Cationic Isozyme Research Articles

Peroxidase-regulated elongation of segments from peanut hypocotyls

Alterations of isoperoxidases were studied in 1.0 cm segments from 3-day-peanut-seedling ( Arachis hypogaea L.) hypocotyls as they were cultured on Linsmayer and Skoog (LS) medium with or without the addition of 5 mM meta-fluoro-tyrosine (MFT). The alterations in peroxidase activity and relative protein levels were correlated with the growth inhibition by MFT, particularly for the cationic isozyme. A further direct link between growth and the peroxidase has been established by the addition of either antibodies or purified isozymes to the culture medium. The enhancement of growth by the antibodies and inhibition by the peroxidase isozymes strongly suggest that peroxidase is directly involved in growth.

Oxidation of tyrosine by peroxidase isozymes derived from peanut suspension culture medium and by isolated cell walls

A comparative study on tyrosine oxidation was made with a pure cationic and anionic peroxidase from peanut cell culture medium. The results showed that both isozymes possessed almost identical capacity to oxidize tyrosine to dityrosine, isodityrosine and polytyrosine with the main difference being the pH optimum (pH 4 for the anionic and pH 7 for the cationic isozyme). Variation of reaction time after 1.5 h incubation had little effect on the quantity and quality of the oxidation products. On the other hand, increase of enzyme units correspondingly increased tyrosine-oxidation. The removal of heme and carbohydrate moieties from the holoenzyme arrested the reaction thereby suggesting the role played by these moieties in stabilizing the active site of peroxidase isoenzymes. Isolated cell wall extracts catalyzed the tyrosine-oxidation equally well as the purified peroxidase. Even though polyclonal antibodies against anionic peroxidase inhibited the in vitro tyrosine reaction they did not affect the tyrosine oxidation by the cell walls, while the cationic antibodies did.

Immunological similarity of the cationic and the anionic peanut peroxidase isozymes

Antibodies against both the native and the deglycosylated cationic peanut peroxidase (C.PRX) were used to probe the structural relationship of this isozyme with its anionic counterpart. Not only the native but also the deglycosylated forms of the cationic and the anionic peroxidases reacted with both antibodies. The activity of the cationic isozymes was inhibited by anti-native C.PRX. Similar but nevertheless distinct immunodetection patterns resulted from reaction of the partially digested cationic and anionic peroxidase peptides with antibodies directed to the deglycosylated as well as to the native C.PRX, suggesting a similarity in their polypeptide structures.

Enzymes in atlatoxin B1 biosynthesis: Strategies for identifying pertinent genes

Recent work on the aflatoxin biosynthetic pathway is reviewed, with special emphasis on the enzymes of the late stages of the pathway involving conversion of sterigmatocystin (ST) to aflatoxin B1 (AFB1) through an O-methylsterigmatocystin intermediate. Two enzyme activities were discovered in subcellular fractions of cell-free extracts of a mutant strain of Aspergillus parasiticus (SRRC 163): 1) A post-microsomal methyltransferase (MT) catalyzed conversion of ST to OMST, and 2) a microsomal-associated activity (oxido-reductase) converted OMST to AFB1. The 168 KDa, anionic MT was purified to homogeneity and characterized (two subunits, 110 KDa and 58 KDa). Preliminary evidence indicated the presence of a cationic isozyme of the MT in mycelial extracts. The oxido-reductase has been partially purified and characterized. Polyclonal antibodies were prepared to the anionic MT and the enzyme's amino acid composition determined. A cDNA library has been constructed from mRNA isolated from Aspergillus parasiticus mycelia during the onset of AFB1 biosynthesis for the purpose of identifying the genes responsible for aflatoxin biosynthesis.

