Isoelectric patterns of peroxidase isoenzymes from tobacco tissue cultures

Summary Peroxidases from tobacco tissue cultures have been separated by thin-layer isoelectric focusing into 12–14 isoenzymes, which have been divided into three groups according to differences in isoelectric points. The isoelectric patterns of callus tissues with and without buds have been compared with those of leaves and stems developed in vitro. Qualitatively, there was a basic similarity of the isoelectric patterns, the same isoenzymes being present in all samples. Distinct quantitative differences in the content and substrate specificity were noted for some of the isoenzymes.     

Malate Dehydrogenase Isoenzymes in Cotton Leaves of Different Ages

Malate dehydrogenase isoenzymes from the blades of different aged leaves of the cotton plant have been investigated. The total extractable malate dehydrogenase activity varied widely between leaves of different ages and different locations on the plant. Malate dehydrogenase zymograms developed from the extracts which contained significantly different levels of enzyme activity appear to indicate the presence of different groups of malate dehydrogenase isoenzymes in leaves of different ages. However, under appropriate conditions of polyacrylamide gel electrophoresis, the same number of malate dehydrogenase isoenzymes with the same relative mobilities were detected in all the leaves studied. These findings are discussed in relation to reports that malate dehydrogenase isoenzymes change with plant development or that they have different roles in the plant.     

Attempts to characterize particular parts of the root tip on the basis of isoenzyme patterns of various glycosidases

Using polyacryl amide gel electrophoresis isoenzyme patterns were studied of α- and β-glucosidase, α- and β-galactosidase, α-fucosidase, β-glucosaminidase, β-glucuronidase, α-man-nosidase and β-xylosidase in whole root tips, in particular growth zones and in the cortex and central cylinder of broad bean and maize. Though considerable differences even in number and position of isoenzymes were found in particular cases in both objects, no general conclusion could be reached. The mentioned differences are not obligatory and have been observed both between zones of division and elongation and between zones of elongation and maturation. This fact is hard to understand on the basis of proteosynthetic processes involved in proliferative and postproliferative cell growth.     

Isoenzymatic variability in seeds of some spanish common beans (Phaseolus vulgaris L. Leguminosae): Relation to their domestication centers

Phaseolus vulgaris is an important crop species. The cultivated common bean derives from wild forms in two independent domestication centers in Mesoamerica and South America. We report a study of electrophoretic patterns of seven isoenzymatic systems in 29 samples of P. vulgaris seeds. Nineteen of them are from northern Spain, four are from Mesoamerican and six are South American forms. The isoenzymatic activity of esterases, cytochrome c oxidase, acid phosphatase, alkaline phosphatase, alcohol dehydrogenase, shikimate dehydrogenase and glutamate oxaloacetic transaminase were studied. On the basis of electrophoretic patterns of seed isoenzymes, four groups can be recognized using clustering procedures (UPGMA). Comparing this biochemical information with previous morpho-agronomic studies, the possible primary domestication centers of the cultivars are discussed.     

Differences in catalytic properties between native isoenzymes of xyloglucan endotransglycosylase (XET)

Four isoenzymes of xyloglucan endotransglycosylase (XET; EC 2.4.1.207) were isolated from sprouting mung bean seedlings (M35, M45, M55a, M55b) and two from cauliflower florets (C30, C45). Purification in each case was by ammonium sulphate precipitation, reversible formation of a covalent xyloglucan-enzyme complex, and cation-exchange chromatography. The isoenzymes differed in pH optimum (range 5.0-6.5), Km for the nonasaccharide XLLGol (Gal2.Xyl3.Glc3.glucitol) as acceptor substrate, ability to utilise diverse oligosaccharides as acceptor substrate, and ability to bind to carboxymethyl-cellulose (and thus possibly to other polyanions such as pectin in the cell wall). None of the isoenzymes was particularly cold-tolerant, unlike one XET (TCH4) of Arabidopsis. The two cauliflower isoenzymes had higher Km values for XLLGol (70-130 microM) than the four mung bean isoenzymes (16-35 microM). We suggest that this difference is related to the major roles of the XETs in these two tissues: integration of new xyloglucan into the walls of the densely cytoplasmic cauliflower florets, and re-structuring of existing wall material in the rapidly vacuolating bean shoots.     

