Purification and characterization of the plasmodial phosphatase that hydrolyses the phosphorylated light chain of Physarum myosin II from Physarum polycephalum

A phosphatase was purified through a combination of ion‐exchange and hydrophobic chromatography followed by native PAGE from Physarum plasmodia. Recently, we demonstrated that this phosphatase isoform has a hydrolytic activity towards the PMLC (phosphorylated light chain of Physarum myosin II) at pH 7.6. The apparent molecular mass of the purified enzyme was estimated at approximately 50 kDa by means of analytical gel filtration. The enzyme was purified 340‐fold to a final phosphatase activity of 400 pkat/mg of protein. Among the phosphorylated compounds tested for hydrolytic activity at pH 7.6, the enzyme showed no activity towards nucleotides. At pH 7.6, hydrolytic activity of the enzyme against PMLC was detected; at pH 5.0, however, no hydrolytic activity towards PMLC was observed. The K _m of the enzyme for PMLC was 10 μM, and the V _max was 1.17 nkat/mg of protein. Ca²⁺ (10 μM) inhibited the activity of the enzyme, and Mg²⁺ (8.5 μM) activated the dephosphorylation of PMLC. Mn²⁺ (1.6 μM) highly stimulated the enzyme's activity. Based on these results, we concluded that the enzyme is likely to be a phosphatase with hydrolytic activity towards PMLC.     

Effects of Salicylic Acid on Heavy Metal-Induced Membrane Deterioration Mediated by Lipoxygenase in Rice

Deterioration of membranes caused by lipoxygenase (LOX) activity under 10 μM PbCl₂ or 10 μM HgCl₂ was partially alleviated by the exogenous application of 100 μM salicylic acid (SA). In two cultivars of rice (Oryza sativa L. cvs. Ratna and IR 36), the presence of SA ameliorated the increased leakage of electrolytes, injury index, and the content of malondialdehyde caused by these heavy metals. Lead decreased H₂O₂ content whereas Hg increased it in both cultivars. Application of SA increased H₂O₂ in presence of Pb, while decreased it in presence of Hg. Both Pb and Hg decreased superoxide dismutase activity, while increased peroxidase activity. The activity of catalase was decreased by Hg but increased by Pb and SA reversed their effects. Thus, SA ameliorated the damaging effects of Pb and Hg on membranes. This revised version was published online in June 2006 with corrections to the Cover Date.     

Similarity in the acute cytotoxic response of mammalian cells to mercury (II) and X-rays: DNA damage and glutathione depletion

Pronounced strand breakage of DNA analyzed by alkaline elution techniques was produced in intact Chinese hamster ovary cells by 25 μM HgCl₂ within 1 hr or 100 μM HgCl₂ within 15 min. HgCl₂-induced strand breakage was directly proportional to concentration up to 100 μM and to time within 1 hr. Levels of reduced glutathione decreased following HgCl₂ in parallel with the induction of DNA strand breakage. Evidence is presented that this rapid and pronounced induction of DNA strand breaks and other cytotoxic responses following acute exposure to HgCl₂ resembles the cellular effects of X-rays.     

The effect of light quality on the induction of efficient photosynthesis under low CO2 conditions in Chlamydomonas reinhardtii and Chlorella pyrenoidosa

The effect of blue and red light on the adaptation to low CO₂ conditions was studied in high-CO₂ grown cultures of Chlorella Pyrenoidosa (82T) and Chlamydomonas reinhardtii(137⁺) by measuring O₂ exchange under various inorganic carbon (Cⁱ) concentrations. At equal photosynthetic photon flux density (PPFD), blue light was more favourable for adaptation in both species, compared to red light. The difference in photosynthetic oxygen evolution between cells adapted to low C_iunder blue and red light was more pronounced when oxygen evolution was measured under low C_i compared to high C_i conditions. The effect of light quality on adaptation remained for several hours. The different effects caused by blue and red light was observed in C. pyrenoidosa over a wide range of PPFD with increasing differences at increasing PPFD. The maximal difference was obtained at a PPFD above 1 500 μmol m^?2s^?1. We found no difference in the extracellular carbonic anhydrase activity between blue- and red light adapted cells. The light quality effect recorded under C_i-limiting conditions in C. reinhardtii cells adapted to air, was only 37% less when instead of pure blue light red light containing 12.5% of blue light (similar PPFD as blue light) was used during adaptation to low carbon. This indicates that in addition to affecting photosynthesis, blue light affected a sensory system involved in algal adaptation to low C_i conditions. Since the affinity for C_i of C. Pyrenoidosa and C. reinhardtii cells adapted to air under blue light was higher than that of cells adapted under red light, we suggest that induction of some component(s) of the C_i accumulating mechanism is regulated by the light quality.     