The Role of Selenium-dependent and Selenium-independent Glutathione Peroxidases in the Formation of Prostaglandin F2α

In recent years, growing evidence suggests that glutathione peroxidases (GSH-Pxs), both selenium-dependent GSH-Px (Se-GSH-Px) and selenium-independent GSH-Px (non-Se-GSH-Px) play an important role in the biosynthesis of prostaglandins and leukotrienes and in the regulation of key enzymes associated with the arachidonic acid cascade. The precise nature of their involvement in eicosanoid metabolism, however, is not yet completely understood. In the study reported here, we have systematically determined the catalytic efficiencies of Se-GSH-Px and non-Se-GSH-Px toward prostaglandin (PG) G2 (PGG2) and PGH2. Se-GSH-Px exhibited high catalytic activity for the reduction of PGG2 as indicated by Km and Vmax values of 12 microM and 78 mumol/min/mg, respectively, whereas PGH2 was found to be a poor substrate, an indication that Se-GSH-Px reduces the hydroperoxide moiety but not the endoperoxide moiety of PGG2. The kinetic constants of Se-GSH-Px toward PGG2 were comparable to those determined for such classical substrates as H2O2 and cumene hydroperoxide. In contrast to Se-GSH-Px, non-Se-GSH-Px associated with cationic isozyme II of glutathione S-transferases (GSTs) from sheep lung cytosol was very active in the conversion of PGH2 to PGF2 alpha with a Vmax of 960 nmol/min/mg and a Km of 77 microM. This study shows that PGF2 alpha formation by non-Se-GSH-Px occurred in a GSH-dependent reduction of either PGG2 or PGH2. When PGG2 was used as the substrate for non-Se-GSH-Px, a novel intermediate compound appeared and was later identified by several methods of structural analysis as 15-hydroperoxy PGF2 alpha. Thus, the reductive cleavage of the endoperoxide occurs faster than the 15-hydroperoxide reduction allowing 15-hydroperoxy PGF2 alpha to accumulate briefly. A study of GSTs from several different tissues and species indicated that the transformation of PG endoperoxides to PGF2 alpha is catalyzed specifically by GST isozymes, which contain Ya size subunits. This specificity of GST isozymes in PG biosynthesis, coupled with their tissue-specific expression, may be a mechanism by which the body modulates the type of PGs produced in these tissues. Also, these results suggest a possible interaction of Se-GSH-Px and non-Se-GSH-Px in the biosynthesis of PGF2 alpha.

Immunochemical relatedness of two peroxidase isozymes from peanut cell culture

The immunological relationships between a cationic and an anionic peroxidase from peanut cell suspension culture were probed with polyclonal antibodies raised against each and with a monoclonal antibody raised against the cationic isozyme. Partial identity of the two isozymes was established by cross-reactivities of the polyclonal antibodies directed to one peroxidase with the alternate isozyme. Some peptides from Western blots of the partially hydrolysed peroxidases were recognized by both antisera, even though the peptide maps of the two isozymes differed. In contrast, the monoclonal antibody reacted more specifically to the fragments derived from the cationic isozyme. The anti-anionic peroxidase serum was passed over a cationic peroxidase (C.PRX) – Sepharose affinity column to separate antibodies cross-reacting with C.PRX from those specific to the anionic isozyme. Deglycosylation of the cationic and the anionic peroxidases eliminated binding of the antibody fraction eluted from the C.PRX–Sepharose column to the deglycosylated forms of peroxidases. These data suggest (i) that complex structural relationships exist between the cationic and the anionic peanut peroxidases, (ii) that the carbohydrate moieties of the two isozymes are antigenically similar to each other, and (iii) that epitope mapping will help in characterizing further each isoperoxidase.Key words: peroxidase, isozymes, ELISA, immunoblotting, peptide mapping.

