Role of extracellular peroxidase in the superoxide production by wheat root cells

Summary Extracellular peroxidase has been shown to contribute to superoxide production in wounded wheat (Triticum aestivum L. cv. Ljuba) root cells. The superoxide-synthesizing system of root cells was considerably inhibited by KCN and NaN₃ and activated by MnCl₂ and H₂O₂. Treatment of roots with salicylic acid and a range of di- and tri-carbonic acids (malic, citric, malonic, fumaric, and succinic acids) stimulated superoxide production in both root cells and extracellular solution. The H₂O₂-stimulated superoxide production in the extracellular solution was much higher when roots were preincubated with salicylic or succinic acid. Exogenous acids enhanced peroxidase activity in the extracellular solution. Pretreatment of root cells with the detergents trypsin and sodium dodecyl sulfate had similar effects on the peroxidase activity. Significant inhibition of both superoxide production and peroxidase activity by diphenylene iodonium suggests that the specificity of the latter as an inhibitor of NADPH oxidase is doubtful. Results obtained indicate that extracellular peroxidase is involved in the superoxide production in wheat root cells. The mobile form of peroxidase can be readily secreted to the apoplastic solution and serve as an emergency enzyme involved in plant wound response.Abbreviations DPI diphenylene iodonium - ECS extracellular solution - ROS reactive oxygen species - SA salicylic acid     

Alterations of wheat root plasma membrane lipid composition induced by copper stress result in changed physicochemical properties of plasma membrane lipid vesicles

A response when wheat is grown in excess copper is an altered lipid composition of the root plasma membrane (PM). With detailed characterisation of the root PM lipid composition of the copper-treated plants as a basis, in the present study, model systems were used to gain a wider understanding about membrane behaviour, and the impact of a changed lipid composition.PMs from root cells of plants grown in excess copper (50 μM Cu²⁺) and control (0.3 μM Cu²⁺) were isolated using the two-phase partitioning method. Membrane vesicles were prepared of total lipids extracts from the isolated PMs, and also reference vesicles of phosphatidylcholine (PC). In a series of tests, the vesicle permeability for glucose and for protons was analysed. The vesicles show that copper stress reduced the permeability for glucose of the lipid bilayer barrier. When vesicles from stressed plants were modified by addition of lipids to resemble vesicles from control plants, the permeability for glucose was very similar to that of vesicles from control plants. The permeability for protons did not change upon stress.Electron paramagnetic resonance (EPR) of the lipid vesicles spin probed with n-doxylstearic acid (nDSA) was used to explore the lipid rotational freedom at different depth of the bilayer. The EPR measurements supported the permeability data, indicating that the copper stress resulted in more tightly packed bilayers of the PMs with reduced acyl chain motion.     

A hypothesis for the minimal overall structure of the mammalian plasma membrane redox system

Summary.  After a long period of frustration, many components of the mammalian plasma membrane redox system are now being identified at the molecular level. Some are apparently ubiquitous but are necessary only for a subset of electron donors or acceptors; some are present only in certain cell types; some appear to be associated with proton extrusion; some appear to be capable of superoxide production. The volume and variety of data now available have begun to allow the formulation of tentative models for the overall network of interactions of enzymes and substrates that together make up the plasma membrane redox system. Such a model is presented here. The structure discussed here is of the mammalian system, though parts of it may apply more or less accurately to fungal and plant cells too. Judging from the history of mitochondrial oxidative phosphorylation, it may be hoped that the development of models of the whole system – even if they undergo substantial revision thereafter – will markedly accelerate the pace of research in plasma membrane redox, by providing a coherent basis for the design of future experiments. Received May 4, 2002; accepted July 26, 2002; published online May 21, 2003 RID="*" ID="*" Correspondence and reprints: Department of Genetics, University of Cambridge, Downing Street, Cambridge CB2 3EH, United Kingdom. E-mail: ag24@gen.cam.ac.uk     

