Cationic Isozyme Research Articles

Enhanced chemiluminescence: A sensitive analytical system for detection of sweet potato peroxidase

3-(10'-Phenothiazinyl)propane-1-sulfonate (SPTZ) was shown to be a potent enhancer of anionic sweet potato peroxidase (aSPP)-induced chemiluminescence. The optimal conditions for aSPP-catalyzed oxidation of luminol were investigated by varying the concentrations of luminol, hydrogen peroxide, Tris, and SPTZ as well as the pH values of the reaction mixture. Addition of 4-morpholinopyridine (MORP) to the reaction mixture markedly increased the light intensity. Using SPTZ and MORP together enhanced the effect 265 times. The lower detection limit (LDL) of SPP was 0.09 pM, approximately in 10 times lower than that for the cationic isozyme c of horseradish peroxidase/4-iodophenol system. It was shown that aSPP in the presence of SPTZ produced a longer lasting chemiluminescent signal.

Structural Influence of Calcium on the Heme Cavity of Cationic Peanut Peroxidase as Determined by 1H-NMR Spectroscopy

The cationic isozyme of peanut peroxidase (CPRx) is one of many peroxidases which requires calcium for enzyme activity. It has been previously shown that it requires 2 mol calcium to coordinate to 1 mol CPRx, and its related peroxidases from the basidiomycete Phanerochaete chrysosporium (LiP) and isozyme C of horseradish (HRPc). X-ray crystallographic studies of LiP have shown that calcium is ligated near the C-terminus of helices proximal and distal to the heme, where it has been suggested to maintain the active site. To determine if such a mechanism was possible in CPRx, high resolution 1H-NMR spectroscopy was used to study the effect of calcium on the environment of its heme group and the coordinating histidine residues. The low-spin cyano complex of the enzyme (CPRxCN) was studied in order to assign the majority of the resonances arising from the protons in the heme pocket in both the presence and absence of bound calcium ions using two dimensional nuclear Overhauser effect spectroscopy (NOESY). The two calcium ions present in CPRxCN were removed by a non-denaturing method and a calcium titration was performed and monitored by 1H-NMR spectroscopy. These studies showed that the binding of both calcium ions in CPRx influenced the heme environment in a similar manner (Kd = 0.1 microM). In particular, calcium-dependent changes in several heme resonances and the proximal and distal histidine residues suggest that calcium binding to CPRx causes some reorientation of these residues with respect to the active site.

Cadmium-Induced Inhibition of Apoplastic Ascorbate Oxidase in Barley Roots

The effect of excess cadmium (Cd: 0.0, 0.25, 0.5, 1.0 and 2.0 mM) on growth and ascorbate oxidase (AO) activity was investigated in barley (Hordeum vulgare L. cv. Jubilant) roots. The study employed a filter-paper technique to germinate and grow the germinating seeds following imbibition with respective Cd treatments for 4 h at 25 °C in darkness. Cd was required at 1.0 mM to affect 50% root growth inhibition 72 h after the treatment. This Cd-induced root growth inhibition was accompanied by a corresponding loss of plasma membrane integrity in root cells as evaluated by Evans blue uptake. Excess Cd (1.0 and 2.0 mM) significantly inhibited the AO activity in all the analysed fractions of barley roots such as extracellular, soluble, cell wall (CW)- and membrane-bound fractions. AO was localized in the apoplast, and its highest specific activity was detected in the CW II fraction obtained by extraction with 1.0 M NaCl from purified cell walls. The analysis of AO isozyme profile showed that besides the reduced activity of two anionic and two cationic isozymes, one cationic AO isozyme was activated during excess Cd treatment, which could be detected in cell wall fractions CW II, III and IV.