Differential expression of α, μ and π classes of isozymes of glutathione S-transferase in bovine lens, cornea, and retina

Isozyme characterization of glutathione S-transferase (GST) isolated from bovine ocular tissue was undertaken. Two isozymes of lens, GST 7.4 and GST 5.6, were isolated and found to be homodimers of a M r 23,500 subunit. Amino acid sequence analysis of a 20-residue region of the amino terminus was identical for both isozymes and was identical to GST ψ and GST μ of human liver. Antibodies raised against GST ψ cross-reacted with both lens isozymes. Although lens GST 5.6 and GST 7.4 demonstrated chemical and immunological relatedness, they were distinctly different as evidenced by their p I and comparative peptide fingerprint. A corneal isozyme, GST 7.2, was also isolated and established to be a homodimer of M r 24,500 subunits. Sequence analysis of the amino-terminal region indicated it to be about 67% identical with the GST π isozyme of human placenta. Antibodies raised against GST π cross-reacted with cornea GST 7.2. Another corneal isozyme, GST 8.7, was found to be a homodimer of M r 27,000 subunits. Sequence analysis revealed it to have a blocked amino-terminus. GST 8.7 immunologically cross-reacted with the antibodies raised against cationic isozymes of human liver indicating it to be of the α class. Two isozymes of retina, GST 6.8 and GST 6.3, were isolated and identified to be heterodimers of subunits of M r 23,500 and 24,500. Amino-terminal sequence analysis gave identical results for both retina GST 6.8 and GST 6.3. The sequence analysis of the M r 23,500 subunit was identical to that obtained for lens GSTs. Similarly, sequence analysis of the M r 24,500 subunit was identical to that obtained for the cornea GST 7.2 isozyme. Both the retina isozymes cross-reacted with antibodies raised against human GST ψ as well as GST π. The results of these studies indicated that all three major classes of GST isozymes were expressed in bovine eye but the GST genes were differentially expressed in lens, cornea, and retina. In lens only the μ class of GST was expressed, whereas cornea expressed α and π classes and retina expressed μ and π classes of GST isozymes.

Patterns of Total Peroxidase Activity and Isozyme Complement During Development of Selected Maturity Genotypes of Sorghum

Changes in peroxidase activity were studied in two maturity genotypes of sorghum [Sorghum bicolor (L.) Moench] grown under 10‐and 14‐h photoperiods. The rationale was that peroxidase activity or isozyme complement may represent a means to characterize developmental changes in the maturity genotypes and, possibly, a system to regulate indoleacetic acid (IAA) levels. The maturity genotypes 100M and 38M are near isogenic except for the differences at three of the maturity gene loci. Tissue extracts were separated into soluble (cytoplasm) and ionically or covalently bound wall fractions, which were assayed for total peroxidase activity detected in the cytoplasm and cell wall fractions but not in organelle fractions. On a fresh weight basis, total peroxidase activity did not show consistent changes with age and there were no differences between genotypes. However, on a specific activity basis, the ionically bound peroxidase exhibited the highest activity, which consistently peaked near the time of floral initiation of 38M under both photoperiods and 100M under 10‐h photoperiods. A similar activity profile was not observed for 100M, which remained vegetative under 14‐h photoperiods. The peroxidase isozyme complement was studied by gel electrophoresis with 100M grown under 10‐h photoperiods as a model system. There were modest quantitative changes; however, qualitative changes were more striking because they occurred near the time of floral initiation on Day 30. In both soluble and covalently bound fractions, the predominant qualitative changes were observed for the cationic isozymes on Days 25 and 30. The obvious changes in specific activity and isozyme pattern imply that peroxidases may play an important role in the control of transition processes from vegetative to reproductive stages.

Comparative biochemistry of mammalian arylsulfatases A and B

1. 1. Arylsulfatases A and B occurred as a major anionic and cationic isozyme, respectively, among eleven eutherian mammalian species. 2. 2. Minor anionic arylsulfatase B isozymes were observed in rodents, dog, whale and pig, and were either monomeric (vole, M r = 67 ± 2 kDa), an apparent aggregate (dog, whale, pig; M r = 192 ± 10 kDa), or both (rat, mouse; monomeric M r = 57 ± 2 kDa; apparent dimeric M r = 114 ± 3 kDa). 3. 3. Minor cationic arylsulfatase A isozymes were isolated from the deer, whale and pig. 4. 4. Opossum arylsulfatase A and B were both anionic, had similar relative molecular weights, were not inhibited by silver, and were not precipitated by anti-murine arylsulfatase B nor anti-bovine arylsulfatase A IgG preparations.

