Anionic Isozyme Research Articles

Comparative analysis of peroxidase profiles in Chinese kale (Brassica alboglabra L.): Evaluation of leaf growth related isozymes

Plant peroxidases (EC 1.11.1.7) with different isoforms catalyze various reactions in plant growth and development. However, it is difficult to elucidate the function of each isozyme in one plant. Here, we compared profiles of entire isozyme in young seedling and mature leaves of Chinese kale (Brassica alboglabra L.) on zymogram and ion exchange chromatography in order to investigate leaf growth related peroxidase isozymes. The results showed that four isozymes were constitutively expressed in kale leaves, whereas other two isozymes were induced in the mature leaves. The Mono Q ion exchange chromatography separated the six isozymes into two major groups due to the difference in their isoelectric points. The results suggested that although there were several isozymes in the leaves of Chinese kale, one isozyme functioned mainly through the leaf development. Two anionic isozymes with molecular weights lower than 32kDa were considered mature related.

Laccase isozymes: production by an opportunistic pathogen, a Fusarium proliferatum isolate from wheat

Laccases often are present in plant tissues colonized by fungal pathogens. Non-denaturing gel electrophoresis revealed laccase isozymes in extracts of wheat leaf sheaths infected by Fusarium proliferatum(Fp). No laccase isozymes were detected in extracts of healthy wheat. The four anionic isozymes (LacA-A to A-D) in the infected wheat extracts corresponded to those produced by the Fp isolate when cultured on low carbon medium. Only two of these isozymes (LacA-C and A-D) were found in the culture filtrates. A single anionic laccase isozyme (LacA-B) was detected on nitrogen-limited medium. Other cationic isozymes produced in vitro were not detected from the diseased sheath tissue. Northern hybridization revealed expression of a transcript corresponding to a potential laccase-gene sequence LacRT-1 in planta and in vitro under conditions of low carbon and nitrogen supply.

The acute effect of copper on the levels of indole-3-acetic acid and lignin in peanut roots

Cu-treated peanut (Arachis hypogaea L.) seedlings showeda significant inhibition in peanut root growth, and a decrease in endogenousindole-3-acetic acid (IAA) content. The decline of IAA content in Cu-treatedtissue was accompanied by an increase in the activity of cationic peroxidase(POD) isozyme P8.5, which was correlated with an increase in cationic PODtranscripts. Cu might suppress the growth of peanut roots by inducing thesynthesis of the cationic POD isozyme that degrades endogenous IAA. Theincrease in the activity of anionic POD isozyme P3.5 was correlated with therise in lignin content in Cu-treated roots. We suggest that the increase inanionic POD isozyme P3.5 induced by Cu might be responsible for ligninsynthesis in peanut roots, and may also remove excess hydrogen peroxide causedby Cu, thus playing a detoxifying role during Cu treatment.

DNA, peroxidase profile, and biochemical composition changes during the treatment in vitro of pollen of Argemone mexicana L. with SO 2

The allergenic pollen of Argemone mexicana L. a common road-side weed was subjected to artificial SO 2 fumigation at a concentration of 100ppm for 24 hours, 48 hours and 72 hours and its effect on the total carbohydrate, lipid, free amino acid, DNA and RNA content as well as peroxidase isozyme and DNA profile studied. In comparison to the control (pollen exposed to charcoal-filtered air), the carbohydrate, lipid, DNA and RNA content decreased with the increase in the time of exposure to SO 2 , where as the percentage composition of free amino acid increased. Contrary, to this, the activity of peroxidase was found to increase with a change in the iso-peroxidase zymogram showing three new anionic isozyme bands. SO 2 affected the total DNA profile leading to the gradual breakdown of DNA with several bands observed on agarose gel electrophoresis with an increase in the time of fumigation.

Involvement of a novel peroxidase isozyme and lignification in hydathodes in resistance to black rot disease in cabbage

The physiological mechanisms associated with resistance of cabbage to black rot disease seem to be associated with the hydathodes. To investigate the role of hydathodes in disease resistance, total peroxidase activities, anionic peroxidase isozyme expression, and lignin deposition were determined in hydathodes of resistant (Hancock and Green Cup), partially resistant (Cheers), and susceptible (Strukton and Perfect Ball) cabbage varieties (Brassica oleracea L.) during pathogenesis with Xanthomonas campestris pv. campestris. Four-week-old plants were petiole-inoculated with a highly virulent strain of X. campestris pv. campestris (FD91L). Hydathodal fluids were collected daily over a 14-day period from infected plants as well as noninfected, mock-inoculated control plants. Hydathodal fluids of resistant varieties had greater peroxidase activity when compared to susceptible ones, with infected plants having higher peroxidase levels than noninfected plants. Isoelectric focusing revealed the presence of four a...