Comparative study on glutathione transferases of rat brain and testis under the stress of phenobarbitol and β-methylcholanthrene

A comparative study was made on the tissue specific expression of glutathione transferases (GST) in brain and testis after exposure of rat to phenobarbitol (PB) and b-methylcholanthrene (MC). Glutathione transferases, a family of multifunctional proteins are involved in intracellular transport processes and in detoxication of electrophilic xenobiotics by catalyzing reactions such as conjugation, isomerization, reduction and thiolysis. On purification, the yield of GST proteins by affinity chromatography was 39% in testis and 32% in brain. The affinity purified testis GSTs were resolved by chromatofocusing into six anionic and four cationic isozymes, and in brain glutathione transferases were resolved into four anionic and three cationic isozymes, suggesting the presence of multiple isozymes with Yc, Yb, Ybeta and Ydelta in both of them. In testis and brain, these isozymes at identical pI values showed variable functions with a battery of substrates and the cationic isozymes of brain and testis showed identical properties in CHP (cumene hydroperoxide) at pH values of above 7.0. Substrate specificity studies and immunoblot analysis of testis and brain proteins revealed that they play a predominant role in the detoxication of phenobarbitol or beta-methylcholanthrene. Expression of the isozymes in testis and brain on exposure to PB and MC indicated elevated subunit variation. In both testis and brain, Ydelta of pi class was expressed on PB treatment and Yc of alpha class and Ybeta of mu class was expressed in MC treated testis and only Yc was predominantly expressed in MC treated brain. Thus these subunits expression is considered as markers for carcinogenesis and specific to chemical toxicity under phenobarbitol and beta-methylcholanthrene stress.

Effect of phenobarbital on the induction of glutathione S-transferases in rat testis

Glutathione S-transferases are a family of multifunctional proteins involved in intracellular transport processes and drug detoxication. In rats, these enzymes are dimeric proteins, and exist as cytosolic and microsomal proteins. The affinity purified rat testicular glutathione S-transferases are comprised of four subunits, Yc of α class, Yb and Yβ of μ class and Yδ of π class. On chromatofocusing, they were resolved into six anionic and four cationic isozymes. The cationic isozymes were found to be abundant. All these isozymes on electrophoresis were found to contain heteromers except for two isozymes. The expression of individual subunits and their activity was elevated on treatment with multiple doses of phenobarbital in rat testis. Among all of these, according to the immunological studies, Yδ, a π class glutathione S-transferase, was induced predominantly in response to phenobarbital.

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.

Changes in chalazal cell walls and in the peroxidase enzymes of the crease region during grain development in barley.

In an investigation of the role of peroxidase enzymes in the differentiation of the tissues of the crease region of barley, plants of winter barley cv. Halcyon were grown from anthesis onwards in controlled conditions at a constant temperature of 16 degrees C. Four ears were harvested at 2-d intervals from 6 d after anthesis (daa) until 50 daa. Grains from mid-ear were used for (i) fresh and dry weight determinations, (ii) extraction of crease tissue for the determination of peroxidase activity and for the separation of isozymes of peroxidase by isoelectric focusing (IEF) and (iii) detection of lignin and suberin in the tissues of the crease using autofluorescence and cytochemistry. Peroxidase activity was located histochemically in the crease tissue of cv. Chariot. Scanning electron microscopy studies were carried out on developing grains of cv. Blenheim. Maximum grain water content was achieved at 14 daa. Lignin and suberin were detected in the walls of the chalazal cells from 18 daa onwards. No changes in the staining of chalazal cell walls were detected at the end of grain filling (32 daa), but loss of autofluorescence and staining were observed at 42 daa, just prior to the final, rapid phase of grain dehydration. Peroxidase activity per fresh weight of crease tissue was high at 6 daa and low at 22 daa. It was also low between 32 and 40 daa, but it rose again from 42 daa onwards. IEF demonstrated that both anionic and cationic isozymes of peroxidase were present in crease tissue, the pattern of bands showing some marked changes during the course of grain development.

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.

