Contribution to the estimation of malic dehydrogenase isoenzymes in the root growth zones ofVicia faba L.

Highly active NAD-MDH (E.C.1.1.1.37) and low activity of NADP-MDH (E.C.1.1.1.40) were found inVicia faba roots. The NAD-MDH activity is associated with 6 to 12 isoenzymes. The number of isoenzymes is dependent on the extraction (use of Triton X-100etc.) and detection procudures (presence of KCN, phenazine methosulphate). The meristematic zone does not contain one isoenzyme (X) which is present in the other two zones. The meristematic zone, elongation zone and zone with the beginning differentiation differ in their activity of individual isoenzymes.     

Glutamate dehydrogenase from pumpkin cotyledons: characterization and isoenzymes

Glutamate dehydrogenase from pumpkin (Cucurbita moschata Pior. cultivar Dickinson Field) cotyledons was found in both soluble and particulate fractions with the bulk of the activity in the soluble fraction. Both enzymes used NAD(H) and NADP(H) but NAD(H) was favored. The enzymes were classified as glutamate-NAD oxidoreductase, deaminating (EC 1.4.1.3). Both enzymes were heat stable, had a pH optimum for reductive amination of 8.0, and were inhibited by high concentrations of NH₄⁺ or α-ketoglutarate. The soluble enzyme was more sensitive to NH₄⁺ inhibition and was activated by metal ions after ammonium sulfate fractionation while the solubilized particulate enzyme was not. Inhibition by ethylenediaminetetraacetate was restored by several divalent ions and inhibition by p-hydroxymercuribenzoate was reversed by glutathione. Particulate glutamate dehydrogenase showed a greater activity with NADP. The molecular weights of the enzymes are 250,000. Separation of the enzymes by disc gel electrophoresis showed that during germination the soluble isoenzymes increased from 1 to 7 in number, while only one particulate isoenzyme was found at any time. This particulate isoenzyme was identical with one of the soluble isoenzymes. A number of methods indicated that the soluble isoenzymes were not simply removed from the particulate fraction and that true isoenzymes were found.     

Purification of glyoxalase I from human erythrocytes by the use of affinity chromatography and separation of the three isoenzymes.

Glyoxalase I has been purified to homogeneity from human erythrocytes. An essential part in the purification procedure involved affinity chromatography on S-hexylglutathione bound to epoxy-activated Sepharose 6B. Three isoenzymes were found, which could be separated by ion-exchange chromatography. Some of the properties of the enzyme are reported.     

Biochemical and genetic studies on alkaline phosphatase ofCeratitis capitata

Two forms of alkaline phosphatase exist in the integument of the “white pupae” (wp) and dark pupae (dp) mutant strains ofCeratitis capitata, during transition from larvae to pupae. They were separated by DEAE-cellulose chromatography. Both isoenzymes have a molecular weight of approximately 180,000 and two pH optima, at 9.4 and at 11.0. The isoenzymes of the “dark pupae” mutant catalyze the hydrolysis of phosphotyrosine and β-glycerophosphate but not phosphoserine, phosphothreonine, ATP, and AMP. In contrast, the isoenzymes of the white pupae mutant hydrolyze all the substrates tested. The ALPase 1 of the dark pupae mutant was inhibited byL-tyrosine, butL-phenylalanine had no effect on either isoenzyme. The effects of divalent cations, EDTA, temperature, urea, and 2-mercaptoethanol were also investigated. Electrophoretic analysis did not reveal any variants of the larval and pupal isoenzymes, but ALPase A, an adult stage-specific isoenzyme, was found to be polymorphic. The electrophoretic variants were shown to be controlled by three codominant alleles located on the third chromosome ofCeratitis capitata. Since we found no hybrid enzyme, we conclude that ALPase A is monomeric.     