Charge and molecular weight differences for fourteen guaiacol peroxidase isozymes in flax indicate that most are encoded by different structural genes.

Ferguson plots, linear regressions of log(relative mobility) against polyacrylamide gel concentration, provide an electrophoretic method of simultaneously obtaining estimates of net charge and molecular weight for nondenatured proteins, and are a powerful method of accurately comparing these characteristics among isozymes within an enzyme system. Here, Ferguson plots compare the characteristics of the nine isozymes (three cationic and six anionic) usually seen in soluble extracts of flax (Linum usitatissimum) seedling tissues, and of five anionic isozymes induced by heavy metal stress. Despite some differences in apparent molecular weight, all but one of the isozymes separated from each other on the basis of net charge and, except for a second isozyme with a comparatively high molecular weight, the net charges of the isozymes ranked in order of their relative mobilities on nondenaturing gels. Differences among the isozymes make it unlikely that any are related, which indicates that they are encoded by different structural genes.

HarpinPSS-induced peroxidase and lignin accumulation in tobacco during the hypersensitive response

Harpinpss, a pathogenic protein encoded by hrpZ in the hrp gene cluster of Pseudomonas syringae pv. syringae, induces the hypersensitive response (HR) in tobacco (Nicotiana tabacum L. cv. Xanthi). An increase in peroxidase activity, lignin content and salicylic acid was observed during the HR elicited by harpin. The increase in anionic, moderately anionic and cationic peroxidase isozymes is positively correlated with the HR in tobacco. In addition, the increase of the anionic peroxidase isozyme (pI 3.5) is correlated with a rise of the transcript of the encoding gene.

Apparent Differences in the Posttranslational Modification of Peroxidase between Flax Genotrophs and Genotypes and between Normal and Stress- Induced Isozymes

Estimates of relative net charge and apparent molecular weight were obtained, using Ferguson plots, for each of the peroxidase isozymes in four completely inbred flax (Linum usitatissimum) plant types. The six anionic and the three cationic isozymes that are normally seen in various seedling tissues were examined. Four anionic isozymes that are seen in stressed seedlings were also included in the study. Charge and molecular weight comparisons detected plant-type differences that apparently resulted from two different forms of posttranslational modification. Plant-type differences in molecular weight for the normal anionic and cationic isozymes were attributable to differences in the degree of glycosylation. However, all of the normal anionic isozymes of one plant type had lower net negative charges than the corresponding isozymes of the other plant types. Therefore, both glycosylation and a second form of common modification, which affects charge but not molecular weight, determine the final structure of these isozymes The stress-induced isozymes did not display the plant-type differences seen in the normal isozymes and, based on charge and weight, none of the induced isozymes appeared to relate to any of the normal isozymes Some stress-induced isozymes seemed to be present in some plant types but not in others. Isozyme interactions that can cause this appearance are described and discussed.

Cadmium and zinc induction of lipid peroxidation and effects on antioxidant enzyme activities in bean ( Phaseolus vulgaris L.)

The relationship between Cd and Zn phytotoxicities and oxidative reactions in bean plants was studied. Ten-day-old bean ( Phaseolus vulgaris L. cv. Morgan) plants treated for 96 h by 5 μM Cd and 100 μM Zn, separately, showed the same reduction of growth. In response to each metal, lipid peroxidation was enhanced in all plant organs, and catalase (CAT) activity was decreased in both roots and leaves, but not in stems. However, Cd and Zn stimulated the activity of guaiacol-dependent peroxidase only in stems, where native electrophoresis revealed, at least, two new anionic isozymes. The induction of one of these iso-guaiacol peroxidase (iso-GPX) was Zn-specific. The exposure to metals did not modify the activity of ascorbate-specific peroxidase either in roots or in stems. In leaves, where the glutathione reductase (GR) activity was assayed, increases of ascorbate peroxidase (APX) and glutathione reductase (GR) activities were recorded. These results suggest that some antioxidant enzymes can be activated, notably in upper plant parts, in response to oxidative stress induced by Cd and Zn.

