Superoxide dismutase and peroxidase are coordinately regulated in differentiated and transformed tissues of Nicotiana tabacum

We used a series of normal and Agrobacterium-transformed, bacteria-free tobacco tissue cultures which differ in their levels of histodifferentiation to test the relationship of superoxide dismutase, peroxidase, and catalase to oncogenic transformation and differentiation. When compared with normal callus, tumor callus contained reduced levels of both superoxide dismutase and peroxidase, and a reduced number of isozymes of both enzymes. Teratomas characterized by limited but abnormal differentiation showed increases in superoxide-dismutase activity and isozymes, but levels of peroxidase activity lower than those found in normal callus despite an increase in the number of peroxidase isozymes. A similar disparity between low peroxidase activity and high isozyme number in the shoot suggests that there are increased levels of peroxidase inhibitors or of molecules which interfere with the spectrophotometric assay for peroxidase in more differentiated tissues. As judged by the number of isozymes of both superoxide dismutase and peroxidase in each tissue, the following conclusions are warranted: first, tobacco copper/zinc superoxide dismutases and peroxidases are encoded in several duplicated loci which are regulated independently. Second, transformation is associated with a decrease in both the specific activity and isozyme number of superoxide dismutase. Third, the partial release from the total inhibition of expression of differentiated function exhibited by teratoma is associated with an increase in both the activity and isozyme number of superoxide dismutase. Finally, the expression of superoxide dismutase and peroxidase isozymes appears to be coordinated during differentiation in a manner that is consistent with their role in an integrated mechanism for the removal of reduced oxygen species.     

Appearance of peroxidase isozymes in floral‐initiated shoot apices of Pharbitis nil

A novel biochemical assay system for detecting the early stage of flowering is reported. Peroxidase isozymes in shoot apices of Pharbitis nil plants that had been exposed to flower‐inducing or non‐inducing conditions were analyzed by isoelectric focusing in polyacrylamide gels and activity staining for peroxidase. Several isozymes with pH 8.5–8.8 appeared for the first time 7 days after the beginning of short‐day treatment, but not after nightbreak (non‐inducing) treatment. When shoot tips were cultured in vitro, these same isozymes also appeared after short‐day treatment but not after night‐break treatment. The extent of the appearance of these isozymes was reduced by exposure to high or low temperature during the inductive dark period and removal of cotyledons after the inductive dark period. Such treatments also reduced the extent of flowering. The appearance of an isozyme with pH 8.5 was more closely correlated with flowering than that of the other isozymes. From these results, the appearance of this peroxidase isozyme in shoot apices is discussed as a biochemical marker of flowering in intact plants and in cultured shoot tips.     

On the multiplicity of rat liver glutathione S-transferases

Rat liver glutathione S-transferases have been purified to apparent electrophoretic homogeneity by S-hexylglutathione-linked Sepharose 6B affinity chromatography and CM-cellulose column chromatography. At least 11 transferase activity peaks can be resolved including five Yb size homodimeric isozymes, two Yc size homodimeric isozymes, one Ya homodimeric isozyme, one Y alpha homodimeric isozyme, and two Ya-Yc heterodimeric isozymes. Distribution of the GSH peroxidase activity among the CM-cellulose column fractions suggests the existence of further multiplicity in this isozyme family. Substrate specificity patterns of the Yb subunit isozymes revealed a possibility that each of the five Yb-containing isozymes is composed of a different homodimeric Yb size subunit composition. Our findings on the increasing multiplicity of glutathione S-transferase isozymes are consistent with the notion that multiple isozymes of overlapping substrate specificities are required to detoxify a multitude of xenobiotics in addition to serving other important physiological functions.     