Testosterone stimulation of mitochondrial aspartate aminotransferase levels and biosynthesis in rat ventral prostate

The effects of testosterone on mitochondrial aspartate aminotransferase (mAAT) synthesis in rat ventral prostate was investigated. Procedures for the isolation, purification and characterization of AAT isozymes were developed and described. Purified mAAT preparations contained no demonstrable contaminating proteins. Prostatic mAAT was characterized as a cationic protein with an estimated mol. wt of 120,000. Cytoplasmic AAT (cAAT) isozyme was identified as an anionic protein with an estimated mol. wt of 132,000. A cytosolic cationic isozyme, similar to mAAT, was also identified as pre-mAAT. Testosterone administration to castrated rats resulted in significant increases in leucine incorporation into mAAT, in the level of mAAT, and in mAAT activity. These effects of testosterone were observed within 2 h of administration. Conversely, testosterone administration had none of these effects on cAAT or on non-AAT protein pool. Testosterone treatment did appear to increase leucine incorporation into pre-mAAT. Testosterone treatment in organ cultures and in prostate epithelial cell cultures resulted in the same stimulatory effects on mAAT as observed in the in vivo studies. The hormone was effective at the physiological concentration of 2 × 10 −9M. These results indicated that testosterone has a rapid and specific effect on the biosynthesis of mAAT. This continues to support our proposal that testosterone regulates prostate citrate production via a stimulatory effect on mAAT which results in increased mitochondrial synthesis of citrate from aspartate.

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.

Comparative biochemistry of fish and amphibian arylsulfatases

1. 1. Arylsulfatases (EC 3.1.6.1) were isolated from several fish and frog tissues, and their properties were compared with those of mammalian arylsulfatases A and B. 2. 2. Fish arylsulfatases were resolved into cationic and anionic fractions by DEAE-Sephacel chromatography. The cationic isozyme possessed properties resembling those of mammalian arylsulfatase A, while the anionic isozyme was arylsulfatase B-like. 3. 3. Both fish arylsulfatase isozymes exhibited relative mol. wts approximating 100,000 ± 10,000 (Sephacryl S-300) to 132,000 ± 7000 (gel electrophoresis). 4. 4. Carp arylsulfatases were inhibited by both cyanide and phosphate; however, neither cationic nor anionic arylsulfatase activity from longear sunfish and largemouth bass was inhibited by cyanide. 5. 5. Both total arylsulfatase activity and proportion of cationic arylsulfatase varied among liver tissues from different fish species. Considerable intertissue arylsulfatase activity variation was also observed within species. 6. 6. Frog liver arylsulfatase was predominantly cationic and possessed many properties characteristic of mammalian arylsulfatase B. The relative mol. wt of the frog cationic isozyme was estimated to be 93,000–139,000, depending upon the analytical method employed. 7. 7. Frog liver contained 10-fold higher total arylsulfatase activity and possessed a much higher proportion of arylsulfatase B-like activity than non hepatic tissues.

Identification of Apple Rootstock Cultivars by Isozyme Analysis

Abstract Thirty-three clonal apple (Malus domestica Borkh.) rootstocks were characterized by their peroxidase, esterase, acid phosphatase, and idoleacetic acid oxidase isozyme banding patterns. Polyacrylamide disk-gel electrophoresis of shoot bark protein extracts, with a 5% to 15% acrylamide concentration gradient in the separating gel, provided adequate resolution for the anionic isozymes for all 4 enzymes, while a 5% to 20% gradient was used to separate the cationic isozymes of peroxidase and acid phosphatase. Growing conditions (greenhouse and 3 different field locations) and sample timing (February, May, August, and November) did not affect banding patterns. Virus-tested clones showed isozymic differences compared with the original contaminated material. Tissue culture propagated selections from EMLA 7 and MAC 9 stoolbeds expressed different banding patterns compared to original stocks.