Expression of a moderately anionic peroxidase is induced by aluminum treatment in tobacco cells: Possible involvement of peroxidase isozymes in aluminum ion stress

To clarify the mechanism of aluminum (Al) toxicity and Al tolerance, we isolated a new clone (pAL201) from a tobacco cDNA library. Northern blot hybridization analysis indicated that the expression of pAL201 is induced by Al treatment and phosphate (P1) starvation. The complete cDNA sequence suggested that this clone encodes a moderately anionic peroxidase (EC 1.11.1.7). Analysis by isoelectric focussing indicated that a moderately anionic peroxidase (approximately pI 6.7) and two cationic peroxidases (pI 9.2 and 9.7) in the soluble fraction are activated by Al treatment and P1 starvation, while two moderately anionic isozymes are repressed by these stresses. We suppose that Al ion stress can control the activity of some peroxidase isozymes, one of which is probably induced by enhanced gene expression of pAL201. There is a possibility that some of these isozymes have some functions in Al ion stress.

Effects of Zn on flax seedlings: differences in the response of the cationic and anionic isozymes of peroxidase

Zinc treatments that affected the growth and development of seedlings, and induced changes in peroxidase in all of the seedlings tissues, were used to examine the relative responses of the soluble, guaiacol peroxidase isozymes in flax ( Linum usitatissimum L.) to stress. In the roots, Zn induced increased activity in all of the normal root isozymes and also the presence of several new activity bands. No new bands were detected in the other tissues. In both the hypocotyls and the cotyledons, the activity of the main cationic isozyme increased relative to the activities of the two slowest migrating anionic isozymes at low Zn concentrations but the reverse occurred at high Zn concentrations. This shift in the balance between anionic and cationic activity was not detected during normal seedling development nor during the developement of nutrient deprived seedlings. However, the increases in the activities of the two slowest anionic isozymes, relative to the cationic activity, appeared to be a generalized response to stress and suggest that these isozymes play a role in detoxification. A third, and different, response was seen in the fast migrating anionic isozyme complex which decreased in activity with increasing treatment concentrations. With the possible exception of this complex, none of the Zn-induced changes in the peroxidase isozymes of flax appeared to be directly related to the induced changes in seedling growth and development.

Relative significance of endothelium and internal elastic lamina in regulating the entry of macromolecules into arteries in vivo.

A role for the internal elastic lamina (IEL), which separates the intima and media of an artery wall, as a restrictive barrier to macromolecular movement has been suggested in atherosclerotic lesion development or restenosis during angioplasty. The permeability coefficient of the IEL, however, has never been quantified in unperturbed vessels in vivo. Using a newly developed technique, we measured the concentration distributions in both intima and media of cationic (pI approximately 8.5) and anionic (pI approximately 6.3) isozymes of the 44-kD macromolecule horseradish peroxidase (HRP). Two mathematical models of arterial wall transport differing in their resolution of the intima were required to simulate the concentration distribution data and to estimate the parameters of interest. Optimal estimates of the permeability coefficients of the endothelium (PE) and IEL (PIEL) to HRP were determined by the best least-squares fit of the two models to experimental data. These estimates (anionic: PE = 0.050 +/- 0.021 microns/min, PIEL = 0.146 +/- 0.082 microns/min, n = 8; cationic: PE = 0.034 +/- 0.018 microns/min, PIEL = 0.110 +/- 0.047 microns/min, n = 8) indicate that the IEL is responsible for approximately 25% (anionic, 26 +/- 9%; cationic, 25 +/- 13%) of the resistance to HRP transport from the blood into the arterial media. Although both parameters were less for the cationic preparation, the differences were not significant, and the relative role of the IEL was similar for both molecules.(ABSTRACT TRUNCATED AT 250 WORDS)

Accumulation of Wall-Associated Peroxidases during Wound-Induced Suberization of Flax

Flax plants deposit ligno-suberin material around wounds. The deposition of this material is first detected histochemically at 3–4 days after wounding. Preceding this, at 2 days after wounding, the levels of ionically bound and covalently bound but Driselase-solubilized wall peroxidase activity are approximately four-fold higher than the control samples from unwounded tissue. Although this increase in wallassociated peroxidase activity is accompanied by an increase in the ability of the wall-associated enzymes to oxidize reduced nicotinamide adenine dinucleotides (NADH) to form hydrogen peroxide (H2O2), the ratio of NADH oxidase to peroxidase activities is lower than the unwounded control samples. These figures suggest that wounding does not result in the accumulation of wall-associated peroxidases that have a particular affinity for the generation of H2O2. It is intriguing, therefore, that cell wall preparations from wounded tissue at 2 days have gained the ability to oxidize peroxidase substrates in the absence of added H2O2. This suggests that these preparations can either generate their own H2O2 or contain covalently bound wall oxidases. An examination of peroxidase isozymes present in the wall-associated fractions suggests that the wound healing process is accompanied by increases in the abundance of specific cationic and anionic isozymes. Some of the cationic peroxidases isozymes can also oxidise peroxidase substrates in the absence of H2O2. The significance of the production of these oxidase/peroxidase isozymes during wound healing is discussed.

