Repression of Proton extrusion from Intact Cucumber Roots and the Proton Transport Rate of Microsomal Membrane Vesicles of the Roots due to Ca2+ Starvation

Proton extrusion from cucumber roots decreased markedly duringCa²⁺ starvation in the presence of KC1. Vesicles with ATP-dependentproton transport activity were prepared from the microsomalmembrane fraction of control and Ca²⁺-starved roots. The protontransport rate of the vesicles from Ca²⁺-starved roots was repressedto less than half of the vesicles prepared from the controlroots. K⁺-Mg²⁺-ATPase activity associated with the vesiclesprepared from Ca²⁺-starved roots was approximately one-thirdof the activity associated with those prepared from controlroots. K_m values of the proton transport rate and K⁺-Mg²⁺-ATPasefor ATP were much higher in vesicles prepared from Ca²⁺-starvedroots. The repression of proton extrusion linked with K⁺ uptake inthe Ca²⁺-starved roots could be largely caused by the reducedproton pumping activity associated with microsomal membranesin the roots. (Received May 25, 1987; Accepted October 14, 1987)     

Effect of Root Zone Temperature on the Mineral Composition of Xylem Sap and Plasma Membrane K+-Mg++-ATPase Activity of Grafted-Cucumber and -Figleaf Gourd Root Systems

Cucumber (Cucumis sativus L.) seedlings were grafted onto cucumber-(CG) or figleaf gourd- (FG, Cucurbita ficifolia Bouché)seedlings in order to determine the effect of solution temperature(12, 22, and 32°C) on the mineral composition of xylem sapand the plasma membrane K⁺-Mg⁺⁺-ATPase activities of the roots.Low solution temperature (12°C) lowered the concentrationof NO^–₃ and H₂PO^–₄ in xylem sap of CG plants butnot of FG plants. Concentrations of K⁺, Ca⁺⁺ and Mg⁺⁺ in xylemsap were less affected than anions by solution temperature.The plasma membrane of FG plants grown in 12°C solutiontemperature showed the highest K⁺- Mg⁺⁺-ATPase activity at allATP concentrations up to 3 mM and at low reaction temperatureup to 12°C, indicating resistance of figleaf gourd to lowroot temperature. (Received December 27, 1994; Accepted March 10, 1995)     

Permeation of Ca2+ and monovalent cations through an outwardly rectifying channel in maize root stelar cells

A depolarization-activated outwardly-rectifying channel (OR),most likely involved in the passive release of K⁺ from the rootsymplasm into the stelar apoplast (for subsequent transportto the shoot via the xylem vessels), has been characterizedin the plasma membrane of maize root stelar cells (Roberts andTester, 1995). In the present study, the selectivity of thischannel was further characterized using single channel current-voltagecurves generated using a voltage ramp protocol. This protocolpermitted the accurate and unambiguous measurement of the reversalpotentials of currents resulting from single channel openings.Using the voltage ramp protocol, it was shown that the OR allowsboth K⁺ efflux and Ca²⁺ influx at potentials positive of E_Kand negative of E_Ca. The OR had a P_Ca/P_K of 1.72–0.21decreasing as extracellular Ca²⁺ was increased. The permeabilityof the OR for monovalent cations other than K⁺ was also investigated.In biionic conditions, a relative permeability sequence of was determined (i.e. Eisenman sequenceIV). The physiological implications of the selectivity of theOR are discussed. Key words: Maize roots, K⁺ channel selectivity, Ca²⁺ permeation     

Characterization of ATPase Activity Associated with Plasma Membrane from Vigna Hypocotyls