Tissue specificity of tobacco peroxidase isozymes and their induction by wounding and tobacco mosaic virus infection

Peroxidases (EC 1.11.1.7) have been implicated in the responses of plants to physical stress and to pathogens, as well as in a variety of cellular processes including cell wall biosynthesis. Tissue samples from leaf, root, pith, and callus of Nicotiana tabacum were assayed for specific peroxidase isozymes by analytical isoelectric focusing. Each tissue type was found to exhibit a unique isozyme fingerprint. Root tissue expressed all of the detectable peroxidase isozymes in the tobacco plant, whereas each of the other tissues examined expressed a different subset of these isozymes. In an effort to determine which peroxidase isozymes from Nicotiana tabacum are involved in cell wall biosynthesis or other normal cellular functions and which respond to stress, plants were subjected to either wounding or infection with tobacco mosaic virus. Wounding the plant triggered the expression of several cationic isozymes in the leaf and both cationic and anionic isozymes in pith tissue. Maximum enzyme activity was detected at 72 hours after wounding, and cycloheximide treatment prevented this induction. Infection of tobacco with tobacco mosaic virus induced two moderately anionic isozymes in the leaves in which virus was applied and also systemically induced in leaves which were not inoculated with virus.     

Heterogeneity and regulation of manganese peroxidases from Phanerochaete chrysosporium.

Lignin and Mn peroxidases are two families of isozymes produced by the lignin-degrading fungus Phanerochaete chrysosporium under nutrient nitrogen or carbon limitation. We purified to homogeneity the three major Mn peroxidase isozymes, H3 (pI = 4.9), H4 (pI = 4.5), and H5 (pI = 4.2). Amino-terminal sequencing of these isozymes demonstrates that they are encoded by different genes. We also analyzed the regulation of these isozymes in carbon- and nitrogen-limited cultures and found not only that the lignin and Mn peroxidases are differentially regulated but also that differential regulation occurs within the Mn peroxidase isozyme family. The isozyme profile and the time at which each isozyme appears in secondary metabolism differ in both nitrogen- and carbon-limited cultures. Each isozyme also responded differently to the addition of a putative inducer, divalent Mn. The stability of the Mn peroxidases in carbon- and nitrogen-limited cultures was also characterized after cycloheximide addition. The Mn peroxidases are more stable in carbon-limited cultures than in nitrogen-limited cultures. They are also more stable than the lignin peroxidases. These data collectively suggest that the Mn peroxidase isozymes serve different functions in lignin biodegradation.     

Isozyme studies in Indian mustard (Brassica juncea L.)

Summary A comparative study of peroxidase and esterase isozymes was carried out at five developmental stages of siliqua in order to characterize twelve genotypes of Indian mustard. The studies showed nearly the same number of isozyme bands at every stage for peroxidase and a varying number of isozyme bands for esterase. The appearance and disappearance of bands, along with their intensity scores, indicated the role of different isozymes at different stages of siliqua development. It has been ascertained that these patterns, especially the intensity scores, can be successfully used to characterize different Indian mustard genotypes.     

Multiple molecular forms of serum α1-antitrypsin

Total soluble proteins, peroxidase, and peroxidase isozymes were examined in polyploid series of fern gametophytes and sporophytes. A distinctive pattern of protein bands was associated with gametophytes and sporophytes and the pattern did not vary within each phenotype with increases in the genome. Peroxidase activity per cell increased in direct proportion to increases in the genome and was determined to be gene dosage related. Slight differences in the patterns of peroxidase isozyme bands were associated with increases in the chromosome complement in both series of plants, but major variations were found between gametophyte and sporophyte. Quantitative analysis of peroxidase activity in each band revealed both increases and decreases in individual isozymes as ploidy increased. These findings suggest the involvement of regulatory mechanisms controlling isozyme activity.     

Isozymes as biochemical and cytochemical markers in embryogenic callus cultures of maize (Zea mays L.)

Isozyme analyses were carried out on protein extracts of non-embryogenic and embryogenic callus fromZea mays L., using polyacrylamide gel electrophoresis. We examined the isozyme patterns of glutamate dehydrogenase, peroxidase and acid phosphatase for their utility as biochemical markers of maize embryogenic callus cultures. These isozyme systems were also used to examine possible correlations between isozymes and different stages of regeneration. The zymograms of peroxidase and glutamate dehydrogenase differed for non-embryogenic and embryogenic callus. Further, some isozymes were correlated with the morphological appearance of the tissue while others seemed to be involved with the duration of the culture period. Using the same enzyme assays on fresh tissue samples we were able to test the three enzymes as cytochemical markers in embryogenic cultures. Glutamate dehydrogenase proved to be most successful to discriminate embryogenic from non-embryogenic cells.     

