Changes In Isoenzyme Patterns Research Articles

Changes in redox regulation during transition from C3 to single cell C4 photosynthesis in Bienertia sinuspersici

Bienertia sinuspersici performs single cell C4 photosynthesis without Kranz anatomy. Peripheral and central cytoplasmic compartments in a single chlorenchyma cell act as mesophyll cells and bundle sheath cells. Development of this specialized mechanism is gradual during plant development. Young leaves perform C3 photosynthesis, while mature leaves have complete C4 cycle. The aim of this work was to investigate changes in redox regulation and antioxidant defence during transition from C3 to single cell C4 photosynthesis in B. sinuspersici leaves. First, we confirmed gradual development of C4 with protein blot and qRT-PCR analysis of C4 enzymes. After this activities and isoenzymes of superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), dehydroascorbate reductase (DHAR) and H2O2 and TBARS and glutathione pool and redox status (GSH/GSSG) were determined in young, developing and mature leaves during transition from C3 to single cell C4 photosynthesis. Activities of SOD, APX and POX decrease, while GR and DHAR were increased. However, most striking results were the changes in isoenzyme patterns of SOD, CAT and GR which were gradual through transition to C4 photosynthesis.

Mg deficiency changes the isoenzyme pattern of reactive oxygen species-related enzymes and regulates NADPH-oxidase-mediated ROS signaling in cotton.

The aim of this work was to investigate changes in isoenzyme patterns of enzymes related to reactive oxygen species (ROS) detoxification such as superoxide dismutase (SOD), catalase (CAT), peroxidase (POX), ascorbate peroxidase (APX), glutathione reductase (GR), and glutathione-S-transferase (GST) in cotton under Mg deficiency. Moreover, we aimed to elucidate how a ROS producer, NADPH oxidase (NOX), responds to changing Mg levels. Cotton plants were grown with different concentrations of MgSO 4 in hydroponic medium to create nutrient deficiency (0, 75, 150, 1000 µM Mg). Gradual decreases in growth and photosynthetic rates were observed with declining Mg concentrations and 0, 75, and 150 µM Mg increased oxidative stress as evidenced by H O and 2 2 lipid peroxidation. Total activities of SOD, CAT, POX, APX, GR, and GST were increased while NOX activity was decreased with Mg deficiency. The activities of GR and GST were highest in plants treated with 0 µM Mg, indicating excess use of glutathione for redox regulation. The most striking results were the changes in isoenzyme patterns of SOD, NOX, POX, and GST. For example, a new Cu/ ZnSOD isoenzyme was induced in plants treated with 0 µM Mg. Cotton plants adapt to Mg deficiency by changing the intensity of existing isoenzymes or inducing new ones.

Microalgae respond differently to nitrogen availability during culturing.

Variations in the exogenous nitrogen level are known to significantly affect the physiological status and metabolism of microalgae. However, responses of red, green and yellow-green algae to nitrogen (N) availability have not been compared yet. Porphyridium cruentum, Scenedesmus incrassatulus and Trachydiscus minutus were cultured in the absence of N in the medium and subsequent resupply of N to the starved cells. Culture growth and in-gel changes in isoenzyme pattern and activity of glutamate synthase, glutamate dehydrogenase, malate dehydrogenase, aspartate aminotransferase, superoxide dismutase, catalase, glutathione peroxidase and glutathione reductase were studied. The results demonstrated that the algae responded to the fully N-depleted and N-replete culture conditions by species-specific metabolic enzyme changes, suggesting differential regulation of both enzyme activity and cellular metabolism. Substantial differences in the activities of the antioxidant enzymes between N-depleted and N-replete cells of each species as well as between the species were also found. In the present work, besides the more general responses, such as adjustment of growth and pigmentation, we report on the involvement of specific metabolic and antioxidant enzymes and their isoforms in the mechanisms operating during N starvation and recovery in P. cruentum, T. minutus and S. incrassatulus.

