The Electrical Potential Difference Across the Tonoplast of Root Cells

Changes in electrical potential, measured as a microelectrodewas advanced into epidermal cells and from cell to cell in rootsof Lolium multiflorum and Zea mays, are described. The recordingssuggest that the electrical potential difference between thecytoplasm and vacuole, E_vc is of the order of a few millivolts,the vacuole tending to be the more positive. E_vc appeared tobe approximately the same for epidermal, cortical, endodermal,and pericycle cells.     

An electrophysiological study of the membrane in aplanospore-like cell of Boergesenia forbesii

Membrane potential and resistance, each of which was the sumof those of the plasmalemma and tonoplast, measured in the coenocyticthallus of Boergesenia forbesii were 6.7 mv inside positiveand 2.8 k.cm², respectively. Protoplasm squeezed from the thallus into artificial sea water(ASW) formed numerous spherical bodies, which are termed aplanospore-likecells (simply "spores"). The following electrical propertiesof the "spores" 20–40 hr after squeezing were obtained:potential difference (p.d.) across plasmalemma (E_co) was –66mv (– means inside negative), plasmalemma resistance 665cm², p.d. across the tonoplast (E_vc) +73 mv, and tonoplast resistance2.6 k.cm². Tenfold increase in external [K⁺] caused +45 mv changein E_co and +17 mv in E_vc. The plasmalemma was entirely depolarizedin Ca⁺⁺-free ASW or ASW containing Triton X-100. When the "spore" was immersed in potassium-rich (277 mil) ASW,E_co was almost zero and the tonoplast showed two states (I andII, E_ve about +70 mv and +20 mv, respectively). E_vc went backand forth between the two states spontaneously or when a smallcurrent was applied. In most cases oscillatory changes in E^vcoccurred after the lapse of a long time in the K+-rich sea water.Membrane resistances in states I and II were 5 and 9 k.cm²,respectively. (Received July 11, 1977; )     

Movement and compartmentation of abscisic acid in guard cells of Valerianella locusta: Effects of osmotic stress,external H+-concentration and fusicoccin

Epidermal peels of Valerianella locusta were acid-treated for 1 h at pH 3.9 to kill all cells other than guard cells. These guard-cell preparations were used to explore the steady-state one-way fluxes and the cytoplasmic and vacuolar contents of abscisic acid (ABA). The method of compartmental analysis has been applied. The intracellular ABA concentrations were surprisingly high. At an external pH of 5.8 the cytoplasm contained 1.28 mmol·dm^-3 of ABA, twice of the amount which accumulated in the vacuoles (0.57 mmol·dm^-3). The fluxes of ABA at the plasmalemma (_oc=_oc=0.43 fmol · cell ^–1 · h ^–1) were higher than those at the tonoplast (_cv=_vc=0.12 fmol · cell ^–1 · h ^–1). Moderate stress (0.1 and 0.3 mol·dm^-3 sorbitol in the medium) caused a change in the kinetics of ABA movement. The rate constants of the fluxes from the cytoplasm into the vacuole (_cv) and into the apoplast (_co) were increased while the rate constant of the flux from the vacuoles into the cytoplasm (_vc) was decreased. As a consequence the amount of ABA sequestered in the vacuole remained unchanged; the cytoplasmic ABA content, however, was reduced to only 20% of that found in the control treatments (no sorbitol in the medium). Under moderate stress, one Valerianella guard cell released rapidly about 0.36 fmol·cell^-1 to its direct cell-wall space. This surprising result is discussed in regard to rapid stomatal closure under reduced water supply.Abbreviations ABA abscisic acid - FC fusicoccin     

Electro-enzymic viologen-mediated stereospecific reduction of 2-enoates with free and immobilized enoate reductase on cellulose filters or modified carbon electrodes