Purification,characterization, and coal depolymerizing activity of lignin peroxidase from <Emphasis Type="Italic">Gloeophyllum sepiarium</Emphasis> MTCC-1170

Lignin peroxidase from the liquid culture filtrate of Gloeophyllum sepiarium MTCC-1170 has been purified to homogeneity. The molecular weight of the purified enzyme was 42 kDa as determined by SDS-PAGE. The K _m values were 54 and 76 μM for veratryl alcohol and H₂O₂, respectively. The pH and temperature optima were 2.5 and 25°C, respectively. Depolymerization of coal by the fungal strain has been demonstrated using humic acid as a model of coal. Depolymerization of humic acid by the purified lignin peroxidase has been shown by the decrease in absorbance at 450 nm and increase in absorbance at 360 nm in presence of H₂O₂. Depolymerization of humic acid by the purified enzyme has also been demonstrated by the decrease in the viscosity with time of the reaction solution containing humic acid, H₂O₂, and the purified lignin peroxidase. The influence of NaCl and NaN₃ and inhibitory effects of various metal chelating agents on the lignin peroxidase activity were studied.     

Rapid purification of an active recombinant His-tagged 2,3-dihydroxybiphenyl 1,2-dioxygenase from Pseudomonas putida OU83

2,3-Dihydroxybiphenyl 1,2-dioxygenase (2,3-DBPD) is an extradiol-type dioxygenase that catalyzes the aromatic ring fission of 2,3-dihydroxybiphenyl, the third step in the biphenyl degradation pathway. The nucleotide sequence of the Pseudomonas putida OU83 gene bphC, which encodes 2,3-DBPD, was cloned into a plasmid pQE31. The His-tagged 2,3-DBPD produced by a recombinant Escherichia coli strain, SG13009(pREP4)(pAKC1), and purified with a Ni-nitrilotriacetic acid resin affinity column using the His-bind Qiagen system. The His-tagged 2,3-DBPD construction, carrying a single 6×His tail on the N-terminal of the polypeptide, was active. SDS-PAGE analysis of the purified active 2,3-DBPD gave a single band of 34 kDa; this is in agreement with the size of the bphC coding region. The K_m for 2,3-dihydroxybiphenyl was 14.5±2 μM. The enzyme activity was enhanced by ferrous ion but inhibited by ferric ion. The enzyme activity was inhibited by thiol-blocking reagents and heavy metals HgCl₂, CuSO₄, NiSO₄, and CdCl₂. The yield was much higher and the time required to purify recombinant 2,3-DBPD from clone pAKC1 was faster than by the conventional chromatography procedures.     

Effects of salicylic acid on ethylene induction and antioxidant activity in peach rootstock regenerants

Ethylene concentration in the culture tubes of peach rootstock regenerants of three genotypes (Cadaman, GF-677, Myrobalan 29C) was increased by the inclusion of 20 µM salicylic acid (SA), methionine (METH) and ethephon (ETH) in the MS medium whereas it was decreased in regenerants exposed up to 20 µM AgNO₃. In leaves of the regenerants the increase of ethylene concentration was accompanied with an increase of non-enzymatic antioxidant activity while remarkable genotype-depended changes in the activities of catalase, peroxidase and their isoenzymes were recorded suggesting that ethylene accumulation imposes oxidative stress responses. However, the results showed that some differences could be observed in the activity of isoenzymes in regenerants exposed to SA in respect to METH and ETH-treated ones.A. Molassiotis is grateful to the State Scholarship’s Foundation of Greece for a fellowship during this work.     