The modifying effect of sucrose on glutamate dehydrogenase (GDH) activity in lupine embryos treated with inhibitors of RNA and protein synthesis

The modifying effect of sucrose on GDH activity and isoenzyme pattern in isolated embryos of lupine subjected to treatments with inhibitors of RNA synthesis (transcription inhibitors: actinomycin D and cordycepin) and protein synthesis (cycloheximide and chloramphenicol) was investigated. Sucrose starvation of embryos caused the increase of total activity of GDH(NADH-GDH and NAD-GDH) more than twice. Supply of sucrose to sucrose starved embryos caused a reduction of enzyme activity by 40 %. Electrophoretic analysis showed the presence of ca 17 isoenzymes of glutamate dehydrogenase in embryos grown for 72 h in medium with sucrose, while sucrose starvation increased the number of isoenzymes up to 22 forms. Addition of sucrose to sucrose starved embryos after 24 h of cultivation caused the inhibition of synthesis of new isoenzymes. This down-regulation by sucrose was blocked when sucrose was added together with cycloheximide, CHX, (0.025 mM). Treatment of sucrose fed and sucrose starved embryos with cycloheximide (0.020 mM) inhibited protein synthesis by 58 % and 24 %, respectively. The addition of cycloheximide (0.025 mM) to sucrose starved embryos decreased by 60 % NADH-GDH and by 30 % NAD-GDH activity and reduced the spectrum of isoenzymes. CHX treatment did not lead to a significant reduction of enzyme activity and isoenzyme pattern in sucrose fed embryos. The chloramphenicol (CMP) treatment (1 mM) stimulated the total GDH activity, 2.5 fold and 1.5 fold, in sucrose fed and sucrose starved embryos, respectively. Addition of CMP (10 mM) to the media did not affect GDH activity. Both concentrations of CMP caused no significant changes in the isoenzymatic pattern of enzyme in sucrose starved embryos, but induction of new isoenzymes was observed in sucrose fed embryos treated with CMP. In the case of using RNA synthesis inhibitors only cordycepin inhibited the total GDH activity (by ca 25 %) but neither actinomycin D or cordycepin caused any changes in isoenzyme pattern of GDH. The possible mechanism of sugar-mediated regulation of GDH activity is discussed.

Changes in activities of superoxide dismutase, peroxidase and catalase from leaves of Impatiens flanaganiae in response to light intensity

Impatiens flanaganiae Hemsl, is a plant species which preferentially grows in shady conditions at an average light intensity of 30–55 microeinsteins m− 2 sec −1 in its natural environment and exhibits strong apical dominance. Previous studies have shown that lateral branching in these plants can be induced by exposing them to a higher light intensity (280 microeinsteins m− 2 sec−1) under controlled laboratory conditions for up to seven weeks. Prolonged exposure to these light conditions lead to abnormal morphological and biochemical changes probably as a result of photo-oxidative stress. Antioxidative enzymes such as, superoxide dismutase (SOD), peroxidase (POD) and catalase (CAT), are known to be involved in protecting plants against oxidative stress, Changes in isoenzyme patterns of SOD, POD and CAT from leaves of Impatiens flanaganiae in response to different light intensities (30 and 300 microeinsteins m−2 sec−1) were investigated over a period of nine weeks. Electrophoretic analysis indicated that the SOD and POD from crude extracts of I. flanaganiae leaves were composed of 5 and 10 distinct bands respectively, whereas catalase (CAT) appeared as a single band with low mobility. Four of the SOD’s showed sensitivity to cyanide, suggesting they were copper-zinc containing. The fifth SOD band was found to be resistant to both cyanide and hydrogen peroxide and was identified as a manganese containing SOD. The SOD, POD and CAT isoenzymes were found to respond differentially to changes in light intensity. Activities of all SOD’s and CAT increased under high light intensity whilst the increase in activities of some isoperoxidases was less pronounced, These results suggest a light dependent induction of SOD and CAT in leaves of I. flanaganiae probably in an attempt to protect the plants from the harmfull effect of superoxide (O2-) and hydrogenperoxide (H2O2) radicals which are considered to be generated at higher levels under photo-oxidative stress. The induced isoperoxidases are considered to be indirectly involved in the regulation of plant growth.

Differential expression of esterase and MDH isozymes during in vitro culture in maize (Zea mays L.)

Isozyme patterns of esterase and malate dehydrogenase were analyzed at different stages of in vitro culture of immature embryos and glumes of Zea mays L. viz. explant, callus formation, root formation and shoot formation. Significant changes in isoenzyme patterns of esterase and MDH were observed besides the appearance of specific and new isozymes. Specific fast migrating isozymes were noted in differentiating calli of embryo and glume calli which were absent at other stages suggesting a possible association of these isozyme patterns with in vitro differentiation.