Vitamin C stabilization as a consequence of the plasma membrane redox system

Summary Ascorbate is stabilized in the presence of HL-60 cells. Our results showed that cAMP derivatives and agents that increase cAMP stimulate the ability of HL-60 cells to stabilize ascorbate. On the other hand, tunicamycin, a glycosilation-interfering agent, inhibited this ability. The ascorbate stabilization in the presence of HL-60 cells has been questioned as a simple chemical effect. Further properties and controls about the enzymatic nature of this stabilization are described and discussed. This data, together with hormonal regulation, support the hypothesis that an enzymatic redox system located at the plasma membrane is responsible of the extracellular ascorbate stabilization by HL-60 cells.Abbreviations AFR ascorbate free radicals - FCS fetal calf serum - Sp-cAMPS Sp-cyclic adenosine monophosphothionate - Rp-cAMPS Rp-cyclic adenosine monophosphothionate     

Evidence for electron transport across the plasma membrane of Zea mays root cells

Exogenous ferricyanide is reduced by roots of Z. mays. In contrast to oxidation of exogenous electron donors, ferricyanide reduction occurs mostly at the apical 5 mm of the root. Using just this portion of the root, it is shown that the activity is neither a consequence of uptake of ferricyanide followed by excretion of its reduced form, nor of leakage of a reductant. Addition of ferricyanide for 40 s or 5 min results in an apparent oxidation of NADPH but not of NADH; rates of ferricyanide reduction vary together with levels of NADPH but not of NADH in the presence or absence of oxygen. It is concluded that an enzyme which can oxidize cytoplasmic NADPH and transfer the electrons to an external acceptor exists at the cell surface of maize roots. This finding extends the results of others who showed similar redox activity at the surface of Fe-depleted dicotyledonous roots, and indicates that an energy source other than ATP exists at the cell surface of a variety of plants under unstressed conditions.     

Plasma membrane redox systems in tumor cells

Summary Plasma membrane redox systems in tumor cells are analyzed, their role in proton flux and tumor cell growth is described, and the modulation of their activity by antitumor drugs and growth factors is presented. As an example of the evolution of studies in the characterization of plasma membrane redox systems in tumor cells, we summarized our own results on the model system Ehrlich ascites carcinoma.     

Molecular characterization of a cold-induced plasma membrane protein gene from wheat

As a means to study the function of plasma membrane proteins during cold acclimation, we have isolated a cDNA clone for wpi6 which encodes a putative plasma membrane protein from cold-acclimated winter wheat. The wpi6 gene encodes a putative 5.9 kDa polypeptide with two predicted membrane-spanning domains, the sequence of which shows high sequence similarity with BLT101-family proteins from plants and yeast. Strong induction of wpi6 mRNA was observed during an early stage of cold acclimation in root and shoot tissues of both winter and spring wheat cultivars. In contrast to blt101 in barley, wpi6 mRNA was also induced by drought and salinity stresses, and exogenous application of ABA. Expression of wpi6 in a Δpmp3 mutant of Saccharomyces cerevisiae, which is disturbed in plasma membrane potential due to the lack of a BLT101-family protein, partially complemented NaCl sensitivity of the mutant. Transient expression analysis of a WPI6::GFP fusion protein in onion epidermal cells revealed that WPI6 is localized in the plasma membrane. Taken together, these data suggested that WPI6 may have a protective role in maintaining plasma membrane function during cold acclimation in wheat. The nucleotide sequence data for wpi6 have been recorded in the EMBL, GenBank and DDBJ Nucleotide Sequence Databases under the accession numbers AB030210 (cDNA) and AB221353 (genomic DNA).     

The NADH oxidizing system of the plasma membrane and metabolic signal control

Summary The development of ideas concerning plasma membrane redox reactions in normal and transformed animal cells is described, with emphasis on transferrin and ceruloplasmin. Control by hormones and growth factors, as well as the NAD⁺/NADH ratio in the cell are important in distinguishing the two types of cells.     

Up-regulation of plasma membrane-associated redox activities in neuronal cells lacking functional mitochondria

Mitochondria-deficient cells (rho(o) cells) survive through enhanced glycolytic metabolism in the presence of pyruvate and uridine. The plasma membrane redox system (PMRS) contains several NAD(P)H-related enzymes and plays a key role in maintaining the levels of NAD(+)/NADH and reduced coenzyme Q. In this study, rho(o) cells were used to investigate how the PMRS is regulated under conditions of mitochondrial dysfunction. rho(o) cells exhibited a lower oxygen consumption rate and higher levels of lactate than parental cells, and were more sensitive to glycolysis inhibitors (2-deoxyglucose and iodoacetamide) than control cells. However, they were more resistant to H(2)O(2), consistent with increased catalase activity and decreased oxidative damage (protein carbonyls and nitrotyrosine). PM-associated redox enzyme activities were enhanced in rho(o) cells compared to those in control cells. Our data suggest that all PMRS enzymes and biomarkers tested are closely related to the ability of the PMs to maintain redox homeostasis. These results illustrate that an up-regulated PM redox activity can protect cells from oxidative stress as a result of an improved antioxidant capacity, and suggest a mechanism by which neurons adapt to conditions of impaired mitochondrial function.     