Parametric equations describing the total reaction rate in an electrochemical cell containing free enoate reductase are presented and their use in determining optimal cell and electrode dimensions discussed. Immobilized enoate reductase from Clostridium tyrobutyricum DSM 1460 was used for the repetitive stereospecific reduction of (E)-2-methyl-3-phenyl-2-propenoate and (E)-2-methyl-2-butenoate to their respective (R)-enantiomeric saturated products. The reducing equivalents were provided by electrochemically regenerated methylviologen. The enzyme immobilized in calcium alginate was used in two different systems: (a) on cellulose filters packed into a reactor outside the electrochemical cell, and (b) in the electrochemical cell on a carbon felt electrode soaked with the enzyme and alginate before it was cross-linked with calcium ions.Cyclic voltammetry indicated that the ionotropic gel increased the concentration of methylviologen close to the electrode surface. Via immobilization the half life of enoate reductase in the electrochemical cell increased from about 8–350 h of continuous operation.     

Redox-state of intactNitella cells: dependency on intracellular pH and photosynthesis

Summary The present paper describes the construction and properties of a Pt/Ir-semi-microelectrode and its application as a redoxsensitive electrode in intact cells of the giant algaNitella. For compartmental analysis of the stationary redox-state voltage (E_RED), a value reflecting the interaction of the dominant redox couples with a Pt/Ir-electrode, the redox-sensitive electrode was inserted into the vacuole of leaf cells or cytoplasm enriched fragments (CEF) fromNitella internodal cells. After correction for the membrane voltage, measured with a second, conventional voltage electrode, E_RED values of+237±93mVand+419±51 mV with respect to a normal H⁺-electrode were obtained for cytoplasm and vacuole, respectively. The redox-state of the cell culture medium was+604 mV. The steady state E_RED in the cytoplasm can be perturbed by experimental treatments: indirect acidification of the cytoplasm by an external pH jump from 7.5 to 5.8 and direct acidification, by acid loading with 5 mM butyrate, both resulted in a positive shift of E_RED, i.e., to an increase in cytoplasmic oxidation. At the same time the membrane depolarized electrically following the external pH jump, but hyperpolarized in response to acid loading. The data demonstrate the direct dependence of cytoplasmic redox state on intracellular pH, probably due to enhanced oxidation of protonated redox couples favoured by mass action. The electrical membrane voltage changes were not correlated with the shift in cytoplasmic E_RED. This demonstrated that redox energy does not determine the electrical membrane voltage. Cytoplasmic E_RED was also affected by photosynthesis. When CEFs were transferred from light to dark, or exposed to 10M 3-(3,4-dichlorophenyl)-1,l-dimethylurea (DCMU), E_RED shifted negatively (more reduced) by 6.4±4.5mV or 4.2±2mV, respectively. These data compare favourably with biochemical estimates of cytoplasmic pyridin nucleotides which also show an increase in cytoplasmic reduction in the dark. Therefore, it is unlikely that diffusable reducing equivalents are supplied to the cytoplasm from photosynthetically-active chloroplasts to act as secondary messengers.Abbreviations E_M transmembrane voltage - E_RED redox-state voltage - E₀ midpoint-redox-voltage - APW artificial pond water - CEF cytoplasm enriched fragment     

Design,synthesis, 3D pharmacophore,QSAR, and docking studies of carboxylic acid derivatives as Histone Deacetylase inhibitors and cytotoxic agents