Isoenzymes of glutamate dehydrogenase in plants

Glutamate dehydrogenase of several different plants was resolved by polyacrylamide gel electrophoresis into separate molecular forms and the isoenzymic patterns detected by the tetrazolium technique were compared. The number of isoenzymes and their electrophoretic mobilities varied among the different plants studied. The isoenzymes were found to have the same coenzyme specificity and to localize in the mitochondrial fraction of the cell in all the plants examined. Electrophoretic heterogeneity in tissue homogenates was observed in some of the plants studied. The pattern of isoenzymes of mungbean hypocotyl was followed and shown to change during germination.     

Activity and isoenzyme composition of peroxidase and esterase in fertile and male-sterile lines of tomatoes (Lycopersicon esculentum Mill.)

A comparative study of the isoenzyme patterns of esterase and peroxidase and overall peroxidase activity in stamens of male-sterile (MS) lines of Pearson ms-35 and P ms-35aa and of the respective male-fertile (MF) tomato plants have been conducted. The study has been made at two stages of stamens development — tetrad and pollen. Higher activities of the esterase isoenzymes in the MF stamens than that of MS in both ontogeny stages have been found. The slow moving esterase isoenzymes both of the MF and the MS stamens are the major isoenzymes in the early stage and are connected with tapetum development while the fast moving esterase isoenzymes are connected with pollen formation in the later ontogeny stage. Overall peroxidase levels in the MS stamens were higher than those of MF. The peroxidase patterns of the MS lines are also characterized by the greater number of isoenzymes and also the presence of specific isoenzymes, the contrast between the MF and the MS stamens being more strongly expressed at the later stage of development. A strong similitude between esterase and peroxidase patterns behaviour in both MS lines has been found.     

Influence des régulateurs de croissance sur la prolifération,l'activité peroxydasique et les isoperoxydases de la suspension cellulaire deSilene alba

Growth ofSilene alba (MILLER) E. H. L. KRAUSE cells, as well as their peroxidase pattern and activity are studied. Cells were grown in the presence and absence either of IAA, or NAA or 2,4-D. The subculture is dependent upon the growth regulator used to sustain the growth of cells. For 14 days' passages, subculture is possible with 2,4-D (5 x 10^-7M) or NAA (10^-5M) but impossible with IAA or without any growth regulators. Cells grown using 2,4-D or NAA in the medium contain a smaller number of isoperoxidases and have lower activities than those grown using IAA or no growth regulator. The nature of growth substances does not affect the compartimontation of the peroxidase; in fact the bulk of the peroxidaso activity is always liable to the ionic wall bound fractions. Tho electrophorotic mobilities of peroxidase isoenzymes detected in the modium are not the same as those of tho eytoplasmic isoenzymes. Cell cultures grown with and without growth regulators show different patterns of modium peroxidase activities. Some forms are present both in cells and media and some other only in the media; this may indicate that there is some selection made in tho cells for retention of particulars forms; the others could be secreted as exoenzymes shortly after they are synthesized in the cells. The nature of the growth regulator used could act on the release of certain isoperoxidases. These results are discussed from the viewpoint of the correlation of isoperoxidase patterns with the possibility of subculture.     

Studies of the development of basic, neutral and acidic isoenzymes of glutathione S-transferase in human liver, adrenal, kidney and spleen.

The ontogeny of basic, near-neutral and acidic glutathione S-transferase isoenzymes was studied by using chromatofocusing and ion-exchange chromatography. These isoenzyme sets demonstrated tissue-specific patterns of expression. For example, whereas basic isoenzymes were identified in all liver and adrenal cytosols obtained after 10 weeks gestation, these forms were not detected in kidney until 10 weeks post-natal age and in spleen until about 40 weeks post-natal age. Our data indicate that the basic monomers B1 and B2 are present in liver cytosol at 21 weeks gestation. Expression of the near-neutral isoenzymes was usually weak; for example, they were not generally expressed in liver until 30 weeks gestation, and no developmental patterns in their expression could be identified in adrenal, kidney and spleen. The acidic isoenzymes were usually strongly expressed in adrenal, kidney and spleen, although there was a decline in the level of expression in kidney after birth.     