Induction of a specific isoenzyme of glutamate dehydrogenase during isolation and the first 48 h of culture of Brassica napus leaf protoplasts

The specific activity of NADH‐glutamate dehydrogenase (GDH, EC 1.4.1.2) in leaf protoplasts (Brassica napus L. cv. Bronowski) was initially low and progressively increased during culture in Murashige and Skoog (MS) medium and MS (−NH4) (ammonium nitrate‐free MS) medium in the dark. Native polyacrylamide gel electrophoresis (PAGE) and tetrazolium staining revealed that the high specific activity of NAD‐GDH (deamination) in leaves correlated with the cathodal isoenzymes, and the high specific activity of NADH‐GDH (amination) in leaf protoplasts to the anodal ones. Changes in isoenzyme pattern were correlated with an increase in the specific activity of NADH‐GDH but not with the NADH‐GDH/NAD‐GDH ratio. The increase in NADH‐GDH (amination) activity of leaf protoplasts was correlated with the occurrence of the isoenzyme GDH7, which was not detected in leaves.

Distinctions between cellulase isoenzyme patterns of five plant-pathogenic Verticillium species

Cellulase isoenzymes produced in liquid shake cultures by isolates of five species of Verticillium were separated by isoelectric focusing. Each species gave a distinctive pattern (or patterns) of isoenzymes which could be used for identification purposes. There were no differences between the isoenzyme patterns of isolates of the same fungal species even from different host species or showing different degrees of virulence. Filtrates from cultures of different ages did not show any qualitative changes in isoenzyme patterns. The same species-specific patterns of cellulase isoenzymes were produced in media containing either a simple carbon source (carboxymethyl cellulose) or a more complex one (solvent extracted plant tissues).

Induction of α-amylase isoenzymes by gibberellic acid in imbibed rice half-seeds

Expression of α-amylase isozymes was studied in germinating intact rice seeds, in embryonated and embryo-less (aleurones) half seeds with or without gibberellic acid treatment. Since most of the available information on rice α-amylases was obtained from scutellar tissues, this report focuses on the aleurone isozymes. Their isoelectrofocusing pattern and immunoreactivity are essentially similar to those of scutellum isoenzymes. Changes in gene expression were studied at various levels including modification in enzyme activity, time course of changes in isoenzyme patterns, immunochemical evaluation of changes in protein content for specific isoenzymes and in vitro translation of mRNA. All these experiments lead to the same conclusion: that in rice aleurone α-amylase isozymes are induced by exogenous gibberellic acid in a similar way as in other studied cereals, although at a lower level. These observations complement previous reports made on the scutellum isoenzymes which indicated that the rice α-amylases in this tissue were insensitive to exogenous gibberellic acid.

Changes in isoenzyme patterns of a cloned culture of nonpathogenic Entamoeba histolytica during axenization.

The axenization of an Entamoeba histolytica isolate with a nonpathogenic isoenzyme electrophoretic pattern (zymodeme) was recently achieved for the first time (15). Forty days after the cells were transferred to the medium used for axenic cultivation, the amebae developed virulence properties, and the zymodeme converted to a pathogenic pattern. To exclude the possibility that the original isolate consisted of two zymodeme populations and that conditions of growth selected for a particular population, the experiment was repeated with a cloned culture of a nonpathogenic (zymodeme III) strain, E. histolytica SAW 1734R clAR, isolated by and obtained from P. G. Sargeaunt. Axenization was accomplished, as before, by transferring trophozoites to TYI-S-33 medium containing a mixture of antibiotics to suppress the growth of the associated bacterial flora and a nutritional supplement consisting of gamma-irradiated bacteria. A change in the hexokinase and phosphoglucomutase isoenzyme pattern was observed 21 days after the amebae had been transferred to the axenic medium but before complete axenization of the amebae had occurred. The change in zymodeme was accompanied by an increase in virulence, as evidenced by the ability of fewer amebae to induce hepatic abscesses in hamsters. A reverse conversion to a nonpathogenic zymodeme was also accomplished by reassociating and subculturing the newly converted pathogenic trophozoites of strain SAW 1734R clAR with the bacterial flora that accompanied this ameba in the original xenic culture. The electromobilities of the hexokinase isoenzymes changed back to their original pattern 7 days after the amebae were returned to xenic growth conditions. Our in vitro results demonstrate that culture conditions and bacterial flora can cause changes in the zymodeme and virulence of a cloned ameba isolate and raise the concern that this could happen also in vivo. Thus, the finding of a particular zymodeme in a culture of E. histolytica isolated from a carrier should not be used to predict a clinical condition or serve as a basis for the recommendation of therapy.