Production of an epigenetic mutant population of Populus nigra: DNA methylation and phenotype analyses

5-Azacytidine (5-AzaC) causes hypomethylation of genomic DNA and induces phenotype variation in many plant species. Modulating the methylation status of cytosine by 5-AzaC to generate novel phenotypes in poplars have not been attempted. In this study, a population of 288 plants was regenerated from leaves of Populus nigra on medium supplemented with 100–1000 µM 5-AzaC. The differentiation of leaves was delayed and the differentiation rate decreased as the 5-AzaC concentration increased. Compared with plants regenerated on 5-AzaC-free medium (control), the plants regenerated on high-5-AzaC medium (600–1000 µM) had significantly higher DNA methylation levels and were shorter, whereas the plants regenerated on low-5-AzaC medium were unchanged; the plants regenerated on 100 µM 5-AzaC medium had significant more leaves; the plants regenerated on 400 µM 5-AzaC medium had significantly higher leaf chlorophyll b and total chlorophyll contents, while the plants on 800 and 1000 µM 5-AzaC medium had a significantly lower chlorophyll a content; most photosynthetic parameters of the plants regenerated on 5-AzaC-treated medium decreased, except for the net photosynthetic rate (P_n) and transpiration rate (T_r) of plants on 100, 200, and 400 µM medium, and the instant water utilization efficiency (WUE_i) of plants on 1000 µM medium. The superoxide dismutase activity of plants on ≥ 400 µM 5-AzaC medium was significantly higher than in the control. Significant weak correlations were found between the methylation level of regenerants and plant height, chlorophyll a content, P_n, T_r, stomatal conductance, intercellular CO₂ concentration, and WUE_i, indicating these phenotypic changes were related to the methylation changes. Our results demonstrated the use of 5-AzaC at the regeneration stage induced epigenetic as well as phynotypic variations in poplar regenerants and generated plant materials for genetic breeding and epigenetic studies.

     

Maize recombinant non-C<Subscript>4</Subscript> NADP-malic enzyme: A novel dimeric malic enzyme with high specific activity

Among the different isoforms of NADP-malic enzyme (NADP-ME) involved in a wide range of metabolic pathways in plants, the NADP-ME that participates in C₄-photosynthesis is the most studied. In the present work, the expression in E. coli of a cDNA encoding for a maize non-photosynthetic NADP-ME is presented. The recombinant NADP-ME thus obtained presents kinetic and structural properties different from the enzyme previously purified from etiolated leaves and roots. Moreover, the recombinant non-photosynthetic NADP-ME presents very high intrinsic NADP-ME activity, which is unexpected for a non-C₄ NADP-ME. Using antibodies against this recombinant enzyme, an immunoreactive band of 66 kDa is detected in different maize tissues indicating that the 66 kDa-NADP-ME is in fact a protein expressed invivo. The recombinant NADP-ME assembles as a dimer, although the results obtained indicate that a higher molecular mass oligomeric state of the enzyme is found in maize roots in vivo. In this way, maize presents at least three NADP-ME isoforms: a 72 kDa constitutive form (previously characterized); the novel non-photosynthetic 66 kDa isoform characterized in this work (which is the product of the ZmChlMe2gene and the likely precursor to the evolution of the photosynthetic C₄ NADP-ME) and the 62 kDa isoform (implicated in C₄ photosynthesis). The contribution of the present work anticipates further studies concerning the equilibrium between the oligomeric states of the NADP-ME isoforms and the evolution towards the C₄ isoenzyme in maize.     

Antioxidant Enzyme Isoforms on Gels in Two Poplar Clones Differing in Sensitivity After Exposure to Ozone