Tissue Specificity of `Chandler' Strawberry Peroxidase Isozymes

Peroxidase activity in extracts from freeze-dried tissue of Fragaria × ananassa Duch. cv. Chandler was highest in tissue-cultured (TC) plants, followed by field-grown (FG) and lowest in greenhouse (GH) plants. Among tissue types, activity was highest in petioles, with leaves second highest. Fruit, root, and crown tissue all exhibited low or no activity. When subjected to isoelectric focusing (IEF), petiole tissue extracts exhibited more isozymes than extracts from other organs regardless of staining substrate. Using 4-chloro-1-naphthol and H2O2 as substrates, anionic and cationic isozymes were observed in TC petiole extract with nine isozyme bands ranging in pI from 3.9 to 9.5. In TC leaf extract an isozyme at pI 7.4 was observed that was not present in other organ extracts when H2O2 and benzidine, p-phenylenediamine or 3-amino-9-ethylcarbazole were used as substrates. Specific isozymes and number of isozymes varied according to plant organ and developmental stage. Mature leaves and over-ripe fruit appeared to exhibit more activity and a larger number of isozymes than developing tissues of those plant organs.

Construction and characterization of a manganese-binding site in cytochrome c peroxidase: towards a novel manganese peroxidase.

Manganese-binding sites are found in several heme peroxidases, namely manganese peroxidase (MnP), chloroperoxidase, and the cationic isozyme of peanut peroxidase. The Mn-binding site in MnP is of particular interest. Oxidation of Mn(II) to Mn(III) is a key step in the biodegradation of lignin, a complex phenylpropanoid polymer, as well as many aromatic pollutants. Cytochrome c peroxidase (CcP), which is structurally homologous to MnP despite a poor sequence homology, does not bind manganese. Thus, engineering a Mn-binding site into CcP will allow us to elucidate principles behind designing metal-binding sites in proteins, to understand the structure and function of this class of Mn-binding centers, and to prepare novel enzymes that can degrade both lignin and other xenobiotic compounds. Based on a comparison of the crystal structures of CcP and MnP, a site-directed triple mutant (Gly41-->Glu, Val45-->Glu, His181-->Asp) of residues near the putative Mn-binding site in CcP was prepared and purified to homogeneity. Titrating MnSO4 into freshly prepared mutant CcP resulted in electronic absorption spectral changes similar to those observed in MnP. The calculated apparent dissociation constant and the stoichiometry of Mn-binding of CCP were also similar to MnP. Titration with MnSO4 resulted in the disappearance of specific paramagnetically shifted nuclear magnetic resonance spectroscopy signals assigned to residues close to the putative Mn-binding site in the mutant CcP. None of the spectral features were observed in wild-type CcP. In addition, the triple mutant was capable of oxidizing Mn(II) at least five times more efficiently than the native CcP. A Mn-binding site has been created in CcP and based on our spectroscopic studies the designed Mn-binding site is similar to the Mn-binding site in MnP. The results provide a basis for understanding the structure and function of the Mn-binding site and its role in different heme peroxidases.

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.

Purification and Characterization of Glutathione S-Transferase of Murine Ovary and Testis

Recent studies have indicated that sex hormones may regulate expression of murine glutathione 5-transferase (GST) isozymes. Therefore, we have purified and compared GST isozymes of murine ovary and testis, two tissues with markedly different hormonal milieu. Isoelectric profiles of the GST isozymes of both these tissues were found to be closely similar. Both expressed one α-class GST (p I 9.8), one π-class GST (p I 8.9), and three μ-class GSTs (p I 8.5, 7.9, and 6.7). In addition, an isozyme (p I 5.8) corresponding to the rat GST 8-8 was also expressed in both these tissues. Total GST protein/g tissue was about 1.7-fold more abundant in testis. The specific activities of the cationic isozymes of testis were 1.2- to 2.4-fold higher as compared to those of ovaries. On the other hand, the specific activities of the anionic testicular isozymes were 6.4- to 10-fold higher compared to the corresponding ovarian isozymes. Structural properties including the N-terminal sequences of the testicular isozymes were indistinguishable from those of their ovarian counterparts. The N-terminal sequence of the π-class GST of both tissues was similar to that of mouse liver GST π. The three μ-class GSTs of testis and ovary arise from the dimeric combinations of two subunits whose N-terminal sequences determined up to 24 residues were similar to those of mouse liver GST subunits μ1 and μ2. Although testicular and ovarian isozymes were structurally similar, K cat values of some of the testicular isozymes were up to 10-fold higher than those of the corresponding ovarian isozymes. The substrate specificities were also significantly different for the corresponding isozymes of testis and ovary.