Pentylenetetrazol modulates redox system by inducing addicsin translocation from endoplasmic reticulum to plasma membrane in NG108-15 cells

Addicsin (Arl6ip5) is a multifunctional physiological and pathophysiological regulator that exerts its effects by readily forming homo- and hetero-complexes with various functional factors. In particular, addicsin acts as a negative modulator of neural glutamate transporter excitatory amino acid carrier 1 (EAAC1) and participates in the regulation of intracellular glutathione (GSH) content by negatively modulating EAAC1-mediated cysteine and glutamate uptake. Addicsin is considered to play a crucial role in the onset of neurodegenerative diseases including epilepsy. However, the molecular dynamics of addicsin remains largely unknown. Here, we report the dynamics of addicsin in NG108-15 cells upon exposure to pentylenetetrazol (PTZ), a representative epileptogenic agent acting on the gamma-Aminobutyric acid A (GABA_A) receptor. Fluorescent immunostaining analysis demonstrated that addicsin drastically changed its localization from the endoplasmic reticulum (ER) to the plasma membrane within 1 h of PTZ exposure in a dose-dependent manner. Moreover, addicsin was co-localized with the plasma membrane markers EAAC1 and Na⁺/K⁺ ATPase alpha-3 upon PTZ stimulation. This translocation was significantly inhibited by a non-competitive GABA_A receptor antagonist, picrotoxin, but not by a competitive GABA_A receptor antagonist, bicuculline. Furthermore, lactate dehydrogenase (LDH) assay and 2,2-diphenyl-1-picrylhydrazyl (DPPH) radical-scavenging assay showed that PTZ-induced addicsin translocation was accompanied by a decrease of radical-scavenging activity and an increase of cytotoxicity in a PTZ dose-dependent manner. These findings suggest that PTZ induces the translocation of addicsin from the ER to the plasma membrane and modulates the redox system by regulating EAAC1-mediated GSH synthesis, which leads to the activation of cell death signaling.     

Characterization and solubilization of residual redox activity in salt-washed and detergent-treated plasma membrane vesicles from spinach leaves

Summary An NADH-hexacyanoferrate(III) oxidoreductase (N-HCF-OR) was purified from spinach leaf plasma membrane (PM) vesicles; detailed biochemical analyses, however, revealed that the purifed protein is an NADH-monodehydroascorbate oxidoreductase (N-MDA-OR) located on the cytoplasmic surface of the PM. After removing all N-MDA-OR activity from the PM vesicles by consecutive treatments with hypoosmotic shock, salt, and detergents, the remaining PM (the stripped PM, SPM) fraction contained about 50% of the protein and 15% of the N-HCF-OR activity of the original PM fraction. The highest redox activity (100%) of the SPM fraction was obtained with NADH as electron donor and hexacyanofer-rate(III) (HCF) as electron acceptor, although redox activity could be measured also with ubiquinone-0 (23%), dichlorophenolindophenol (16%), cytochromec (9%), and Fe³⁺-EDTA (2%) as electron acceptors. The followingK _m values were obtained for the N-HCF-OR activity of SPM:K _m(NADH)=66.5 ± 3.8 M [with 200 M HCF(III)],K _m[HCF(III)]=11.1 ± 1.1 M (with 150 M NADH). NAD⁺ competitively inhibited the activity. Under special conditions, SB-16 (palmityl sulfobetaine, a zwitterionic detergent with a C-16 hydrocarbon chain) solubilized about 50% of the protein and more than 90% of the N-HCF-OR activity of the SPM fraction. Redox activity of the solubilized fraction with dichlorophenolindophenol as electron acceptor was 45% of that with HCF(III). The SB-16-solubilized fraction containedb-type cytochrome(s) which could be reduced by dithionite> ascorbate > NADH. Silver-stained sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the SB-16-solubilized SPM fraction revealed numerous polypeptides between 17 and 95 kDa. Further purification steps are needed to match the redox activities and spectrophotometric data to one or more of the polypeptides seen on the gel.Abbreviations c.m.c. critical micellar concentration - DCPIP 2,6-dichlorophenolindophenol - HCF(III) hexacyanoferrate(III) - MDA monodehydroascorbate - N-DCPIP-OR NADH-2,6-dichlorophenol-indophenol oxidoreductase - N-HCF-OR NADH-hexacyanoferrate(III) oxidoreductase - N-MDA-OR NADH-monodehydro-ascorbate oxidoreductase - PM plasma membrane - SB-16 N-hexadecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (palmityl sulfobetaine, a zwitterionic detergent with a C-16 hydrocarbon chain) - SPM stripped plasma membrane     