Elicitation of lignin biosynthesis and isoperoxidase activity by pectic fragments in suspension cultures of castor bean

Suspension cultures of castor bean (Ricinus communis L.) which have been treated with pectic fragment elicitor rapidly accumulate lignin as measured by derivatization with thioglycolic acid. The responsiveness of cultured cells to elicitor is dependent on the stage of culture growth. In 6-day (maximally responsive) cultures, increases in lignin are first evident 3 hours after addition of pectic fragment elicitor with maximal rates of lignin synthesis between 4 and 10 hours. The abundance of lignin in cultures after 12 hours of elicitor treatment is 10- to 20-fold higher than in untreated control cultures and can thereby account for as much as 2% of the dry cell weight. Only intermediate sizes of pectic oligomer are active as elicitors of lignin. Half-maximal accumulation of lignin occurs at 250 to 300 micrograms per milliliter of an optimal elicitor preparation with an average degree of polymerization of seven. We consider the synthesis of lignin in elicited cultures to be a mechanism of plant disease resistance which is induced by the elicitor. Plant peroxidases have been proposed to catalyze the last enzymatic steps in the biosynthesis of both lignin and hydrogen peroxide. Six extracellular isoenzymes of peroxidase (two anionic, designated A1 and A2, and four cationic, designated C2, C3, C4, and C7) are detectable in healthy suspension cultures of castor bean by native gel electrophoresis. Treatment of cultures with elicitor causes substantial changes in the activity of four of these species (A1, C2, C3, and C7). Elicitor treatment also results in the appearance of three new peroxidase isoenzymes that are not readily detectable in healthy cultures (C1, C5, and C6). Increases in the activities of these isoenzymes are concurrent with or slightly precede the accumulation of lignin in elicited 6-day cultures. By 12 hours after addition of elicitor, C1 becomes the most abundant extracellular isoperoxidase. The differential regulation of expression of peroxidase isoenzymes following elicitor treatment suggests that individual isoenzymes of peroxidase may have specific functional roles in the biosynthesis of disease-lignin.     

Lack of host specialization in Aspergillus flavus

Aspergillus spp. cause disease in a broad range of organisms, but it is unknown if strains are specialized for particular hosts. We evaluated isolates of Aspergillus flavus, Aspergillus fumigatus, and Aspergillus nidulans for their ability to infect bean leaves, corn kernels, and insects (Galleria mellonella). Strains of A. flavus did not affect nonwounded bean leaves, corn kernels, or insects at 22 degrees C, but they killed insects following hemocoelic challenge and caused symptoms ranging from moderate to severe in corn kernels and bean leaves injured during inoculation. The pectinase P2c, implicated in aggressive colonization of cotton balls, is produced by most A. flavus isolates, but its absence did not prevent colonization of bean leaves. Proteases have been implicated in colonization of animal hosts. All A. flavus strains produced very similar patterns of protease isozymes when cultured on horse lung polymers. Quantitative differences in protease levels did not correlate with the ability to colonize insects. In contrast to A. flavus, strains of A. nidulans and A. fumigatus could not invade living insect or plant tissues or resist digestion by insect hemocytes. Our results indicate that A. flavus has parasitic attributes that are lacking in A. fumigatus and A. nidulans but that individual strains of A. flavus are not specialized to particular hosts.     

Red cell lactic acid dehydrogenase in the family Macropodidae

Lactic acid dehydrogenase isoenzyme patterns from red cells of members of the family Macropodidae are described as they appear in disc acrylamide gels. The reproducibility of patterns, which are determined by the relative intensities of LDH isoenzymes, is noted. Of the 473 animals tested, 471 had red cell LDH patterns belonging to one of five patterns. The distribution of these five patterns among the different genera and species is discussed, and it is suggested that investigation of LDH patterns by this method might assist in the classification of members of the family Macropodidae.     