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.

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.

Peanut peroxidase and the anionic component

Secreted peroxidase (EC 1.11.1.7) from cultured peanut (Arachis hypogaea L.) cells has two main isozymes, one cationic and one anionic. The cationic isozyme has been studied intensively owing to its greater availability. However, the several-fold lower yield of anionic peroxidase has limited study of this isozyme. In the current investigation, the yield of this isozyme from the spent medium is quantified. In addition, feedback by cationic peroxidase on its own synthesis is investigated. Key words: peanut, peroxidase, anionic, cationic, yield, feedback.

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.

Glutathione S-transferases of mouse liver: sex-related differences in the expression of various isozymes

Sex-related differences in the expression of glutathione S-transferase (GST) isozymes of mouse liver have been described. There were no apparent qualitative differences in the isoelectric focusing profiles of the GST isozymes from male and female mouse liver. Both male and female mice have at least four GST isozymes in their liver with p I values of 9.8, 8.7, 6.4 and 5.7. Kinetic, immunological, and structural properties including the N-terminal region amino acid sequences of these isozymes have been determined and they have been classified into α, μ, and π classes. The most cationic isozyme (p I 9.8) belongs to the α class and is comparatively more abundant in female liver. The isozyme having p I 8.7 belongs to the π class and is more abundant in male liver. The μ class GST p I 6.4 as well as the isozyme having p I 5.7 which corresponded to the a class isozyme GST 8-8 of rat liver were more abundant (about 1.5-fold) in male mouse liver as compared to the female. Interestingly, present studies reveal sex-related differences in the heat stabilities of the α and π class GSTs of mouse liver. The α class GST p I (9.8) isolated from female mouse liver was more thermostable as compared to the corresponding enzyme from male mouse liver. On the contrary, the π class GST (p I 8.7) from male mouse was more thermostable as compared to the corresponding enzyme from the female mouse.

Characterization of two forms of cationic peroxidase from cultured peanut cells.

Two forms of cationic peroxidase from peanut cells were differentiated by concanavalin A affinity chromatography. They differed in molecular mass as well as concanavalin A binding, leading to the initial suggestion that they represented two isozymes of peroxidase. However, similar values for the specific activity, Soret absorption, calcium content, and peptide molecular mass were observed for each of the forms. Therefore, the binding and nonbinding fractions most likely represent two molecular forms of cationic peanut peroxidase, rather than two distinct cationic isozymes. The difference between these two forms is discussed in terms of glycosylation. Through the amino acid sequence analysis of the formic acid treated peptide, the cationic isozyme has been shown to be identical in amino acid sequence to the cDNA clone PNC1.

Preliminary crystallographic study of peanut peroxidase.

The cationic isozyme of peroxidase isolated from suspension cultures of peanut cells is a heme-containing and calcium-dependent glycoprotein having four covalently attached oligosaccharide chains. Attempts were made to crystallize the glycoprotein for X-ray diffraction analysis, and these have met with some success. Crystals have now been grown that are suitable for a full three-dimensional structural analysis. The crystals are thin plates and we have shown them to be of the orthorhombic space group P2(1)2(1)2(1) with a = 48.1, b = 97.2, c = 146.2 A. The crystals diffract to beyond 2.8 A resolution, appear to be stable to lengthy X-ray exposure, and contain two molecules of 40,000 daltons each in the asymmetric unit.