The role of peroxidase isozymes in resistance to wheat stem rust disease

In common with other disease situations, rust-resistant wheat leaves show a large increase in peroxidase activity during infection. Peroxidase isozymes from healthy or infected lines of wheat (Triticum aestivum L.) near isogenic for resistance and susceptibility to race 56 of Puccinia graminis tritici were separated by gel electrophoresis and the activity of each was estimated by photometric scanning. In order to ensure that the activity of isozymes observed on gels reflected the changes found in peroxidase enzymes assayed spectrophotometrically in extracts, a study was made of extraction procedures, substrates, and reaction conditions for both types of enzyme measurements. Of the 14 isozymes detected in both healthy and infected leaves, increases in only 1 (isozyme 9) were associated consistently with the development of resistant disease reaction at 20 C. Additional evidence was obtained to show that this isozyme can account for the increased peroxidase activity observed in extracts from resistant plants. When plants with high induced peroxidase activity due to resistance at 20 C were treated with ethylene or transferred to 25 C, they reverted to complete susceptibility. However, the disease-induced activity of isozyme 9 did not fall. The data suggest that, in this case, the association of peroxidase with resistance was a consequence of, not a determinant in, resistance.     

苜蓿组织培养中球形胚发生时特异蛋白质和同工酶分析

试验在苜蓿组织培养中,对球形胚形成过程中特异蛋白质表达的模式、过氧化物酶及酯酶同工酶酶谱变化进行研究,结果表明：苜蓿组织培养中从胚性愈伤组织到球形胚发育的进程中,顺序消失和出现了11种中小分子量多肽;过氧化物酶同工酶酶谱发生了显著的变化;酯酶同工酶酶谱变化不大,但其总活力对于维持体细胞胚胎发生是必须的。     

On the determination of isozyme levels in preparations containing cytoplasmic and mitochondrial aspartate aminotransferase.

A spectrophotometric assay has been developed for the determination of the content of each isozyme of aspartate transaminase (L-aspartate:2-oxoglutarate aminotransferase, EC 2.6.1.1) in physiological fluids or tissue extracts. The methods relies on the ability of adipate, at low pH and ionic strength to inhibit the cytoplasmic isozyme but not the one from mitochondria. Two assays are necessary, one at pH 8.0 which measures the content of both isozymes and another at low pH which measures primarily the amount of mitochondrial isozyme. Results obtained by this simple procedure match those in which each isozyme is inhibited by its antibody. The validity of the results obtained by the new method was tested at different ratios of cytoplasmic:mitochondrial isozyme and with tissue extracts. Since the amounts of each isozyme determined by radial immunodiffusion match those values gathered by following enzymatic activity, it is concluded that the quantity of each isozyme obtained from its respective catalytic activity must represent the total protein content of each isozyme in a given sample.     

Lignin peroxidase from Phanerochaete chrysosporium. Molecular and kinetic characterization of isozymes

Five isozymes of lignin peroxidase from Phanerochaete chrysosporium were purified and their physical, molecular and kinetic properties determined. The isozymes differ from each other in terms of their isoelectric point, molecular mass, sugar content, spectral characteristics, substrate specificity and stability. The N-terminal sequence of amino acids was different for each isozyme suggesting they are different gene products. The isozyme with the highest carbohydrate level was most sensitive to changes in environmental factors. The kinetic behaviour of the isozymes varied clearly when tert-butyl hydroperoxide instead of hydrogen peroxide was used as the oxidant. Two out of five isozymes had very similar substrate specificity. The results are discussed in relation to the role which lignin peroxidase isozymes may play in lignin biodegradation.     

Alterations in peroxidase activity and peroxidase isozymes in virus-infected plants

Peroxidase activity and isozyme patterns were investigated in two leguminous species infected with viruses which produced either local necrotic or systemic chlorotic symptoms. Highest peroxidase activity was recorded when the hosts reacted to infection with necrotic local lesions. No new virus-specific isozymes were found as a result of infection, but some isozymes, apparently associated with senescence, appeared earlier in extracts from leaves showing necrosis than in extracts from healthy leaves, or from infected leaves showing only very mild chlorosis. Increase in peroxidase activity was accompanied by alteration in isozyme pattern.     