The effect of acute ozone (O₃) fumigation on isozyme patterns of superoxide dismutase (SOD), peroxidase (POD) and ascorbate peroxidase (APX) in mature (ML) and young leaves (YL) of two poplar clones, contrasting in O₃-sensitivity was analysed. Untreated leaves of both the O₃-sensitive (O₃-S) clone Eridano of Populus deltoides×P. maximowiczii and the O₃-resistant (O₃-R) clone I-214 of P.×euramericana showed four distinct SOD isoforms with a relative mobility (R_f) of 0.54 (MnSOD), 0.60 (Cu/ZnSOD), 0.65 (unidentified), and 0.71 (Cu/ZnSOD). After O₃-fumigation the activity of the SOD isoforms showed only quantitative variations with respect to control plants. In ML of untreated O₃-R plants seven POD isoforms (R_f= 0.13, 0.19, 0.34, 0.59, 0.64, 0.70 and 0.75) were found, while in YL one isoform (R_f= 0.34) was undetected. Only three POD isoforms in both ML and YL of untreated O₃-S plants were resolved. The electrophoretic pattern of POD in O₃-S leaves was greatly modified by acute O₃-fumigation with the appearance of new isoforms in both YL and ML and the disappearance of an isoform (R_f= 0.13) in YL. Additionally, O₃-exposure induced the appearance of two APX isoforms in YL (R_f= 0.66 and 0.70), and one isoform in ML (R_f= 0.70) of the O₃-S clone. By contrast, the activity of the three APX isoformes (R_f= 0.64, 0.70 and 0.76) detected in O₃-R leaves showed only quantitative variation with respect to untreated plants. From these data it is concluded that: 1) in these poplar hybrids antioxidant enzyme activity is developmentally regulated and greatly affected by acute O₃ stress treatments and 2) the different enzymes activity displayed by the two poplar clones, especially for POD and APX isoformes, could partly explain their distinct O₃-sensitivity.     

KINETICS OF SILICON-LIMITED GROWTH IN THE FRESHWATER DIATOM ASTERIONELLA FORMOSA1,2

Growth rates of two clones of the freshwater planktonic diatom Asterionella formosa Hass. were measured under conditions in which external silicon concentrations controlled growth. Clone AfOH2 from Lake Ohrid, Yugoslavia, had a higher maximum growth rate (μ_max= 1.11 doublings/day) and apparent half-saturation constant (K_si] + Si_o= 1.93 μM Si) than clone L262 from Lake Windermere, England. (μ_max= 0.61 doublings/day; K_si+ Si_o= 1.09 μM Si). K_lim, the silicon concentration at μ= 0.9 μ_max, is 13.8 μM Si for clone AfOH2 and 6.5 μM Si for clone L262. These values agree well with published field observations showing A. formosa populations decreasing below 0.5 mg/l SiO₂ (= 8.4 μM Si). Calculations of yield gave a range of 0.5–1.5 μM Si/10⁶ cells for clone AfOH2 and 0.6–1.9 μM Si/10⁶ cells for clone L262.     

Isolation and characterization of Glyceraldehyde-3-phosphate dehydrogenase from the common octopus (<Emphasis Type="Italic">Octopus vulgaris </Emphasis>Cuvier, 1797)

The NAD⁺ dependent cytosolic Glyceraldehyde-3-phosphate dehydrogenase (GAPDH, EC 1.2.1.12) from arms of Octopus vulgaris, Cuvier, 1787, (Octopoda, Cephalopoda) was purified to homogeneity and its kinetic properties investigated. The purification method consisted of ammonium sulfate fractionation followed by Blue Sepharose CL-6B chromatography resulting in a 26-fold increase in specific activity and a final yield of approximately 16%. The apparent molecular weight of the purified native enzyme was 153 kDa. The protein is an homotetramer, composed of identical subunits with an apparent molecular weight of approximately 36 kDa. The Michaelis constants K_m for both NAD⁺ and d-G3P were 66 μM and 320 μM, respectively. The maximal velocity V_max of the purified enzyme was estimated to be 21.8 U/mg. Only one GAPDH isoform (pI 6.6) was obtained by isoelectrofocusing in polyacrylamide slab gels holding ampholyte generated pH gradients. Under the conditions of assay, the optimum activity occurs at pH 7.0 and at temperature of 35°C. Polyclonal antibodies raised in rabbits against the purified GAPDH immunostained a single 36 kDa GAPDH band on crude extract protein preparations blotted onto nitrocellulose.     