Changes in isoenzyme patterns expressed by the erythroleukemia cell lines K-562 and HEL after induction of differentiation.

Stable human leukemia-lymphoma cell lines provide model systems to study the processes involved in leukemic and normal cell differentiation [10]. Leukemic cells, arrested at a certain stage of differentiation, can be triggered to differentiate to functionally and morphologically more mature cells [7]. Both “fresh” leukemic cells and cells maintained in long-term culture are sensitive to in vitro induction of differentiation. Furthermore, cell differentiation is a novel concept in the treatment of acute leukemias and several substances such as low dose Ara-C and the physiologic Compound retinoic acid have been shown to act as differentiating agents in vivo [7].KeywordsAcute LeukemiaErythroleukemia Cell LineCarboxylic EsteraseMyelogenous Leukemia Cell LineHuman Erythroleukemia Cell LineThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Red and far-red effect on the isoenzyme composition of young fern gametophytes

Summary Changes in isoenzyme patterns, induced by active phytochrome were studied in germinating fern spores of Pteridium aquilinum and Dryopteris filixmas. The functional analysis of soluble proteins by a sensitive electrophoretic method supplemented with enzyme specific staining showed characteristic phytochrome effect in NADP-IDH of D. filix-mas and G6PD of both species. This effect was virtually prevented by inhibitors of protein synthesis.

Amelioration of cardiotoxic effects of alcohol by vitamin E.

Injection of mice with a single ip dose of 2g/kg of ethanol leads to time dependent increases of lactic dehydrogenase plasma isoenzymes indicative of myocardial damage. Electron microscopic analysis of the myocardium shows changes in mitochondrial structure, endoplasmic reticulum and myofibrils. Pretreatment of the animals with 86 units of alpha tocopherol partially prevented the changes in isoenzyme patterns and reduced the electron microscopic evidence of myocardial damage. The study supports previous findings that some of the toxic effects of alcohol might be mediated through free radical mechanisms leading to lipid peroxidation and that the ameliorating effect of alpha tocopherol could relate to its function as antioxidant and free radical scavenger.

Kinetic constants of lactase of the small intestine of growing rats fully recovered from protein depletion, in relation to dietary protein and lactose concentration.

The effect of dietary composition on the kinetic constants of intestinal lactase was studied using rats depleted of protein by feeding protein-free diet from the weanling stage to 34 days of age and subsequently allowed to recover on diets containing 11.5 or 17% of protein calories (P%) and different levels of lactose (L%; 1, 15, 27 and 37%). After four days of refeeding, rats were decapitated and lactase activity was determined at different substrate concentrations by the method of Dahlqvist using homogenate of intestinal mucosa. Maximum velocity (Vmax) and Michaelis constant (Km) were calculated according to Eisenthal and Cornish-Bowden. At both levels of P%, Vmax tended to increase with the dietary lactose concentration. With diets containing 37% lactose, at P% 11 Vmax was about 60% of that at P% 17.0 Km tended to increase with L% in groups given the 17.0% protein calorie diets, but no difference was observed between groups fed at the lower level of protein. These results can be explained on the basis of interactions between dietary protein at different concentrations and inducer substrate which results in changes in isoenzyme patterns.

Changes in Isoenzyme Patterns during the Ontogeny of Silene alba

Summary Protein variation within and between populations can be caused by genetic differences or by variation during ontogeny. We have observed enzyme variation during the development of 42 individuals of Silene alba. For the 7 enzymes (AcP, EST, GOT, LAP, MDH, NADH DH and PO) variation was found in all sta,dia of development. Only in the stem leaves of the young plant the pattern was constant within a plant. However, the isoenzyme patterns of the stem leaves of senescent plants deteriorated. Therefore only young plants can be used to estimate the genetic variation in S. alba.

On the ecogenetics of Stellaria media (L.) Vill. and Stellaria pallida (Dum.) pire from abandoned arable field.