A link between transport and plasma membrane redox system(s) in carrot cells

Carrot (Daucus carota L.) cells grown in suspension culture oxidized exogeneous NADH. The NADH oxidation was able to stimulate K⁺ (⁸⁶Rb⁺) transport into cells, but it did not affect sucrose transport.N,N'-Dicyclohexyl-carbodiimide, diethylstilbestrol, and oligomycin, which only partially inhibited NADH oxidation, almost completely collapsed the K⁺ (⁸⁶Rb⁺) transport. Vanadate, which is less effective as an ion transport inhibitor, was less effective in inhibiting the NADH-driven transport of K⁺ (⁸⁶Rb⁺).p-Fluormethoxycarbonylcyanide phenylhydrazone inhibits the K⁺ transport over 90% including that induced by NADH. The results are interpreted as evidence that a plasma membrane redox system in root cells is closely associated with the ATPase which can drive K⁺ transport. Because of the inhibitor effects, it appears that membrane components common to the redox system and ATPase function in the transport of K⁺.     

Plasma membrane redox and regulation of cell growth

Summary Gene expression can be activated by external oxidants which are reduced at the cell surface by plasma membrane electron transport. The signals generated in response to the plasma membrane electron transport include activation of proton release, internal calcium changes, and change in reductant/oxidant ratio in the cytosol. H₂O₂ generated in response to ligands which bind to plasma membrane receptors can also activate protein tyrosine kinases and gene expression. Inhibition of oxygen radical generation at the cell surface in response to the mitogen, phorbol myristate acetate by retinoic acid is consistent with a role for the plasma membrane electron transport as the source for H₂O₂ in Balb 3T3 cells. Agents which affect the binding of coenzyme Q to redox sites in the plasma membrane electron transport may increase formation of semiquinone radicals in the membrane which can be a source of oxygen radicals and H₂O₂. The generation of H₂O₂ by transformed cells indicates that oncogene product expression in the plasma membrane may also increase quinone-based oxygen radical generation.     

Iron at the cell surface controls both DNA synthesis and plasma membrane redox system

Summary Addition of the impermeable iron II chelator bathophenanthroline disulfonate (BPS) to cultured Chinese hamster lung fibroblast (CCL 39 cells) inhibits DNA synthesis but not protein synthesis or cytoplasmic alkalinization, when cell growth is initiated with growth factors such as EGF plus insulin, thrombin, or ceruloplasmin. The BPS inhibition is reversed by addition of stoichiometric ferrous iron at stoichiometric concentration. BPS does not inhibit cell growth stimulated by fetal calf serum. The effect of the BPS differs from the inhibition of growth by hydroxyurea which acts on the ribonucleotide reductase. The BPS treatment leads to release of iron from the cells as determined by BPS iron II complex formation over 90 min. Cells treated with BPS just during starvation period cannot re-initiate DNA synthesis after mitogen stimulation even if BPS is removed from the medium and cells are previously washed. BPS treatment also inhibits transplasma membrane electron which is restored by incubation of cells with 10 M ferric ammonium citrate. Growth factor stimulation of DNA synthesis is restored by addition of 1 M ferrous ammonium sulfate or ferric ammonium citrate, or 0.1 M diferric transferrin. Copper, cobalt, nickel, zinc, gallium, aluminum, or apotransferrin cannot restore the activity. The BPS effect is consistent with removal of iron from a site on the cell surface which controls electron transport and DNA synthesis.Abbreviations BCS bathocuproine disulfonate - BPS bathophenan-throline disulfonate - CUP ceruloplasmin - FCS fetal calf serum - Fe₂Tf diferric transferrin - EGF epidermal growth factor - HU hydroxyurea - THR -thrombin     