Regulation of biosynthesis of secondary metabolites

Two partly purified malate dehydrogenase (EC 1.1.1.37) isoenzymes were isolated fromStreptomyces aureofaciens. This is the first example of a non-homogeneous enzyme in actinomycetes and one of the very few cases in bacteria in general. The characteristics of the enzymatic reaction were studied for each enzyme in relation to the concentration of both substrates and cofactors and the apparent Michaelis constant was calculated. It was found that the reaction was affected by Mg²⁺ ions and that SH-groups could be specifically inhibited. The optimal pH and the influence of temperature changes were also determined. In all the parameters, one of the isoenzymes resembled mitochondrial MDH, while the other resembel the supernatant MDH described in the literature in the tissues of higher organisms. The functional relationship of the two MDH isoenzymes inStreptomyces aureofaciens is discussed.     

Analyses of Soluble Protein,Total and Free Amino Acids in Leaves of Different Ecotypes of Phragmites communis Growing in the Hexi Corridor

The difference of total and free amino acids and protein extracted from the leaves of four different reed ecotypes growing in Hexi corridor of Gansu Province were investigated. In all of the different reed ecotypes, the content of Asp, Glu, Gly, Leu and Ala in total amino acids were high, while the contents of Ala, Phe, Met and Thr, Pro in total amino acids varied among different reed ecotypes. Albeit Ala, Glu, Asp, Gly and Ser were the chief composition of free amino acids in leaves of all reed ecotypes, but temarkble difference was found in the content of each free amino acid from different reed ecotypes. The content of free Pro in leaves of salt meadow and salt meadow-sand dune transitional zone reed were 3.5 and 1.6 times respectively as much as in leaves of swamp reed. Swamp reed had 11 soluble proteins whereas other three reed ecotypes show that each has 13 soluble proteins. Three “salt adaptation proteins” (66 kD, 40.3 kD, 16.5 kD) were found in leaves of three reed ecotypes with varying degree of salt stress, however, the contents of 3 “salt adaptation protens” showed a negative correlation with the degree of salt stress. There was a large amount of “special protein” (11.7 kD) in leaves of sand dune reeds. These results suggest that the difference in cytogene expression takes a priority basis of adaptation of reed plants to different habitats, while a closer relationship of reeds tolerance to salt or drought stress with Pro accumulation in cells is seen than with the of accumulation stress adaptation protein.     

The O-diphenol-oxygen-oxidoreductase of Agaricus bisporus: Activity and multiple forms during ageing

Mushroom o-diphenol oxidase was separated into multiple forms by isoelectric focusing. Three major bands, as opposed to the four isoenzymes previously found, were separated over the pH range 3.5–9.5. A fourth form was obtained when the pH range was narrowed to 5.0–8.0. Changes in the enzyme activity were investigated during post-harvest ageing at different temperatures. Rapid ageing using tissue discs with or without inhibitors of protein synthesis showed that an increase in activity of the enzyme took place during this time, but was prevented by actinomycin D and 6 methyl purine.     

Activity of NAD- and NADP-dependent malate dehydrogenase isoenzymes in the myocardium of rabbits with alloxan diabetes]

Isoenzymes NAD-and NaDP MDH were detected in the cardiac muscle of rabbits by disc electrophoresis in polyacrylamide gel. Alloxan diabetes proved to be accompanied by a significant reduction in the activity of mitochondrial NADP MDH (in the reaction of malic decarboxylation) and its increase in cytozol. The activity of NAD-MDH (in the reaction of oxyacetate reduction) was also decreased in various isoenzymes in the myocardium (particularly in the mitochondria) in diabetes. Insulin restored the correlation of the activities of the isoenzymes NAD- and NADP-MDH in the cytostructures of the myocardium disturbed in diabetes.     