Isoenzyme analyses were carried out on the hypotetraploid Stellaria media and the diploid Stellaria pallida occurring on abandoned sandy fields in the coastal dunes of the Netherlands. Amylase isoenzymes in germinating seeds of Stellaria media showed a striking intrapopulational variability, which is in accordance with the germination behaviour of the different subpopulations. In contrast to these findings no intrapopulational variation nor developmental change in isoenzyme pattern could be detected on basis of 14 other analysed leaf enzymes. For five enzymes there was a difference in isoenzyme pattern between the species Stellaria media and Stellaria pallida. Of these five enzymes two showed an interpopulational variation in Stellaria media. The fact that there was almost no variability of the isoenzyme pattern in Stellaria pallida could be explained by the permanent autogamous (cleistogamous) state of the species. Effects of polyploidy on the isoenzyme pattern and its activity could not be demonstrated. Consequences of the extreme difference between the monomorphic pattern of the leaf enzymes as opposed to the polymorphism of the amylase are discussed in view of the difference in germination ecology and lifecycle strategy of Stellaria media subpopulations.

Origin of the Heterogeneity of Peroxidase Isoenzyme Group Gr from Nicotiana tabacum. I. Conformation

Summary By several methods of de- and renaturation (treatment with urea, guanidine and mercaptoethanol, freezing-thawing, heating-cooling etc.) it was studied whether the multiple molecular forms of tobacco peroxidases, group GI, are conformers or not. Most of the applied methods resulted in inactivation of the enzymes only, or did not show any effect. Only heating-cooling resulted in distinct denaturation-renaturation curves but there was no change in isoenzyme pattern at all. Also treatment of GI-isoenzymes with ion-exchange resins or glycosidases did not influence the molecules in any way. Therefore it was concluded that GI-isoenzymes are not conformational variants of a single polypeptide chain and that the origin of heterogeneity might be -due to different primary structures of the isoenzymes.

Differential Changes in Isoenzyme Pattern of 6-Phosphogluconate Dehydrogenase in Individual Plants of Senecio sylvaticus L. during Development

The isoenzyme patterns of individual plants from a population of the annual Senecio sylvaticus was determined at four stages of development, i.e. small rosette stage, large rosette stage, shooting stage and flowering stage, by gelelectrophoresis. Changes of isoenzyme pattern occurred only in 6-phosphogluconate dehydrogenase, the other six enzymes tested, remained constant throughout life cycle. The isoenzymes of 6-phosphogluconate dehydrogenase were compartmentated in the cytosol and chloroplast. With ageing activity of the isoenzymes in the chloroplast decreased and that of those in the cytosol increased.

Effects of morphine on isoenzymes of pyruvate kinase and tyrosine aminotransferase in rat

The acute and chronic effects of morphine on isoenzymes of pyruvate kinase and tyrosine aminotransferase in rat liver have been studied in vivo. Acute administration of morphine inhibits the L-type pyruvate kinase but increases forms II, III and IV of tyrosine aminotransferase in rats which have received a single intraperitoneal injection of morphine 6 hr prior to sacrifice. However both effects are lost after 24 hr. Morphine starts to regain its stimulatory effect on tyrosine aminotransferase at the hr 48 and reaches a maximum at the hr 72. Chronic morphinization leads to similar changes in the total activities of pyruvate kinase and tyrosine aminotransferase but the change in isoenzyme pattern of tyrosine aminotransferase concerns only forms II and III. Results from this study support the hydrocortisone-like property of morphine and indicate that morphine may enhance gluconeogenesis in vivo.

Regulation und Bedeutung der Peroxidase-Muster�nderungen in spro�differenzierenden Kalluskulturen von Nicotiana tabacum L.

Peroxidase activity and isoenzyme pattern were studied during dedifferentiation of tobacco stem-sections leading to callus formation and during redifferentiation of tobacco callus leading to formation of shoots. These processes are both accompanied by an increase in total peroxidase activity and by characteristic changes in isoenzyme pattern. The isoenzyme pattern of tobacco callus differs from that of tobacco stem-tissue. The plantlets differentiated from the callus show the same pattern as seedlings do. During the differentiation process, before any buds are visible, the callus shows a peroxidase pattern that is determined by a reduction of fast-migrating anodic isoenzymes and by an increase of activity in all the other peroxidase isoenzymes. The formation of this pattern is independent of the growth regulators responsible for the differentiation: only the kind of differentiation itself determines the pattern. By artificial inhibition of callus growth it is possible to induce an isoenzyme pattern very similar to that of differentiation; the fast-migrating anodic enzymes are reduced in activity but the others are not increased as they are during differentiation. Therefore the question arises whether there are two independent processes taking place in differentiating callus. The one process, inhibition of growth in the cells that do not differentiate, is accompanied by a reduction of fast-migrating anodic isoenzymes. The other process, formation of meristemoids in the callus, is accompanied by a sharp rise in peroxidase activity of the other anodic and cathodic isoenzymes.