Fingerprinting Apple Cultivars by Electrophoretic Isozyme Banding Patterns

Anionic electrophoretic isozyme patterns of peroxidase, esterase and acid phosphatase and cationic peroxidase isozyme patterns from shoot bark protein extracts were used to identify clonal apple scion cultivars. Each of the 21 cultivars included in this study developed a unique combination of isozyme patterns which allowed it to be distinguished from the others. Sports within cultivars exhibited identical patterns of enzymes, with the exception of ‘Wijcik’, a natural compact mutant of ‘McIntosh’ which could be distinguished from the latter, although it was indistinguishable from the cultivar ‘Spartan’. Isozyme patterns remained constant when samples were taken from wood of different ages, at several times of the year and with trees growing in different locations and on different rootstocks.

Possible Involvement of Lipoxygenase in the Mechanism of Resistance of Oats to Puccinia coronat avenae

AbstractPossible participation of the peroxidation system in the cultivar‐race specificity was studied for the oat‐crown rust system. The levels of lipid peroxidation and total lipoxygenase (LOX) activity were extensively increased in leaves of cv. Shokan l responding with resistance to race 226. One anionic and one cationic LOX isozyme was detected in the extract of Shokan 1 leaves inoculated with race 226. In addition, three anionic and one cationic isozymes were consistently found in the extract of uninoculated and compatible race 203‐inoculated leaves. The blockage experiments with race 226‐inoculated leaves using RNA and protein synthesis inhibitors indicated that the two LOX isozymes characteristic of the incompatible combination are de novo synthesized and their activity is causally linked to the resistance expression. Production of the two isozymes was also demonstrated in the five resistant Pc oat lines, but not in the two susceptible Pc lines.

Genetical and biochemical comparisons of alcohol dehydrogenase isozymes from Anastrepha fraterculus and A. obliqua (Diptera: Tephritidae): evidence for gene duplication.

The electrophoretic patterns of the enzyme alcohol dehydrogenase (ADH) from Anastrepha fraterculus and A. obliqua were studied. Two loci were found to code for the enzyme in A. fraterculus, and three in A. obliqua. In both species, all isozymes were active in third-instar larvae. A cationic isozyme (Adh-1) was active mainly in the visceral fat body of both species. In A. fraterculus, the locus had an anionic polymorphic isozyme (Adh-3) that was detected in the parietal fat body. In addition to these two loci, a third locus for an anionic isozyme (Adh-2), which was active in the digestive tube of larvae, was present in A. obliqua and probably resulted from gene duplication. For both species, multiple forms of the isozymes are formed by binding of an NAD-carbonyl compound, as in Drosophila melanogaster. Both larvae and early pupae of A. obliqua had almost twice the specific ADH activity as A. fraterculus. The ethanol content of the host fruit infested with A. obliqua (red "mombim") was also higher than that of the host fruit infested with A. fraterculus (guava).

Purification and characterization of an abscisic acid-inducible anionic peroxidase associated with suberization in potato ( Solanum tuberosum)

An anionic peroxidase (EC 1.11.1.7), thought to be involved in suberization, was purified 110-fold from wound-healing slices of Solanum tuberosum by a combination of ammonium sulfate fractionation, Sephadex G-100 gel filtration, isoelectric focusing, and phenyl-Sepharose CL-4B chromatography in 24% yield. The purified enzyme was homogeneous as judged by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and horizontal thin-layer polyacrylamide gel electrophoresis. The molecular weight of the enzyme was estimated to be 47,000 by both Sephadex G-100 gel filtration and sodium dodecyl sulfate-polyacrylamide gel electrophoresis. This peroxidase was found to be a glycoprotein containing about 17% carbohydrate, approximately one-quarter of which was shown to be glucosamine residues. It was found to have an isoelectric point of 3.15. An anionic peroxidase was also isolated from abscisic acid-treated callus tissue culture of S. tuberosum by the above purification procedure. The two enzymes were shown to be immunologically similar, if not identical, based on their crossreactivity with rabbit antibody prepared against the peroxidase from wound-healing slices, whereas the major cationic peroxidase from wound-healing slices did not crossreact with this antibody. The anionic enzyme from both sources showed very similar specific activities when assayed with a range of substrates, whereas the specific activities found for the cationic isozyme isolated from wound-healing slices were quite different.