Association of Calcium and Calmodulin to Peroxidase Secretion and Activation

Summary The effect of the addition of EGTA, chlorpromazine, or trifluoperazine to the medium of cultured peanut cells for 14 d caused alterations in the patterns of released proteins. The observations on the peroxidases, identified amongst these proteins, suggested that the cationic peroxidase (C.PRX) is probably calcium controlled and that the anionic peroxidase (A.PRX) is under control of calmodulin. These suggestions are further supported by in vitro experiments with EGTA lnormal enzyme activity of C.PRX at (A407nm = 0.395 x 106; Umol = 5.36 x 1011) to (A404-407nm = 0.467 x 106; Umol = 1.07 x 1012). Subsequent reconstitution of the enzyme with calcium restores activity to the basal level. Cadmium is capable at physiological ranges (µM) to substitute effectively for the calcium in C.PRX since it restores the enzyme activity as well as calcium does. Since the anionic isozyme is not so affected, it suggests a major difference between peroxidase isozymes.

Alterations in surface-associated peroxidases during in vitro root development of explants of Linum usitatissimum

Hypocotyl explants of Linum usitatissimum were induced to form roots without an intermediate eallus phase by incubation on a defined medium. Loosely bound and ionically bound surface-associated proteins were extracted from the explants during root development by sequential vacuum infiltration using distilled water and 100 mM calcium chloride solution. The ionically extracted samples generally had higher peroxidase activity than the secreted samples, but both had reached maxima after 28 days culture. In contrast, the secreted samples were more able to oxidise indole-3-acetic acid (IAA) than the ionically-extracted samples. After 14 days culture the peroxidase and IAA-oxidase activities of the two samples were approximately equal, but by 35 days the secreted sample was twice as effective in oxidising IAA as the ionically extracted sample. The results suggest an accumulation of a loosely associated IAA-oxidase/peroxidase on the surfaces of the explants during root growth and development. Five anionic (A1–A5) and five cationic (C1−C5) isozymes were identified using non-denaturing PAGE. All five anionic isozymes were present throughout the development of roots and became more abundant from 14 days to 35 days culture. In contrast, root development was accompanied by a reduction in the levels of cationic isozymes that are characteristic of hypocotyl tissue. Two cationic isozymes (C3 and C4) were exclusively present during the early phases of root development (14 days) and the other three cationic isozymes were present at 14 days, dropped in abundance at 21 days and then recovered to higher levels after 35 days. The possible roles and consequence of these cationic isozymes and the significance of their removal from the explant surface during root development is discussed.

Changes in cell wall-associated peroxidases during the lignification of flax fibres

Cell wall-associated peroxidases were extracted selectively from fibre-bearing tissue dissected from the stem of Linum usitatissimum and their activities correlated with the extent of fibre lignification. The onset of fibre lignification was accompanied by increases in the levels of the peroxidases both ionically and covalently bound to the wall. However, the rise in the levels of covalently bound wall peroxidases was more dramatic. A portion of the covalently bound peroxidases could be isolated for study by solubilization with the glycanase mixture, driselase, whilst others remained firmly attached to the wall residue. The ability of driselase-solubilized wall peroxidases to oxidize NADH to form hydrogen peroxide rose sharply as the fibre cells became progressively lignified. This suggests that this process is accompanied by an increase in peroxidases covalently bound to the wall that have an affinity for the generation of hydrogen peroxide. A survey of the peroxidase isozymes present in both fractions using non-denaturing polyacrylamide gel electrophoresis identified a number of cationic and anionic isozymes that may have specific roles in the lignification of flax fibres.