A plasma membrane fraction was isolated from the hypocotylsof cowpea {Vigna unguiculata) by a combination of differentialcentrifugation and sucrose density gradient centrifugation.The ATPase activity of this fraction was dependent on divalentcations (Mn²⁺>Mg²⁺>Co²⁺>Ca²⁺>Fe²⁺>Zn²⁺>Ni²⁺)but was not further stimulated by monovalent cations (K⁺ and/orNa⁺). The pH optimum for the activation of ATPase by Mg²⁺ was7.0. This fraction hydrolyzed ATP or UTP as a substrate andthe ATPase activity obeyed a Michaelis-Menten type of kinetics.The K_m for MgATP ranged from 0.65 to 1.1 mM. The ATPase activitywas inhibited by inhibitors such as N, N'- dicyclohexylcarbodiimide,diethylstilbestrol and triphenyltin chloride, all of which arereported to block proton (H⁺) transport in plant cells, butwas insensitive to those of mitochondrial ATPase such as oligomycinand sodium azide. The ATPase activity was not stimulated bytreatment with ionophores (e.g., carbonyl cyanide p-trifluoromethoxyphenylhydrazone,3,5-di-ter-butyl-4-hydroxybenzilidenemalononitrile and valinomycin⁺KCl)which would be expected to dissipate the electrochemical potentialdifference of H⁺ or the membrane potential difference. The characteristics of the ATPase are compared with those ofplasma membrane ATPases of other plants and its possible rolein H⁺-transport is discussed. ¹ Present address: Institute of Applied Biochemistry, Yagi MemorialPark, Mitake, Gifu 505-01, Japan or Laboratory for Plant EcologicalStudies, Faculty of Science, Kyoto University, Kyoto 606, Japan. (Received April 20, 1984; Accepted August 14, 1984)     

Effects of cations on the sugar receptor-site of the blowfly, Phormia

1) Ca^{+ +} (1 to 10 mM) lowered the binding affinity of sugarreceptor-site for sucrose in the labellar sugar receptor ofthe blowfly, Phormia regina, without changing the maximum-responseamplitude. It also elevated the values of the Hill coefficient(n_H) in some degrees. 2) Other divalent cations such as Mg^{+ +}, Ba^{+ +} or Cd^{+ +} alsoshowed almost the same property as above. The sequence of theeffect is as follows: Ba^{+ +}, Mg^{+ +} x Ca^{+ +} x Cd^{+ +}. Trivalentcation, La^{+ + +} (1 mM), changed the value of n_H from 1 (La^{++ +}-free) to 2. 3) On the contrary, the action of monovalent cations such asK⁺ or Na⁺, of which ionic strength was made the same as thatof the divalents hardly suppressed the response. 4) The results obtained do not support the hypothesis, at leaston the sugar receptor of the fly, that the receptor potentialis attributable to a change of the surface potential (zeta potential)as is proposed for the frog sugar receptor.     

Non-specificity of Salt Effects on Mg2+dependent ATPase from Grass Roots

The effect of tris, choline, and ethanolamine chlorides on theactivity of Mg^2–dependent ATPase in membrane fractions(cell walls, mitochondria, and microsomes) of Zea mays L. (cv.Neve Yaar 22), Avena saliva L. (cv. Mulga), and Hordeum vulgareL. (cv. Omer) was compared with the effect of KC1 and NaCl.Considerable salt effects on apparent Mg²⁺ATPase activity werefound only at relatively high pH values (8.2) at which Mg²⁺.ATPaseactivity was low in the absence of monovalent cation salts.The Mg²⁺-dependent ATP hydrolysis by ATPases from all the membranefractions increased in the presence of at least one of the organiccations to the same extent as in the presence of KCI or NaCl.The monovalent organic cations are only very slowly absorbedby corn roots in comparison with K⁺ and Na⁺. It is concluded that monovalent salt effects on ATPase fromthese plant roots are not cation specific and not related tothe capability of root cells to absorb cations. Present evidencefor the existence of a cation-transport ATPase in plant tissueis critically reviewed.     