In this study, five series of (E)-6-(4-substituted phenyl)-4-oxohex-5-enoic acids IIb–f (E), (E)-3-(4-(substituted)-phenyl)acrylic acids IIIa–g (E), 4-(4-(substituted)phenylamino)-4-oxobutanoic acids VIa,b,e, 5-(4-(substituted)phenylamino)-5-oxopentanoic acids VIIa,f and 2-[(4-(substituted)phenyl) carbamoyl]benzoic acids VIIIa,e were designed and synthesized. Selected compounds were screened in vitro for their cytotoxic effect on 60 human NCI tumor cell lines. Compound IIf (E) displayed significant inhibitory activity against NCI Non-Small Cell Lung A549/ATCC Cancer cell line (68% inhibition) and NCI-H460 Cancer cell line (66% inhibition). Moreover, the final compounds were evaluated in vitro for their cytotoxic activity on HepG2 Cancer cell line in which histone deacetylase (HDAC) is overexpressed. Compounds IIc (E), IIf (E), IIIb (E), and IIIg (E) exhibited the highest cytotoxic activity against HepG2 human cancer cell lines with IC₅₀ ranging from 2.27 to 10.71 μM. In addition, selected compounds were tested on histone deacetylase isoforms (HDAC1–11). Molecular docking simulation was also carried out for HDLP enzyme to investigate their HDAC binding affinity. In addition, generation of 3D-pharmacophore model and quantitative structure activity relationship (QSAR) models were combined to explore the structural requirements controlling the observed cytotoxic properties.     

Nucleic Acid Similarities Among Pseudomonas pseudomallei, Pseudomonas multivorans, and Actinobacillus mallei

Annealing experiments on membrane filters were carried out with deoxyribonucleic acids (DNA) from selected strains of the nomen-species of Pseudomonas, Actinobacillus, Chromobacterium, and Micrococcus, with the use of DNA of Pseudomonas pseudomallei and Actinobacillus mallei as reference materials. Under the usual conditions employed in these experiments, the results were not quantitatively reproducible. Incorporation of dimethylsulfoxide (DMSO) into the incubation medium greatly increased differences in comparative binding. DNA binding in agar matrices was examined in the presence and absence of DMSO at various incubation temperatures. It was found that the greatest specificity, stability, and total binding for DNA containing high amounts of guanine and cytosine occurred in the presence of DMSO. Under the most stringent annealing conditions permitted in agar, DNA species from P. pseudomallei and A. mallei in the presence of DMSO demonstrated interspecific relative bindings of 76 to 86% when compared to the homologous reactions. The thermal elution midpoints (E_m) of these duplexed interspecific DNA species were quite close to the homologous E_m values. The relative bindings of P. multivorans DNA types to either reference DNA ranged between 6 to 27%, and the E_m values were 4 to 7 C less than those for the homologous reactions.     

Extracellular charge adsorption influences intracellular electrochemical homeostasis in amphibian skeletal muscle

The membrane potential measured by intracellular electrodes, E_m, is the sum of the transmembrane potential difference (E₁) between inner and outer cell membrane surfaces and a smaller potential difference (E₂) between a volume containing fixed charges on or near the outer membrane surface and the bulk extracellular space. This study investigates the influence of E₂ upon transmembrane ion fluxes, and hence cellular electrochemical homeostasis, using an integrative approach that combines computational and experimental methods. First, analytic equations were developed to calculate the influence of charges constrained within a three-dimensional glycocalyceal matrix enveloping the cell membrane outer surface upon local electrical potentials and ion concentrations. Electron microscopy confirmed predictions of these equations that extracellular charge adsorption influences glycocalyceal volume. Second, the novel analytic glycocalyx formulation was incorporated into the charge-difference cellular model of Fraser and Huang to simulate the influence of extracellular fixed charges upon intracellular ionic homeostasis. Experimental measurements of E_m supported the resulting predictions that an increased magnitude of extracellular fixed charge increases net transmembrane ionic leak currents, resulting in either a compensatory increase in Na⁺/K⁺-ATPase activity, or, in cells with reduced Na⁺/K⁺-ATPase activity, a partial dissipation of transmembrane ionic gradients and depolarization of E_m.     