Identification of catalytic residues of a very large NAD-glutamate dehydrogenase from Janthinobacterium lividum by site-directed mutagenesis

We previously found a very large NAD-dependent glutamate dehydrogenase with approximately 170?kDa subunit from Janthinobacterium lividum (Jl-GDH) and predicted that GDH reaction occurred in the central domain of the subunit. To gain further insights into the role of the central domain, several single point mutations were introduced. The enzyme activity was completely lost in all single mutants of R784A, K810A, K820A, D885A, and S1142A. Because, in sequence alignment analysis, these residues corresponded to the residues responsible for glutamate binding in well-known small GDH with approximately 50?kDa subunit, very large GDH and well-known small GDH may share the same catalytic mechanism. In addition, we demonstrated that C1141, one of the three cysteine residues in the central domain, was responsible for the inhibition of enzyme activity by HgCl₂, and HgCl₂ functioned as an activating compound for a C1141T mutant. At low concentrations, moreover, HgCl₂ was found to function as an activating compound for a wild-type Jl-GDH. This suggests that the mechanism for the activation is entirely different from that for the inhibition.     

A Branched-chain Amino Acid Aminotransferase from the Rumen Ciliate Genus Entodinium

A branched-chain amino acid aminotransferase was extracted from rumen ciliates of the genus Entodinium and was partially purified by Sephadex G-200, DEAE-cellulose and DEAE-Sephadex A-50 column chromatography. The purified enzyme was active only with leucine, isoleucine and valine, and required pyridoxal phosphate as cofactor. The amino acids competed with each other as substrates. The enzyme had optimal activity at pH 6.0 in phosphate buffer. The K_m values for the substrates and cofactor are as follows: 1.66 for leucine; 0.90 for isoleucine; 0.79 for valine; 0.29 mM for α-ketoglutarate: and 0.1 μM for pyridoxal phosphate. Enzyme activity was inhibited by p-chloromercuribenzoate and HgCl₂. Gel filtration indicated the enzyme to have a molecular weight of 34,000.     

Effects of mercury and cadmium on the activities of antioxidative enzymes in the seedlings ofPhaseolus aureus

Phaseolus aureus Roxb. was exposed to HgCl₂ and Cd(NO₃)₂ either at the germination stage in concentration 0.5, 5 and 25 μM for 48 and 96 h, or at the seedling stage (5^th day of germination) in concentration 0.5, 5 and 20 μM for 6, 24 and 48 h. The germination and the growth of roots (germination stage treatment) were less in Hg than in Cd treatment. The seedlings (seedling stage treatment) were, however, more susceptible to Cd than Hg. Both root and leaf tissues of the plant treated at the germination stage showed enhanced lipid peroxidation and activities of the antioxidative enzymes (catalase, guaiacol peroxidase and ascorbate peroxidase), except the catalase in leaf in 25 μM Cd treatment. At seedling stage the content of malondialdehyde increased significantly only in the leaf tissue, during 6 h exposure. The activities of all the enzymes exhibited an increasing trend in both the tissue of the seedlings, particularly the leaf, at least after 24 and 48 h, except the catalase whose activity declined in response to Cd. Active involvement of the guaiacol and ascorbate peroxidases, rather than catalase, in scavenging cellular H₂O₂ was indicated. It was concluded that the two metals had little primary damaging effect on membranes.     

DMPS reverts morphologic and mitochondrial damage in OK cells exposed to toxic concentrations of HgCl<Subscript>2</Subscript>

Mercuric chloride (HgCl₂) is a highly toxic compound, which can cause nephrotoxic damage. In the present study effects of HgCl₂ on mitochondria integrity and energy metabolism, as well as antidotal effects of 2,3-dimercaptopropane-1-sulfonate (DMPS) were investigated in the opossum kidney derived cell line (OK). OK cell monolayers were incubated during 0, 1, 3, 6, and 9 h in serum-free culture medium containing 15 μM HgCl₂, either in the absence or in the presence of 60 μM DMPS in a 1:4 ratio. Intracellular ATP content, MTT reduction, and HSP70/HSP90 induction were studied; confocal, transmission electron microscopy, and light microscopy studies were also performed. For confocal analysis, a mitochondrial selective probe (MitoTracker Red CMXH2Ros) was used. Antioxidant activity of DMPS was also studied by the scavenging of the free radical 2, 2-diphenyl-1-picrylhydrazyl (DPPH) technique. A decrease of ATP content, an impaired ability to reduce tetrazolium, and dramatic changes on cellular and mitochondrial morphology, and energetic levels were found after either 6 or 9 h of HgCl₂ exposure. Increased expression of HSP90 and HSP70 were also seen. When OK cells were co-incubated with HgCl₂ and DMPS, cellular morphology, viability, intracellular ATP, and mitochondrial membrane potential were partially restored; a protective effect on mitochondrial morphology was also seen. DMPS also showed potent antioxidant activity in vitro. Mitochondrial protection could be the cellular mechanism mediated by DMPS in OK cells exposed to a toxic concentration of HgCl₂.     

