the metabolism of H(2)O(2) in higher plants. APX is also found in eukaryotic algae. The characterization of APX isoenzymes and the sequence analysis of their clones have led to a number of investigations that have yielded interesting and novel information on these enzymes. Interestingly, APX isoenzymes of chloroplasts in higher plants are encoded by only one gene, and their mRNAs are generated by alternative splicing of the gene's two 3'-terminal exons. Manipulation of the expression of the enzymes involved in the AOS-scavenging systems by gene-transfer technology has provided a powerful tool for increasing the present understanding of the potential of the defence network against oxidative damage caused by environmental stresses. Transgenic plants expressing E. coli catalase to chloroplasts with increased tolerance to oxidative stress indicate that AOS-scavenging enzymes, especially chloroplastic APX isoenzymes are sensitive under oxidative stress conditions. It is clear that a high level of endogenous ascorbate is essential effectively to maintain the antioxidant system that protects plants from oxidative damage due to biotic and abiotic stresses.     

Biochemical and ecological characterization of two peroxidase isoenzymes from the mangrove, Rhizophora mangle

This study examines phenolic peroxidase (POX) in Rhizophora mangle L. leaves in order to assess its role in phenolic manipulation and H₂O₂ scavenging. Sun-exposed and understorey leaves experiencing varying degrees of nutrient stress were analysed from an oligotrophic cay off the coast of Belize. POX activity was unaffected by growth environment, but increased throughout leaf development and persisted through senescence and after abscission. Histochemical analyses indicated POX activity throughout leaf tissues, especially in the apoplast. Phenolics were similarly broadly distributed. Two isoenzymes of POX were partially characterized with pIs of 4.1 and 6.3 and masses of 65.5 and 54.3 kDa, respectively. The larger, more acidic isoenzyme showed especially high heat stability, showing no reduced activity after 24 h at 60 °C. Rhizophora mangle POX oxidized quercetin preferentially, and, to a lesser extent, coniferyl alcohol, caffeic acid, chlorogenic acid, and p-coumaric acid. It did not oxidize ascorbate, but ascorbate could act as a secondary electron donor in the presence of a phenolic substrate and H₂O₂. However, because quercetin and other aglycones were not present in R. mangle leaves, and because POX showed no activity with the most abundant leaf flavonoid, rutin, it was concluded that detoxification of H₂O₂ is secondary to the other roles of POX in manipulation of phenolics.     

Genetics of the peroxidase isoenzymes inPetunia

Summary The structural geneprxD inPetunia codes for a slow moving anodic peroxidase whose activity is sensitive to high concentrations of hydrogen peroxide. The PRXd enzyme could be found in mature and old leaf and stem tissue of full-grown flowering plants. PRXd was found to be absent in tissues from flower corolla and root. The geneprxD is the fourth gene that codes for peroxidases in leaf and stem. Two mobility variants of the PRXd enzyme have been found among our inbred lines using starch gel system II electrophoresis. The geneprxD could be located on chromosome III by a four-point-cross involving the genesprxA, prxD, Mf1 andHt1. The order of the genes established is:Ht1 — Mf1 — prxD — prxA.     

Transcriptome profiling reveals similarities and differences in plant responses to cadmium and lead

We analyzed the influence of salts of two heavy metals—lead and cadmium (Pb²⁺ and Cd²⁺) on plants, including plant and root size, plant genome stability as well as global genome expression. Measurement of the metal uptake showed that there was a significantly higher incorporation of Cd than of Pb, 0.6 and 0.15 uM per gram of dry weight, respectively. The analysis of the root length and plant size showed a dose dependent decrease in plants exposed to cadmium. In contrast there was little difference in the size of plants exposed to lead, although there was nearly four-fold increase of the root length. Analysis of the genome stability revealed that cadmium led to a dose dependent increase of homologous recombination whereas lead had no effect.