A facilitated electron transfer of copper–zinc superoxide dismutase (SOD) based on a cysteine-bridged SOD electrode

The direct electrochemical redox reaction of bovine erythrocyte copper–zinc superoxide dismutase (Cu₂Zn₂SOD) was clearly observed at a gold electrode modified with a self-assembled monolayer (SAM) of cysteine in phosphate buffer solution containing SOD, although its reaction could not be observed at the bare electrode. In this case, SOD was found to be stably confined on the SAM of cysteine and the redox response could be observed even when the cysteine-SAM electrode used in the SOD solution was transferred to the pure electrolyte solution containing no SOD, suggesting the permanent binding of SOD via the SAM of cysteine on the electrode surface. The electrode reaction of the SOD confined on the cysteine-SAM electrode was found to be quasi-reversible with the formal potential of 65±3 mV vs. Ag/AgCl and its kinetic parameters were estimated: the electron transfer rate constant k_s is 1.2±0.2 s⁻¹ and the anodic (α_a) and cathodic (α_c) transfer coefficients are 0.39±0.02 and 0.61±0.02, respectively. The assignment of the redox peak of SOD at the cysteine-SAM modified electrode could be sufficiently carried out using the native SOD (Cu₂Zn₂SOD), its Cu- or Zn-free derivatives (E₂Zn₂SOD and Cu₂E₂SOD, E designates an empty site) and the SOD reconstituted from E₂Zn₂SOD and Cu²⁺. The Cu complex moiety, the active site for the enzymatic dismutation of the superoxide ion, was characterized to be also the electroactive site of SOD. In addition, we found that the SOD confined on the electrode can be expected to possess its inherent enzymatic activity for dismutation of the superoxide ion.     

Requirements for obtaining maximum indices of photosynthesis and transpiration in attached leaves of willow plants grown in short-rotation forest

The results of multiyear studies of gas exchange in intact attached leaves of several willow species (Salix sp.) were analyzed. Measurements were performed with a portable Li-6400 infrared gas analyzer both on plants in their natural environment and on rooted cuttings grown in a greenhouse. Individual attached leaves were placed into the leaf chamber where climatic conditions were either similar to or different from those outside the chamber. The maximal rates of net photosynthesis (P _n) and transpiration (E) were only observed with the provision that the environmental variables inside and outside the chamber were identical. On rainy or cloudy days, the P _n and E values observed under optimum conditions inside the leaf chamber were lower than their potential maxima by 12–18% and 35–45%, respectively. Deviation of temperature in the chamber by 5–7°C from the external level and fluctuations of ambient temperature affected P _n but not E rates of tested leaves. Variations in relative air humidity in the chamber directly influenced E but had no effect on P _n of attached leaves. It was shown that the maximum rates of gas exchange in the attached willow leaf could be only attained by providing optimum conditions for the whole plant.     

Click synthesis of estradiol–cyclodextrin conjugates as cell compartment selective estrogens

Cyclodextrin (CD) is a well known drug carrier and excipient for enhancing aqueous solubility. CDs themselves are anticipated to have low membrane permeability because of relatively high hydrophilicity and molecular weight. CD derivatization with 17-beta estradiol (E₂) was explored extensively using a number of different click chemistries and the cell membrane permeability of synthetic CD–E₂ conjugate was explored by cell reporter assays and confocal fluorescence microscopy. In simile with reported dendrimer–E₂ conjugates, CD–E₂ was found to be a stable, extranuclear receptor selective estrogen that penetrated into the cytoplasm.     

Effect of estrogen-treated porcine ampulla oviductal epithelial cells on early embryonic development in vitro and characterization of their protein synthetic activity