Deletion map of the Escherichia coli K-12 dnaB gene

Summary Acid phosphatase isoenzymes of Chlamydomonas reinhardii were investigated by isoelectric focusing in polyacrylamide gel systems. In this paper we describe in detail an original method for isoelectric focusing of acid phosphatases extracted from wildtype and acid phosphatase-lacking mutant algae, obtained from Laboratoire de Génetique of University of Liège. Three isoenzymes can be separated from the buffer-soluble components of these cells. An additional isoenzyme type can be visualized using the nonionic detergent NP40 as solubilizer. We conclude that these four isoenzymes are releated to the structural gene of the soluble constitutive acid phosphatase, which was shown by their appearance in P ₂ and their total absence in mutant P _a. The pl values of soluble constitutive acid phosphatase isoenzymes range between pH 5.2 and 6.2. As a result of treatment with NP40 the extracts from both wild-type and mutant lines contain two additional active phosphatase forms which can be characterized by their high heat resistance and low pI values. These enzymes are fully active using either -naphthyl phosphate or different acetate esters as substrates.     

Properties of three sets of isoenzymes of alcohol dehydrogenase isolated from barley (Hordeum vulgare)

Three sets of isoenzymes of alcohol dehydrogenase were separated from root and shoot tissue of Hordeum vulgare by DEAE-cellulose chromatography. Set I showed only one band of ADH activity after polyacrylamide gel electrophoresis; Set II—two and Set III—three, making a total of six discernable bands. Only one set (I) was detected in the dry seed and one set (III) in the M9 (Adh-1-null) mutant available in tissue culture. The sets were found to have identical molecular weights (90 000), were all located in the cytoplasm but showed small differences in pH optima and substrate specificity. The affinity for ethanol (K_m value, mM) varied between Set I (27.5), Set II (7.2) and Set III (3.5), whilst the affinity for NADH varied five-fold between the three sets. A dimeric quaternary structure was inferred from the random reassociation of enzyme subunits after dissociation in high ionic strength buffer.     

Variations des zymogrammes du tube digestif et du muscle chez Cyclonassa neritea en fonction de la température d''acclimatation

Soluble proteins, esterases 2C, acid phosphatases of the digestive gland and foot muscle of Cyclonassa neritea, were compared using polyacrylamide gradient gels. α-Glucosidases, alkaline phosphatases, l-leucine aminopeptidase and peptidase were studied from digestive gland extracts. Molecular weights of isoenzymes were evaluated with 5000 d accuracy. Variation in activity of the most important isoenzymes of each enzyme under the influence of acclimation temperature was measured. In both muscle and digestive gland, the concentration of soluble proteins is stable. Through the whole acclimation temperature range, esterase activity per mg protein decreased with increased temperature. l-Leucine aminopeptidase activity decreases steadily from 10 to 25°, even though the two alkaline phosphatase isoenzyme activities increase. The other enzymes have their maximum activities at 20°.     

Characterization of aspartate aminotransferase isoenzymes from leaves of Lupinus albus L. cv Estoril

Two aspartate aminotransferase (EC 2.6.1.1) isoenzymes (AAT-1 and AAT-2) from Lupinus albus L. cv Estoril were separated, purified, and characterized. The molecular weight, pI value, optimum pH, optimum temperature, and thermodynamic parameters for thermal inactivation of both isoenzymes were obtained. Studies of the kinetic mechanism, and the kinetics of product inhibition and high substrate concentration inhibition, were performed. The effect of some divalent ions and irreversible inhibitors on both AAT isoenzymes was also studied. Native PAGE showed a higher molecular weight for AAT-2 compared with AAT-1. AAT-1 appears to be more anionic than AAT- 2, which was suggested by the anion exchange chromatography. SDS-PAGE showed a similar sub-unit molecular weight for both isoenzymes. The optimum pH (between 8.0 and 9.0) and temperature (60-65 degrees C) were similar for both isoenzymes. In the temperature range of 45-65 degrees C, AAT-2 has higher thermostability than AAT-1. Both isoenzymes showed a high affinity for keto-acid substrates, as well as a higher affinity to aspartate than glutamate. Manganese ions induced an increase in both AAT isoenzymes activities, but no cooperative effect was detected. Among the inhibitors tested, hydroxylamine affected both isoenzymes activity by an irreversible inhibition mechanism.     