The Relationship Between Peroxidase Activity and Resistance to Sphaerotheca fuliginea in Melons

The levels of peroxidase activity in leaves of non-infected melon plants resistant to Sphaerotheca fuliginea (Schl. ex. Fr.) Poll. were considerably higher than those in the leaves of susceptible plants. After infection the ratio of peroxidase activity to that in non-infected plants reached a value of up to 47.5 in susceptible plants whereas in resistant plants it remained between 1 and 2. Changes in isozyme pattern were investigated by polyacrylamide gel electrophoresis. In non-infected resistant leaves slowly migrating isozymes were found. Their intensities increased with time following infection. These isozymes were absent in the susceptible leaves but appeared after the leaves had been infected with S. fuliginea. The role of these isozymes in the resistance mechanism is discussed.     

Isozymal differention between two species of Prosopis

The patterns of five multilocus isozyme systems were investigated in seed, shoot and cotyledon tissue of two species of mesquite, Prosopis glandulosa var. glandulosa and P. pallida. The isozymes of malate dehydrogenase, peroxidase, esterase, alcohol dehydrogenase and acid phosphatase from each of these tissues were analysed by starch gel electrophoresis and specific histochemical stains. In the case of each enzyme system examined, there were distinctly different isozymes which could be utilized to differentiate between these two species.     

Peroxidase Isozymes from Meloidogyne spp. and Their Origin

Two peroxidase isozymes (Ef 0.43 and 0.53) were detected by electrophoretic analysis in homogenates of Meloidogyne arenaria, M. hapla, M. javanica, and M. incognita females reared on tomato. No peroxidase isozymes were detected electrophoretically in homogenates of adult males, preparasitic larvae, or eggs. Peroxidase isozymes from females reared on bean, eggplant, or tobacco differed from those from females reared on tomato. Bean and eggplant populations had a single peroxidase isozyme each, respectively Ef 0.21 and 0.28. No peroxidase isozymes were detected in tobacco populations under the conditions used, although total activity assays did reveal low levels of peroxidase activity in homogenates of tobacco populations. The peroxidase isozymes detected in females reared on tomato or bean appear similar to the peroxidase isozymes present in root-knot galls, adjacent ungalled roots, and roots from uninoculated plants of the corresponding hosts. The probability is discussed that most of the peroxittase activity associated with Meloidogyne spp. females is of host origin.     

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.     

Multiple roles of phosphoinositide-specific phospholipase C isozymes

Phosphoinositide-specific phospholipase C is an effector molecule in the signal transduction process. It generates two second messengers, inositol-1,4,5-trisphosphate and diacylglycerol from phosphatidylinositol 4,5-bisphosphate. Currently, thirteen mammal PLC isozymes have been identified, and they are divided into six groups: PLC-beta, -gamma, -delta, -epsilon, -zeta and -eta. Sequence analysis studies demonstrated that each isozyme has more than one alternative splicing variant. PLC isozymes contain the X and Y domains that are responsible for catalytic activity. Several other domains including the PH domain, the C2 domain and EF hand motifs are involved in various biological functions of PLC isozymes as signaling proteins. The distribution of PLC isozymes is tissue and organ specific. Recent studies on isolated cells and knockout mice depleted of PLC isozymes have revealed their distinct phenotypes. Given the specificity in distribution and cellular localization, it is clear that each PLC isozyme bears a unique function in the modulation of physiological responses. In this review, we discuss the structural organization, enzymatic properties and molecular diversity of PLC splicing variants and study functional and physiological roles of each isozyme.     

Comparison of anionic with cationic peroxidase from cultured peanut cells

A cationic and an anionic peanut peroxidase were isolated to purity as shown by 2D electrophoresis. Amino acid analysis offered evidence for differences. Variations between the isozymes were also noted in a slight difference in the heme absorption maxima, specific enzyme activity and particularly in the relative amount of each in the suspension medium measured by the heme absorption. In contrast the two isozymes were at least partially similar in their structure as demonstrated by the crossreaction with the antisera. The percent crossreactions were used in turn to amend the calculation for the synthetic rate of each isozyme. In spite of the difference in amount secreted in the suspension medium, the in vivo biosynthetic rate of the two isozyme measured cellularly is much the same.