Recent studies by Buhi et al. have demonstrated that estrogen (E₂) is responsible for the induction of de novo synthesis and secretion of certain oviductal secretory proteins (OSP) and inhibition of other OSP in porcine oviductal explant cultures. The present work was undertaken to evaluate the effect of E₂-treated oviductal epithelial cell coculture on the development of early porcine embryos derived from in vitro matured and fertilized oocytes. In vitro synthesis of secretory proteins by E₂-treated oviductal cells used for coculture was also investigated by one-dimensional (1D) and two-dimensional (2D) sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis (PAGE). The results showed that the cleavage rate was significantly enhanced by coculturing fertilized eggs with E₂-treated oviductal epithelial cells. The in vitro protein synthetic pattern of oviductal secretory proteins was influenced by E₂ treatment. These variations included the disappearance of one protein (82 000 M_r) and the appearance of another (33 000 M_r) in the E₂-treated group as assessed by 1D-SDS-PAGE. Additional proteins of M_r 97 000 and an M_r 36 000–45 000 complex were increased in abundance by the E₂ treatment. Analyses by 2D-SDS-PAGE revealed three major E₂-dependent proteins, of M_r 45 000 (pI 5.5), 43 000 (pI 5.5) and a 36 000–45 000 M_r (pI 4.8) protein complex, whereas polypeptides of M_r 97 000 (pI 5.1), 36 000 (pI 8.0) and 25 000 (pI 6.8) were inhibited by E₂ treatment. The results demonstrated that porcine epithelial cell protein synthetic patterns are influenced by E₂ treatment and that estradiol treatment of oviductal cells may increase the rate of zygote cleavage during early development in vitro in pigs.     

Evidences and magnitude of nighttime transpiration derived from <Emphasis Type="Italic">Populus euphratica</Emphasis> in the extreme arid region of China

Extensive research has found that nighttime transpiration (E _n) is positively correlated to the vapour pressure deficit (VPD), that suggested E _n was highest during the night under high temperatures and low humidity along with high soil water availability, typically for the riparian forest in the extreme arid region of China. This study used the heat ratio method to measure sap velocity (V _s) for mature and saplings Populus euphratica Oliv., and then E _n was conservatively calculated as total nocturnal sap flow (F _s, the product of V _s and sapwood area A _s) between 01:00 to 06:00. A gas exchange system was used to measure the leaf transpiration rate (T _r) and stomatal conductance (g _s) of saplings. For mature trees, nighttime V _s was extensive and logarithmic correlated to VPD (similar to daytime). For saplings, g _s and T _r was extensive in different months, and also a strong logarithmic relationship was found between V _s and VPD for both daytime and nighttime periods. Both of stem sap flow and leaf gas exchange suggusted the occurrence of E _n, whether mature or sapling trees. E _n contribution to daily transpiration (E _d) was high just as expected for P. euphratica, which was confirmed by proportional E _n to E _d (E _n/E _d) means taken in 2012 (24.99%) and 2013 (34.08%). Compared to mature trees, E _n/E _d of saplings in 2013 was lower with means of 12.06%, that supported further by the shorter duration times and less T _r,n (16.64%) and g _s,n (26.45%) of leaf, suggesting that E _n magnitude is associated to individual the tree size, that effect to stored water of individual trees, although this hypothesis requires further research.     

Efflux and Influx of Erythrocyte Water

Rabbit erythrocytes were washed in buffered NaCl solutions isotonic with rabbit serum (Δ^t -0.558°C.) and suspended in buffered NaCl solutions of tonicity equidistant from intracellular tonicity (Δ^t = -0.558°C. ± 0.112°C.) of varying pH and incubated at varying temperatures. After incubation, the freezing point depression (Δ^t) was measured on the supernatant. Change in the Δ^t measured change in the water content of the extracellular solutions—water being withdrawn by erythrocytes (W_I) from the hypotonic solutions and added (W_E) to the hypertonic solutions. W_E was always less than W_I and was inversely proportional to the pH in the range 6.5–8.0. W_E was significantly increased by lowering the temperature of the cell suspension to 4°C. W_I was increased by raising or lowering the pH or raising the temperature of the cell suspension. W_E x W_I ≠ k. W_E and W_I were affected differently by changes in pH and temperature. It was concluded that W_E and W_E were probably under different physicochemical control.     