Lactate dehydrogenase isoenzymes in the eye, cardiac and skeletal muscles of several decapods]

Properties of lactate dehydrogenase (LDH) in the eye, heart and muscles of Hemigrapsus sanguineus, Paralithodes camtschatica, Erimacrus isenbeckii, Pandalus latirastrus, Pagurus brachiomastus have been studied with acrylamide gel electrophoresis and kinetics analysis. LDH in all the tissues of all the representatives studied was found to be specific for L-pyruvate and lactate; it migrated in electrophoresis as a single band revealing low mobility towards anode. The isoenzyme from P. camtschatica and P. latirastrus differed from the isoenzymes of other animals studied by higher mobility towards anode that reflected higher negative value of its total charge. The LDH isoenzymes in all the animals studied resembled the A4 (LDH5) of the vertebrates being unstable to the denaturing action of high temperature and being unaffected by high concentrations of pyruvate up to 1.0.10-3M. On the other hand, in conrast to the A4 of mammals, the LDH in question displayed enhancement of the reaction rate and decrease of the Km values upon increase in the NAD+ and NAD.H concentrations both in the presence of high or low lactate and pyruvate concentrations. The isoenzymes displayed catalytic activity also in the presence of NADP, the Km values for pyruvate in the presence of equimolar (2.25 mM) concentrations of NAD.H or NADP.H were practically identical and were found to be within the limits of 14-26.10-5 M. Molecular weight of the LDH studied assessed by the gel filtration method was found to be 130-140,000. It is suggested that the LDH isoenzyme from the representatives of the decapod crayfish studied is homologous in its certain properties to the homotetrameric A4 form of the vertebrates.     

Effect of temperature on ascorbate peroxidase activity and flowering of Arabidopsis thaliana ecotypes under different light conditions

Four Arabidopsis thaliana ecotypes were grown at 14 degrees C and 22 degrees C under two light conditions (300 microE m-2 s-1 or 150 microE m-2 s-1) and the effect of temperature on their growth and flowering time was studied. Flowering occurred within 31 days (experimental period) at 22 degrees C, whereas a decrease in growth temperature resulted in a delay in flowering (63 days) under both light conditions. At 14 degrees C, membrane-bound APX (tAPX) activity decreased and total chlorophyll (Chl) content increased with growth under both light conditions. However, at 22 degrees C, the tAPX activity increased and total Chl content decreased with growth under both light conditions. These results suggest that at 22 degrees C oxidative stress was high under both light conditions and consequently Chl content decreased under stressful conditions or vice versa for all the four A. thaliana ecotypes studied. Under both the temperature and light conditions, soluble APX activity showed an irregular pattern of growth. The increase in tAPX activity, with growth only at 22 degrees C but not at 14 degrees C, suggests increased H2O2 formation in flowering plants at 22 degrees C for all the four A. thaliana ecotypes studied. Before flowering, the tAPX activity showed a significantly negative correlation with flowering time. Higher oxidative stress in the lower-latitude ecotypes might induce earlier flowering than the higher-latitude ecotypes. From these results, we propose a hypothesis that H2O2 is one of the possible factors in flower induction.     

Two isoenzymes each of glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase in spinach leaves