Studies on preferential binding to glucocorticoid of rat liver anionic glutathione S-transferase

A new isozyme of Glutathione-S-transferase (GST) with more acidic pI (6.7) than other forms of GST hitherto reported was isolated from rat liver cytosol by consecutive chromatographies on a DEAE cellulose column, lysyl-GSH affinity column and Sephadex G-100 column. This anionic form of GST represented approximately one third of total GST activity in rat liver cytosol. Amino acid composition, immunological reactivity, enzymatic properties, and secondary structure as measured by circular dichroism of this form were distinct from those of cationic isozymes (GST-AA, GST-B, GST-X), presently investigated. The stoichiometric ratio of high affinity site for bilirubin to GST molecule differs amongst isozymes. The anionic form of GST bound two bilirubin per molecule whereas cationic GSTs bound only one bilirubin per two subunits. The most distinguished property of anionic GST was its strong affinity for glucocorticoid. The dissociation constant of anionic GST-corticosterone complex was as low as 2.0 X 10(-8) M. Corticosterone inhibited the enzyme activity of anionic GST in a noncompetitive fashion with an apparent Ki value of 8.6 X 10(-5) M and 1.1 X 10(-6) M for 1-chloro-2.4-dinitrobenzene and GST respectively. The anionic GST-corticosterone complex bound to DNA coupled Sepharose at 25 degrees C and passed through the column at 4 degrees C. Conversely, the complex bound to a DEAE cellulose column at 4 degrees C but passed through at 25 degrees C. These properties of anionic GST are quite similar to those of glucocorticoid receptor of rat liver cytosol reported previously.

Purification and characterization of the individual glutathione S-transferases from sheep liver

The glutathione S-transferases (EC 2.5.1.18) have been purified to electrophoretic homogeneity from 105,000 g supernatant of sheep liver homogenate by employing a combination of gel filtration on Sephadex G-150 and affinity chromatography on S-hexylglutathione-linked Sepharose-6B columns. Approximately 70% of the original glutathione S-transferase activity toward 1-chloro-2,4-dinitrobenzene and glutathione peroxidase activity toward cumene hydroperoxide could be recovered by this purification method. Of particular importance in developing this procedure was the fact that the enzyme preparation obtained after affinity column chromatography represented all the isozymes of sheep liver glutathione S-transferases. Further purification by CM-cellulose and DEAE-cellulose column chromatography resolved the glutathione S-transferases into seven distinct cationic isozymes designated C-1, C-2, C-3, C-4, C-5, C-6, and C-7 and five overlapping anionic transferases designated A-1, A-2, A-3, A-4, and A-5, respectively, in the order of their elution from the ion-exchange columns. The sodium dodecyl sulfate SDS-gel electrophoretic data on subunit composition revealed that cationic enzymes are composed of two subunits with an identical M r of 24,000 whereas a predominant subunit with M r of 26,000 was observed in all anionic isozyme peaks except A-1. Cationic isozymes accounted for approximately 98% of the total peroxidase activity associated with the glutathione S-transferase whereas only A-1 of the anionic isozymes displayed some peroxidase activity. Isozyme C-4 was found to be the most abundant glutathione S-transferase in the sheep liver. Characterization of the individual transferases by their specificity toward a number of selected substrates, subunit composition, and isoelectric points showed some similarities to those patterns for human liver glutathione S-transferases.

Isolation of a cationic peroxidase from cultured peanut cells

Medium that had supported the growth of a peanut cell suspension culture was found to be a rich source of peroxidase. Precipitation with acetone and ammonium sulfate, followed by a pH shift and ion exchange chromatography, led to the isolation of a single cationic peroxidase isozyme of high specific activity and high RZ of 2.91. The isolated peroxidase was shown to be a single peptide chain.