Characterization of Phytomonas isolated from fruits by electrophoretic isoenzymes and kinetoplast-DNA analysis

Abstract Two flagellates of the family trypanosomatidae were isolated from the fruits of Lycopersicon esculentum (tomato) and Annona cherimolia (cherimoya) in the southeastern region of Spain. The isolates were characterized by isoenzyme analysis using nine different isoenzymes and by analysis of kinetoplast DNA (kDNA) restriction fragment length polymorphism using four different restriction endonucleases. Most of the isoenzymes were unable to distinguish between the two fruit isolates, while they were all able to distinguish these two from four other Phytomonas isolates, three of which were from laticiferous plants i.e. Euphorbia characias E. hirta and E. hyssopifolia , and one was a phloem-restricted isolate associated with Hartrot disease. Only the enzyme Superoxide dismutase was able to differentiate between the two fruit isolates. Electrophoretic and restriction endonuclease analysis of kDNA minicircles, using four restriction enzymes, showed similar if not identical restriction cleavage patterns of the minicircles of the two isolates from fruits, while the patterns were different for the other isolates. These results confirm the hypothesis that the two isolates from fruits constitute a group of trypanosomatids that are the same or closely related and that this group can parasitize more than one host plant.     

Organspezifische multiple Formen der Glutamatdehydrogenase in Medicago sativa

Summary The alteration of the multiple forms of NAD-dependent glutamic dehydrogenase (GDH) during the development of Medicago sativa is investigated by means of polyacrylamide electrophoresis. Seed germination is accompanied by a characteristic change of the GDH-isoenzyme pattern. Seeds contain seven isoenzymes, which gradually decrease in number during germination. At the same time a pattern of new isoenzymes becomes visible. The seed pattern is called GDH-I and the later appearing pattern GDH-II. GDH-I is characteristic for the cotyledons, whereas GDH-II is the typical pattern of the root system. Shoots produce a mixed pattern composed of the GDH-II isoenzymes as well as some GDH-I isoenzymes.These isoenzyme patterns are organ specific. No qualitative change occurs during further development of the plants and during growth in the presence of different inorganic and organic N-sources in the culture medium.All the individual isoenzymes are found predominantly in the particulate fraction. They represent stable forms which are not altered by variation of the conditions of enzyme extraction or during enzyme purification. Re-electrophoresis of the individual isoenzymes following elution from the polyacrylamide gels reveals only one specific band. The molecular weights of all the distinctive isoenzymes are identical.There is some evidence that the different isoenzymes represent conformational forms of one enzyme, and it is postulated that the GDH-I isoenzymes are correlated to a normal metabolic (or catabolic) function of the enzyme, whereas the GDH-II isoenzymes are responsible for a primarily anabolic function of glutamic dehydrogenase.     

6-Phosphogluconate Dehydrogenase Isoenzymes from the Developing Endosperm of Ricinus communis L

The cytosolic and proplastid isoenzymes of 6-phosphogluconate dehydrogenase were purified from the developing endosperm of the castor bean (Ricinis communis L.). No differences in physical or kinetic properties were found for the purified isoenzymes. Each was composed of two identical 55,000 subunits. They had identical pH optima of 7.8 to 8.0 and similar MgCl₂ stimulation for the oxidative decarboxylation of 6-phosphogluconate. The Km values for 6-phosphogluconate were 12 and 9.6 micromolar and for NADP⁺ were 4.1 and 5.4 micromolar for the cytosolic and proplastid isoenzymes, respectively. Therefore, the synthesis of two distinct 6-phosphogluconate dehydrogenase isoenzymes does not appear to have any kinetic significance for the developing seed. However, changes in the proplastid contribution toward carbohydrate metabolism occur in the developing seed and may necessitate independent gene expression to allow for a unique and flexible subcellular distribution of isoenzymes during development.     

Do S allele-specific peroxidase isoenzymes exist in self-incompatible Nicotiana alata?