Isoenzymes of glucose-6-phosphate dehydrogenase and 6-P-gluconate dehydrogenase from a 70% ammonium sulfate precipitate of spinach leaf homogenate were separated by differential solubilization in a gradient of 70-0% ammonium sulfate and analyzed by disc gel electrophoresis. Isolated whole chloroplasts contained isoenzyme 1 of both glucose-6-phosphate dehydrogenase and 6-phosphogluconate dehydrogenase 1, whereas isoenzyme 2 of each was found in the soluble cytosol fraction. Both isoenzymes of each dehydrogenase were present in about equal amounts. Glucose-6-phosphate dehydrogenase isoenzymes 1 and 2 had pH optima of 9.2 and 9.0 and K_m values of 400 and 330 μm, respectively. Molecular weights for both isoenzyme of glucose-6-phosphate dehydrogenase were very similar at about 105,000 ± 10% as estimated by sedimentation velocity measurements. For 6-phosphogluconate dehydrogenase isoenzymes 1 and 2 the pH optima were 9.0 and 9.3, respectively, the K_m values were 100 and 80 μm, and the apparent molecular weights were also nearly identical at about 110,000 ± 10%. The data support the hypothesis that leaf cells have two oxidative pentose phosphate pathways, one in the chloroplast and the other in the cytosol.     

Biochemical composition of temperate and Arctic populations of Saccharina latissima after exposure to increased pCO2 and temperature reveals ecotypic variation

Previous research suggested that the polar and temperate populations of the kelp Saccharina latissima represent different ecotypes. The ecotypic differentiation might also be reflected in their biochemical composition (BC) under changing temperatures and pCO₂. Accordingly, it was tested if the BC of Arctic (Spitsbergen) and temperate S. latissima (Helgoland) is different and if they are differently affected by changes in temperature and pCO₂. Thalli from Helgoland grown at 17 °C and 10 °C and from Spitsbergen at 10 °C and 4 °C were all tested at either 380, 800, or 1,500 µatm pCO₂, and total C-, total N-, protein, soluble carbohydrate, and lipid content, as well as C/N-ratio were measured. At 10 °C, the Arctic population had a higher content of total C, soluble carbohydrates, and lipids, whereas the N- and protein content was lower. At the lower tested temperature, the Arctic ecotype had particularly higher contents of lipids, while content of soluble carbohydrates increased in the Helgoland population only. In Helgoland-thalli, elevated pCO₂ caused a higher content of soluble carbohydrates at 17 °C but lowered the content of N and lipids and increased the C/N-ratio at 10 °C. Elevated pCO₂ alone did not affect the BC of the Spitsbergen population. Conclusively, the Arctic ecotype was more resilient to increased pCO₂ than the temperate one, and both ecotypes differed in their response pattern to temperature. This differential pattern is discussed in the context of the adaptation of the Arctic ecotype to low temperature and the polar night.     

Purification and Properties of Three Pig Erythrocyte Carronic Anhydrases

Three distinct forms of the zinc containing enzyme carbonic anhydrase were isolated from pig erythrocytes. One low activity type enzyme and two genetic variants of the high activity type enzyme with identical CO2 hydratase activities which were 8 times as high were isolated from Danish Black and White Swine. In the isolation procedure described, the hemoglobin was eliminated by precipitation with chloroform-ethanol, and the isoenzymes were separated by DEAE-Sephadex chromatography. A number of enzymatically active minor components were separated. They were apparently all genetically linked to one of the three major components. The three purified isoenzymes behaved as homogeneous components during isoelectric focusing and electrophoresis at different pH values. They were characterized in terms of molecular weight, isoelectric pH, zinc content, amino acid composition, and enzymatic activity against CO2, p-nitrophenyl acetate, and β-napthyl acetate. The circular dichroism of the enzymes in the ultraviolet region was studied. The properties of the enzymes were similar to those of carbonic anhydrases of corresponding types isolated from other mammalian species. Sulphur containing amino acid residues were absent in the low activity type enzyme. The amino acid composition of the two high activity mutants deviated only in that an arginine residue in the most widespread genetic variant was replaced by a histidine residue in the less frequent variant. Otherwise the two mutants showed identical properties.     