The functional group on (E)-4,4′–disubstituted stilbenes influences toxicity and antioxidative activity in differentiated PC-12 cells

This work probes the relationship between stilbene functional group and biological activity. The biological activity of synthesized stilbenes (E)-4,4′-dicyanostilbene, (E)-4,4′-diacetylstilbene, (E)-4,4′-diaminostilbene, a novel stilbene, 1,1′-(vinylenedi-p-phenylene)diethanol, and (E)-stilbene was assessed at biologically relevant nanomolar concentrations using the MTS cell viability assay in differentiated PC-12 cells under optimal culture conditions and conditions of oxidative stress. Under optimal culture conditions the synthesized stilbene derivatives were found to be non-toxic to cells at concentrations up to 10 μg/ml. To mimic oxidative stress, the activity of these stilbene derivatives in the presence of 0.03% H₂O₂ was investigated. Stilbene derivatives with electron-withdrawing functional groups were 2–3 times more toxic than the H₂O₂ control, indicating that they may form toxic metabolites in the presence of H₂O₂. Fluorescence data supported that stilbene derivatives with electron-withdrawing functional groups, (E)-4,4′-dicyanostilbene and (E)-4,4′-diacetylstilbene, may react with H₂O₂. In contrast, the stilbene derivative with a strong electron-donating functional group, (E)-4,4′-diaminostilbene, rescued neurons from H₂O₂-induced toxicity. The DPPH assay confirmed that (E)-4,4′-diaminostilbene is able to scavenge free radicals. These data indicate that the Hammett value of the functional group correlates with the biological activity of (E)-4,4′-disubstituted stilbenes in differentiated PC-12 cells.     

Stereocontrolled synthesis of (E)-stilbene derivatives by palladium-catalyzed Suzuki-Miyaura cross-coupling reaction

A general procedure for the stereocontrolled synthesis of (E)-stilbene derivatives by palladium-catalyzed Suzuki-Miyaura cross-coupling reaction of (E)-2-phenylethenylboronic acid pinacol ester with aryl bromides was investigated. (E)-2-Phenylethenylboronic acid pinacol ester was prepared by 9-BBN-catalyzed hydroboration of phenylacetylene with pinacolborane. This reagent undergoes facile palladium-catalyzed cross-coupling with a diverse set of aryl bromides to provide the corresponding (E)-stilbene derivatives in moderate to good yield. The use of the sterically bulky t-Bu₃PHBF₄ ligand was crucial to the successful coupling of electron-rich and electron-poor aryl bromides. Complete stereochemical retention of the (E)-2-phenylethenylboronic acid pinacol ester alkene geometry was observed in all of the (E)-stilbene derivatives synthesized.     

Structure guided inhibitor designing of CDK2 and discovery of potential leads against cancer

On the basis of stereo specific information obtained from crystal structures of CDK2, indole and chromene analogues were designed by suitably substituting the pharmacophores on their moiety and docked with target protein for calculating binding affinities. The binding affinities are represented in glide score. (5E)-5-[(1-methyl-1H-indol-3-yl)methylidene]-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide (I₁), (5E)-5-(1H-indol-3-ylmethylidene)-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide (I₂) and 2-amino-4-(4-methyl phenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (C₉) were selected for synthesis and biological testing based on vital interactions. (5E)-5-(1H-indol-3-ylmethylidene)-2,4,6-trioxotetrahydro-2H-pyrimidin-1-ide(I₂) and 2-amino-4-(4-methyl phenyl)-5-oxo-5,6,7,8-tetrahydro-4H-chromene-3-carbonitrile (C₉) were proved to be active against MCF-7 and HeLa cell lines.     

Direct electron transfer and bioelectrocatalysis of hemoglobin at a carbon nanotube electrode

A stable suspension of carbon nanotube (CNT) can be obtained by dispersing the CNT in the solution of the surfactant cetyltrimethylammonium bromide. CNT has promotion effects on the direct electron transfer of hemoglobin (Hb), which was immobilized onto the surface of CNT. The direct electron transfer rate of Hb was greatly enhanced after it was immobilized onto the surface of CNT. Cyclic voltammetric results showed a pair of well-defined redox peaks, which corresponded to the direct electron transfer of Hb, with the formal potential (E^0′) at about −0.343 V (vs. saturated calomel electrode) in the phosphate buffer solution (pH 6.8). The electrochemical parameters such as apparent heterogeneous electron transfer rate constant (k_s) and the value of formal potential (E^0′) were estimated. The dependence of E^0′ on solution pH indicated that the direct electron transfer reaction of Hb is a one-electron transfer coupled with a one-proton transfer reaction process. The experimental results also demonstrated that the immobilized Hb retained its bioelectrocatalytic activity to the reduction of H₂O₂. The electrocatalytic current was proportional to the concentration of H₂O₂ at least up to 20 mM.     