Summary In order to test Pandey's hypothesis that peroxidase isoenzymes determine S-gene specificity in Nicotiana alata, peroxidase isoenzymes in styles and pollen from various plants of an inbred- and a cross progeny were compared by means of starch gel electrophoresis and electrofocusing.No relation between the S-genotype and the peroxidase isoenzyme patterns of pollen or of styles could be established. The differences between the isoenzyme patterns of different S-genotypes were ascribed to differences in the genetic background of various plants that had the same S-genotype.     

Bio-functional legumes with nutraceutical, pharmaceutical, and industrial uses

To help or prevent certain health problems and adequately feed people, there is a need for added contributions from legumes. Legumes produce primary and secondary metabolites and other phytochemicals such as nutraceuticals, pharmaceuticals, pesticides, and industrial products. In addition, legumes such as hyacinth bean seed contain nearly 10% more fiber while winged bean contains three times more fiber than common bean. The potential breast cancer fighting chemical known as kievitone is found in hyacinth bean but not in common bean nor soybean. Both agmatine and isovitexin are potential combatants of microbial organisms in mammals including humans. Agmatine and isovitexin are not found in soybean nor common bean, however they exist in winged bean. Studies regarding value added traits such as the bio-functional and biologically active components of legumes have only recently begun because most specialty phytochemicals are extracted from other plant sources. Not only can bio-functional legumes provide healthy food constituents for use as nutraceuticals, pharmaceuticals, and pesticidals, but they can increase healthy food resources worldwide. Bio-functional legumes have been used in the past primarily for forage, pasture, minor food, green manuring, and erosion control. Current uses include these previously mentioned plus some fairly new ones such as hyacinth bean used as an ornamental and wildlife food. The future for these common bean relatives is for use in the health markets as new medicines or nutraceuticals and to provide farmers with additional crop production as phytopharmaceutical or nutraceutical crops.     

Glucose-6-Phosphate Dehydrogenase in Plastids from Developing Castor Bean Seeds

Glucose-6-phosphate dehydrogenase, together with the other enzymesof pentose phosphate pathway, was found in the cytosol as wellas in the plastid from developing castor bean (Ricinus communisL.) seeds. The plastid enzyme was found in both the matrix andthe membrane. The plastid enzyme has a sharp pH profile withthe optimum at 8.5, while the cytosolic enzyme has a broad pHprofile, optimum at 7.5. The plastid enzyme was inactivatedby storage at 0°C and by detergents such as Triton X-100,Brij and Nonidet, but the cytosolic enzyme was not. Slab geldisc electrophoresis indicated that three isoenzymes of glucose-6-phosphatedehydrogenase were found in the plastid but one enzyme in thecytosol of developing castor bean seed. From the presence ofglucose-6-phosphate dehydrogenase in the plastid, the operationof whole pentose phosphate pathway in this organelle of developingcastor bean seeds is suggested. (Received September 21, 1982; Accepted January 17, 1983)     

Diagnostic and prognostic criteria for heat stroke with special reference to plasma enzyme and isoenzyme release patterns.

1. The aim of this study was to investigate the potential value of isoenzyme release patterns as parameters of hyperthermic injury in improving the diagnostic protocol for heat stroke. 2. Rats were exposed to combinations of exercise and hyperthermic stress. Following exposure, respective release patterns of isoenzymes were determined. 3. Isoenzyme release patterns following hyperthermic injury portray both the type and extent of tissue damage more clearly than do plasma total enzyme release patterns. 4. The sensitivity of isoenzymes as a diagnostic criterion is extended to include both a temporal component (i.e. an accurate diagnosis will be possible at an earlier stage subsequent to hyperthermic exposure) and at the same time to be indicative of the exposure.     

Isolation and characterization of three alpha-glucosidases from the Japanese quail

We have defined one type of acid alpha-glucosidase and two types of neutral alpha-glucosidases from quail skeletal muscle on the basis of differences in the elution patterns on a DEAE-cellulose column. The appearance of the two neutral alpha-glucosidase isoenzymes was age-dependent. A decrease in acid alpha-glucosidase activity was demonstrated in Japanese quails with glycogenosis type II. The characteristics of these three alpha-glucosidase isoenzymes are described.