Characterization of NAD-dependent malate dehydrogenases from spinach leaves

Summary. Spinach leaves were used to extract isoforms of NAD-dependent malate dehydrogenase (NAD-MDH) (EC 1.1.1.37), either soluble or bound to microsomal, plasma, or chloroplast envelope membranes. All fractions were subjected to isoelectric focusing analysis, which showed that purified chloroplast envelopes contain an NAD-MDH isoform tightly bound to the membranes, since treatment with 0.5 or 1% Triton X-100 was not able to release the enzyme from the envelopes. In contrast, plasma membranes released an isoform with a pI of 3.5 following treatment with 0.5% Triton X-100. The most abundant soluble leaf isoform had a pI of 9, while the chloroplast stroma contained an isoform with a pI of 5.3. Kinetic analysis of oxaloacetate (OAA)-dependent NADH oxidation in different fractions gave different K _m values for both substrates, the envelope- and plasma membrane-bound NAD-MDH exhibiting the highest affinities for OAA. Leaf plasma membrane-bound MDH exhibited a high capacity for both reaction directions (malate oxidation and OAA reduction), while the two chloroplast isoforms (stromal and envelope-bound) preferentially reduced OAA. Our results indicate that the chloroplast envelope contains a specifically attached NAD-MDH isoform that could provide direct coupling between chloroplast and cytosol adenylate pools. Correspondence: T. Cvetić, Institute of Botany and Botanical Garden, Faculty of Biology, University of Belgrade, Takovska 43, 11000 Belgrade, Serbia.     

不同生境甘草的生态型研究

采用SDS-PAGE电泳的方法,对5种不同生境的甘草(G ly cy rrh iza ura lensis F isch.)幼苗进行了同工酶[过氧化物酶(POD)、酯酶(EST)、乳酸脱氢酶(LDH)和苹果酸脱氢酶(M DH)]研究,并利用PEG 6000进行了水分胁迫的生理响应实验.同时,对种植在相同生境下的2年生子代植株的地上部形态特征进行了观测.结果表明,5种生境的甘草至少已分化为2种生态型,且与它们的生境有显著的相关性,生长在生境干燥度较大的民勤甘草(M G)和酒泉甘草(JG)为一类,其幼苗的耐旱能力较强,而生长在干燥度相对小的大兴甘草(DG)、沙坡头甘草(SG)以及内蒙甘草(NG)归为另一类,幼苗的耐旱能力也低于前者,甘草生境中气候因素的水因子可能是影响其生态型分化的主导因子.由本研究结果得出以下推论,不同生境甘草的生态型分化是其适应环境进化的结果,甘草可能是甘草属的先锋种,由于长期适应不同的生态环境而产生了趋异变化,并形成了不同的生态型乃至新种.     

Differential physiological and antioxidative responses to drought stress and recovery among four contrasting Argania spinosa ecotypes

Our study was undertaken to ascertain whether the change of the water status and the activation of superoxide dismutase and their isoenzymes in Argan tree can support edaphic drought tolerance and its recovery under rehydration. An experiment was conducted on four contrasting ecotypes of Argania spinosa plants: two contrasting coastal ecotypes (Admine (Adm) and Rabia (Rab)) and two contrasting inland ecotypes (Aoulouz (Alz) and Lakhssas (Lks)). Drought stress significantly decreased the leaf water potential and stomatal conductance in the four contrasted ecotypes. In terms of biochemical responses, significant accumulation of carbonyl groups, hydrogen peroxide and superoxide radical has been recorded in the leaves of stressed plants reflecting oxidative stress. In parallel, the activities of total superoxide dismutase (SOD) and their isoenzymes Cu/Zn-SOD, Cu/Zn-SOD and Fe-SOD were also found to have increased to scavenging ROS and protecting the cell against induced oxidative stress. The recovery kinetics of A. spinosa, as a response to rehydration, were significant and rapid. According to the traits having the most discriminating power, both inland ecotypes (Lks and Alz) showed a better upregulation of its protective mechanisms compared to coastal ecotypes (Rab and Adm). All these adaptive traits make the inland ecotypes as an elite resource of drought tolerance and might become the new focus of domestication research of argan tree in arid and semi-arid environments.