Alterations in Surface-Associated Peroxidases during Callus Development and Shoot Formation in Explants of Linum usitatissimum

Two commercial cultivars of Linum usitatissimum differed in their ability to form callus and then undergo shoot formation on inductive media. The linseed cultivar, Antares, produced a greater amount of callus at an earlier stage than the fibre cultivar, Belinka. After 28 days of incubation, explants of Antares formed large calli, of which over 80 % had developed a shoot-like structure. At the same stage, Belinka calli were smaller and only 30–40 % had developed shoot-like structures. Surface-associated peroxidases were extracted from calli at stages throughout their development by sequential vacuum infiltration/elution with water, followed by 100 mM CaCl2. This procedure gave samples of ‹secreted› and ‹ionically-bound› surface-associated peroxidases. These extracts exhibited activity against the peroxidase substrates o-dianisidine, tetramethylbenzidine and guaiacol. Total peroxidase activity of both cultivars was generally highest after 14 days when the explants were developing callus. After this point, the activity dropped to a minimum after 21 days and recovered to near initial levels between 28 and 35 days when shoot formation was in progress. These two stages of differentiation, stem explant to callus and callus to shoot, may involve the production of specific peroxidases. Using non-denaturing PAGE, seven cationic isozymes (C1-C7) and five anionic isozymes (A1-A5) were detected in extracts of Antares and Belinka. Of these, one cationic isozyme (C6) and one anionic isozyme (A1) were exclusively present in the early samples (14 and 21 days) during the dedifferentiation of explant to callus. Other isozymes were either absent in early samples (e.g. C5-Antares; C1-Belinka) or increased markedly in later stages (e.g. C7, C2 and C5 — Antares and Belinka) during differentiation of callus to shoots. Possible functions for these differentiation-specific peroxidases are discussed.

Anionic peroxidase catalysed ascorbic acid and IAA oxidation in the presence of hydrogen peroxide: A defence system against peroxidative stress in peanut plant

In vitro experiments show that anionic peroxidase has a higher affinity for both ascorbic acid and IAA and a greater V max than cationic peroxidase in the oxidation reaction with the presence of H 2O 2. The peanut hypocotyls cultured in a medium with double amount of ascorbic acid (0.4 mg 1 −1) and IAA (4.0 mg 1 −1) show an increase of 104.3% and 128.4%, respectively, in anionic isozyme compared to the control, and only an increase of 22.6% and 12.8% in cationic isozyme. Treatment of entire plants with H 2O 2 (7.5%, v/v) demonstrated that a higher per cent of anionic isozyme over cationic is induced. These results suggest that cytoplasm-located anionic peroxidase probably plays a key role in the defence system against peroxidative stress through the detoxification of H 2O 2 during the processes of peroxidation of ascorbic acid and IAA.

A novel model to assess developmental toxicity of dihaloalkanes in humans: bioactivation of 1,2-dibromoethane by the isozymes of human fetal liver glutathione S-transferase.

Glutathione S-transferase (GST) isozymes from human fetal liver (16-18 weeks gestation) were purified by affinity chromatography followed by ion-exchange high performance liquid chromatography (HPLC). The purified isozymes were used to investigate toxicity of 1,2-dibromoethane(EDB) in an in vitro model of rat embryos in culture as passive targets. At least five isozymes of GST were found in the human fetal liver. Two anionic forms [pI values 5.5 (P-2) and 4.5 (P-3)] and one basic form [pI value 8.7 (P-6)] were clearly separated. The presence of two near-neutral forms was also identified. All the isozymes of the human fetal liver GSTs tested metabolized EDB (specific activities were 2.1, 7.0, and 2.0 mumol of GSH consumed/min/mg protein for P-2, P-3, and P-6 isozymes, respectively). Covalent binding of EDB to DNA and protein was 144% and 212% higher, respectively, with the P-3 anionic isozyme when compared to the P-6 basic isozyme of GST. No covalent binding to either protein or DNA was observed with the P-2 isozyme. EDB bioactivation by the GST isozyme P-3 (15 units; 1 unit = 1 nmol of GSH consumed/min) resulted in toxicity to cultured rat embryos. Significant reductions of crown rump length, yolk sac diameter, and the composite score of morphological parameters (Brown and Fabro method) were observed. The central nervous system, optic and olfactory systems, and the hind limb were most significantly affected. The results of this investigation suggest that EDB may be classified as a suspected developmental toxicant in humans.

Immunological Assays on the Relation of Culture Medium Peroxidases to Those in Fractions Isolated from Peanut Cells

Summary Through the means of Ouchterlony gel diffusion, Western blot and competitive ELISA it was determined that A. PRX or C. PRX from culture medium and their corresponding isozymes from the cytoplasm and cell wall have a higher than 80% similarity. Quantitative assays indicated that the cell-wall fractions were largely represented by the major cationic peroxidase isolated from spent medium of peanut cells. Conversely the cytoplasmic- or soluble-cell fraction was mainly represented by the anionic isozyme isolated from that medium. Consequently the enriched medium peroxidases may be used for studies of events in the cytoplasm and cell wall.