Analysis of the global genome expression of plants chronically exposed to 50 uM of Cd and Pb revealed 65 and 338 up- and down-regulated genes by Cd and 19 and 76 by Pb, respectively. Interestingly, half of the genes that changed their expression in Pb-treated plants also changed their expression in Cd-treated ones. The greater number of genes regulated by Cd reflects generally higher genome instability of plants as well as higher uptake as compared to Pb.     

Germinating barley selenium-containing peroxidase is one of the peroxidase isoenzymes

A selenium-containing peroxidase from the germinating barley grown on a selenium-containing artificial medium was isolated and purified by means of cold acetone precipitation, Sephadex-G150 filtration, followed by DEAE-Sepharose chromatography and sodium dodecyl sulfate — polyacrylamid gel electrophoresis. The form of selenium existing in the peptide assayed with paper chromatography was selenomethionine. The amino acid composition of this enzyme was similar to those peroxidases from other sources except amino acids Glu, Val Phe, Lys, and Arg. Electron-spin resonance (ESR) spectra recorded at −136°C showed that both the selenium-containing peroxidase from germinating barley and horseradish peroxidase had same the ESR signals as iron protoporphyine. Those results suggested that the germinating barley selenium-containing peroxidase is one of the peroxidase isoenzymes.     

Structures of two classes of MHC molecules elucidated: crucial differences and similarities

New structures of MHC molecules have significantly improved our understanding of molecular recognition in cellular immunology. Highlights include the first structure of a class II MHC molecule, complexed with a viral peptide and with a bacterial superantigen. A structure of an MHC-like Fc receptor is expected soon. Interesting comparisons can now be made between the recognition properties of MHC and MHC-like proteins.     

Acetylcholinesterases--the structural similarities and differences

Acetylcholinesterase (AChE) is a widely spread enzyme playing a very important role in nerve signal transmission. As AChE controls key processes, its inhibition leads to the very fast death of an organism, including humans. However, when this feature is to be used for killing of unwanted organisms (i.e. mosquitoes), one is faced with the question - how much do AChEs differ between species and what are the differences? Here, a theoretical point of view was utilized to identify the structural basis for such differences. The various primary and tertiary alignments show that AChEs are very evolutionary conserved enzymes and this fact could lead to difficulties, for example, in the search for inhibitors specific for a particular species.     

Genetics of the peroxidase isoenzymes in Petunia

Summary As detected by starch gel electrophoresis, the fast moving anodal group of peroxidase isoenzymes, the PRXa complex, of a Petunia homozygous for the encoding gene can be made up of one to four bands, depending on the tissue sampled, the age of the tissue and of the plant, and the genetic background. Additional evidence is presented showing that the PRXa complex is encoded by one structural gene, prxA, rather than by tandem duplicated genes. On the basis of electrophoretic variation in Petunia hybrida and related species, five prxA alleles were found. A prxA internal site mutation was found recognized by the absence of recombination between the mutation that affected the temporal programme of the gene and the mutation that altered the mobility of the enzyme. By a three-point test, the gene prxA was located on chromosome III and found to be linked to the genes Mf1 and Ht1 in the order prxA-Mf1-Ht1. The construction of a trisomic III triply heterozygous for prxA confirmed the location of prxA.     

Genetics of the peroxidase isoenzymes in Petunia

Theoretical and Applied Genetics - In Petunia four alleles of the gene prxB could be identified by starch gel electrophoresis. Investigation of PRXb allozyme balance during development of tissue...     

Genetics of the peroxidase isoenzymes in Petunia

Summary Antibodies were raised against the peroxidases encoded by the allele prxA1 to determine the specific activities of the peroxidases encoded by the alleles prxA1, prxA2, prxA3, and prxA5. The results from double diffusion experiments indicated that all peroxidases encoded by the four alleles are antigenically identical. By rocket immuno electrophoresis it was shown that the peroxidases encoded by the alleles prxA1, prxA2, prxA3, and prxA5 have different specific activities. The results presented are discussed in relation to differential expression of the alleles involved.