Spontaneous Spatial Correlation of Elastic Modulus in Jammed Epithelial Monolayers Observed by AFM

For isolated single cells on a substrate, the intracellular stiffness, which is often measured as the Young’s modulus, E, by atomic force microscopy (AFM), depends on the substrate rigidity. However, little is known about how the E of cells is influenced by the surrounding cells in a cell population system in which cells physically and tightly contact adjacent cells. In this study, we investigated the spatial heterogeneities of E in a jammed epithelial monolayer in which cell migration was highly inhibited, allowing us to precisely measure the spatial distribution of E in large-scale regions by AFM. The AFM measurements showed that E can be characterized using two spatial correlation lengths: the shorter correlation length, l_S, is within the single cell size, whereas the longer correlation length, l_L, is longer than the distance between adjacent cells and corresponds to the intercellular correlation of E. We found that l_L decreased significantly when the actin filaments were disrupted or calcium ions were chelated using chemical treatments, and the decreased l_L recovered to the value in the control condition after the treatments were washed out. Moreover, we found that l_L decreased significantly when E-cadherin was knocked down. These results indicate that the observed long-range correlation of E is not fixed within the jammed state but inherently arises from the formation of a large-scale actin filament structure via E-cadherin-dependent cell-cell junctions.     

Neutralization of the Charge on Asp369 of Na+,K+-ATPase Triggers E1 ? E2 Conformational Changes

This work investigates the role of charge of the phosphorylated aspartate, Asp³⁶⁹, of Na⁺,K⁺-ATPase on E₁ ↔ E₂ conformational changes. Wild type (porcine α₁/His₁₀-β₁), D369N/D369A/D369E, and T212A mutants were expressed in Pichia pastoris, labeled with fluorescein 5′-isothiocyanate (FITC), and purified. Conformational changes of wild type and mutant proteins were analyzed using fluorescein fluorescence (Karlish, S. J. (1980) J. Bioenerg. Biomembr. 12, 111–136). One central finding is that the D369N/D369A mutants are strongly stabilized in E₂ compared with wild type and D369E or T212A mutants. Stabilization of E₂(Rb) is detected by a reduced K_0.5Rb for the Rb⁺-induced E₁ ↔ E₂(2Rb) transition. The mechanism involves a greatly reduced rate of E₂(2Rb) → E₁Na with no effect on E₁ → E₂(2Rb). Lowering the pH from 7.5 to 5.5 strongly stabilizes wild type in E₂ but affects the D369N mutant only weakly. Thus, this “Bohr” effect of pH on E₁ ↔ E₂ is due largely to protonation of Asp³⁶⁹. Two novel effects of phosphate and vanadate were observed with the D369N/D369A mutants as follows. (a) E₁ → E₂·P is induced by phosphate without Mg²⁺ ions by contrast with wild type, which requires Mg²⁺. (b) Both phosphate and vanadate induce rapid E₁ → E₂ transitions compared with slow rates for the wild type. With reference to crystal structures of Ca²⁺-ATPase and Na⁺,K⁺-ATPase, negatively charged Asp³⁶⁹ favors disengagement of the A domain from N and P domains (E₁), whereas the neutral D369N/D369A mutants favor association of the A domain (TGES sequence) with P and N domains (E₂). Changes in charge interactions of Asp³⁶⁹ may play an important role in triggering E₁P(3Na) ↔ E₂P and E₂(2K) → E₁Na transitions in native Na⁺,K⁺-ATPase.