Regulated redox processes at the plasmalemma of plant root cells and their function in iron uptake

Plants take up iron as ferric chelates or, after reduction, as ferrous ions. Ferric reduction takes place at the plasma membrane of the root epidermis cells by a transmembrane redox system, which can be activated when iron is getting short. It is proposed that this inducible system, with NADPH as electron donor, is separate from a system, presumably present in all plant cells, which transports electrons from NADH or NADPH to ferricyanide, or,in vivo, oygen.     

春小地片质膜氧化还原系统及其对缓慢干旱胁迫的响应

研究了抗旱性不同的2个品种小麦（Triticum aestivum L.)叶片质膜氧化还原系统的部分性南及其在田间缓慢干时时下氧化还原活力的变化。结果显示,2个品种小麦叶片质膜氧化还原活性的最适pH为8．0,最适温度在40℃左右,Mg^2 对其活性有刺激作用,Ca^2 对其活性没有影响。但这2个品种叶片的质膜氧化还原系统对K^ 和Na^ 的响应不尽相同：在品种定西24中,K^ 刺激作用不太明显,Na^ 有一定的抑制作用;而在品种8139中,这两种离子都有明显的刺激作用。干旱降低了小麦叶片的水势和水分含量,影响了小麦的生长发育;在缓慢干旱下,小麦叶片质膜氧化还原活力在生长发育的前期上升;在后期,其活性不变或下降,这与前人在实验室内以植物幼苗进行短期而剧烈的模拟干旱下所观察的结果不同。这种差异的原因除了与植物材料不同有关外,主要与胁迫方式及植物的发育阶段有关。     

Drought stress stimulates p-nitrophenyl phosphate hydrolysis rate of the plasma membrane H+-ATPase from wheat leaves

The influence of drought stress on the ATP and p-nitrophenyl phosphate (PNPP) hydrolysis activity by plasma membrane H⁺-ATPase was investigated using purified plasma membrane vesicles from wheat leaves by two-phase partitioning. Drought stress increased the ATPase activity, and the optimal pH was shifted from 6.5 to about 7.0. Drought stress also stimulated the PNPP hydrolysis rate. The K_m for PNPP hydrolysis was moved from 4.49 ± 0.33 mM to 3.64 ± 0.12 mM. In addition, the PNPP hydrolysis was more sensitive to vanadate under drought compared to the control. However, the inhibitory effect of hydroxylamine on the ATPase was not changed by the present drought stress. In addtion, drought stress also decreased the trypsin activation of PNPP hydrolysis by PM H⁺-ATPase. These results suggested that drought stress altered the catalytic mechanism of the plasma membrane H⁺-ATPase, and the stimulation of its activity by drought stress was mainly due to increase of the catalytic activity of its phosphatase domain. It is also suggested that drought stress might alter the structure or property of the C-terminal end of PM H⁺-ATPase, therefore increasing the catalytic activity of the phosphatase domain.     

Contribution of plasma membrane Ca ATPase to cerebellar synapse function

The cerebellum expresses one of the highest levels of the plasma membrane Ca(2+) ATPase, isoform 2 in the mammalian brain. This highly efficient plasma membrane calcium transporter protein is enriched within the main output neurons of the cerebellar cortex; i.e. the Purkinje neurons (PNs). Here we review recent evidence, including electrophysiological and calcium imaging approaches using the plasma membrane calcium ATPase 2 (PMCA2) knockout mouse, to show that PMCA2 is critical for the physiological control of calcium at cerebellar synapses and cerebellar dependent behaviour. These studies have also revealed that deletion of PMCA2 throughout cerebellar development in the PMCA2 knockout mouse leads to permanent signalling and morphological alterations in the PN dendrites. Whilst these findings highlight the importance of PMCA2 during cerebellar synapse function and development, they also reveal some limitations in the use of the PMCA2 knockout mouse and the need for additional experimental approaches including cell-specific and reversible manipulation of PMCAs.