Phycobiliproteins from <Emphasis Type="Italic">Pseudanabaena tenuis</Emphasis> rich in c-phycoerythrin protect against HgCl<Subscript>2</Subscript>-caused oxidative stress and cellular damage in the kidney

Our objective was to study if the phycobiliproteins of the cyanobacterium Pseudanabanea tenuis rich in phycoerythrin protect renal cells against mercury-caused oxidative stress and cellular damage in the kidney. We used 40 male mice that were assigned into five groups: a control group that received phosphate buffer (PB) and saline and four treatment groups which received either PB+HgCl₂, PB+phycobiliproteins, or HgCl₂+phycobiliproteins. The kidneys of the mice were used to determine lipid peroxidation and quantification of reactive oxygen species, oxidized glutathione, and peroxidase activities (catalase and glutathione peroxidase) and were also examined histologically. Our results demonstrated that HgCl₂ causes oxidative stress and cellular damage and that all doses of phycobiliproteins prevented the increase of oxidative markers and partially protected against HgCl₂-caused cell damage. This is the first report which applied phycobiliproteins of P. tenuis rich in c-phycoerythrin, like antioxidants against mercury chloride-caused oxidative stress and renal damage.     

Purification and characterization of a lectin from seeds and cotyledonary callus of Zizyphus mauritiana

A lectin from Zizyphus mauritiana lamk. has been purified from the 25–50% (NH₄)₂SO₄ fraction of crude seed and cotyledonary leaf callus extracts. The lectins purified from the two sources had the same structure and properties. Upon specific adsorption on Sephadex G-100, the lectin (ZML) could be displaced with 0.1 m d-glucose. ZML yielded a single band corresponding to a M_r of 66 kDa both in the absence and presence of β-mercaptoethanol on native- as well as in SDS-PAGE. It is thermostable but pH sensitive and agglutinates human erythrocytes only. Lectin activity could also be detected in the cotyledons, leaf, and stem, and in their cultures, as well as in in vitro regenerants. Cotyledons, cotyledonary leaf and its callus showed much higher lectin activity than seeds. Received: 5 April 1997 / Revision received: 30 June 1997 / Accepted: 31 October 1997     

The Expression Pattern of NAD(P)H Oxidases and the Cyclic Electron Transport Pathway around Photosystem I of Synechocystis sp. PCC6803 Depend on Growth Conditions

Cyclic electron transport and NADH and/or NADPH (NAD(P)H)-oxidizing activities were investigated in Synechocystis sp. PCC6803 grown under various stressed conditions and in ndhB-less (M55) and ycf33-deletion mutants. Activity staining and inhibitor data suggested that the ferredoxin-quinone reductase (FQR) route is the main pathway in ycf33-deletion and high-light (300 μE m^?2 s^?1)-grown cells as well as in M55 cells. The FQR route was highly sensitive to HgCl₂, but not to diphenyleneiodonium (DPI). On the other hand, cells grown under low CO₂ (0.03%) or normal (100 μE m^?2 s^?1, 3% CO₂) conditions were found perhaps to use the complex I-type NAD(P)H dehydrogenase route, which was found to be highly sensitive to DPI but not to HgCl₂. In high-salt (0.55 M NaCl)-grown cells, the amount of ferredoxin-NADP⁺ oxidoreductase (FNR) increased, and the main cyclic electron flow was perhaps the FNR route. Both DPI and HgCl₂ were strong inhibitors of the FNR route.