Measurement of Changes in Cytosolic Ca2+ in Arabidopsis Guard Cells and Mesophyll Cells in Response to Blue Light

Phototropins (phot1 and phot2) are blue light (BL) receptorsthat mediate responses including phototropism, chloroplast movementand stomatal opening, and increased cytosolic Ca²⁺. BL absorbedby phototropins activates plasma membrane H⁺-ATPase in guardcells, resulting in membrane hyperpolarization, and drives K⁺uptake and stomatal opening. However, it is unclear whetherthe phototropin-mediated Ca²⁺ increase activates the H⁺-ATPase.Here, we determined cytosolic Ca²⁺ concentrations in guard cellprotoplasts (GCPs) from Arabidopsis transformed with aequorin.Cytosolic Ca²⁺ increased rapidly in response to BL in GCPs fromboth the wild type and phot1 phot2 double mutants, but was mostlysuppressed by an inhibitor of photosynthetic electron flow (DCMU).With depleted external K⁺, we observed another slower Ca²⁺ increase,which was phototropin- dependent. Fusicoccin, a H⁺-ATPase activator,mimicked the effect of BL. The slow Ca²⁺ increase thus appearsto result from membrane hyperpolarization. The slow Ca²⁺ increasewas suppressed by external K⁺ and was restored by blockers ofinward-rectifying K⁺ channels, CsCl and tetraethylammonium,suggesting the preferential uptake of K⁺ over Ca²⁺. Such efficientK⁺ uptake in response to BL was not found in mesophyll cells.Both the fast and the slow Ca²⁺ increases were inhibited byCa²⁺ channel blockers (CoCl₂ and LaCl₃) and a chelating agent(EGTA). These results indicate that the phototropin-mediatedCa²⁺ increase was not observed prior to H⁺-ATPase activationin guard cells and that Ca²⁺ entered guard cells via Ca²⁺ channelsthrough photosynthesis and phototropin-mediated membrane hyperpolarization.     

Repression of Asolectin-Dependent Activation of Partially Lipid Depleted ATPase Prepared from the Plasma Membrane-Enriched Fraction of Cucumber Roots due to Ca2+ Starvation

The ATPase activity of the plasma membrane-enriched fractionwas severely inhibited by withdrawal of Ca²⁺ from the mediumfor 5 days, although the root system appeared to be unaffectedto visual inspection. Partially lipid-depleted ATPases withsimilar ratios of phospholipid to protein were prepared fromthe plasma membrane-enriched fraction of cucumber roots culturedwith control medium and one lacking Ca²⁺, and their propertieswere compared. SDS disc polyacrylamide gel electrophoresis showedthat the polypeptide components were essentially similar betweencontrol and Ca²⁺-starved roots. Partially lipid-depleted ATPasereassociated with asolectin, the lecithin from soybean, showedtypical characteristics of plasma membrane type ATPase; pH optimumat 6.5, high specificity for ATP as substrate and strong inhibitionby vanadate but not nitrate. The activity of reassociated ATPaseobtained from the control roots was apparently higher than theactivity obtained from Ca²⁺-starved roots. The amount of asolectinrequired for maximum activation of the partially lipid-depletedATPase prepared from control roots was much lower than thatprepared from Ca²⁺-starved roots. Reassociation of partiallylipid-depleted ATPase with asolectin produced higher ATPaseactivity than that with individual phospholipids. The activationof partially lipid-depleted ATPase prepared from control rootswith asolectin was not inhibited by addition of a sample preparedfrom Ca²⁺-starved roots. Thus, a decrease in the functionalassociation of ATPase with phospholipids might be one of thephysiological injuries in root cell membranes of cucumber causedby Ca²⁺ starvation. ¹Permanent address: Department of Horticulture, College of Agriculture,Chonnam National University, Chonnam 500, Korea. (Received February 23, 1988; Accepted August 18, 1988)     

Modeling transmural heterogeneity of K(ATP) current in rabbit ventricular myocytes

To investigate the mechanisms regulating excitation-metabolic coupling in rabbit epicardial, midmyocardial, and endocardial ventricular myocytes we extended the LabHEART model (Puglisi JL and Bers DM. Am J Physiol Cell Physiol 281: C2049–C2060, 2001). We incorporated equations for Ca²⁺ and Mg²⁺ buffering by ATP and ADP, equations for nucleotide regulation of ATP-sensitive K⁺ channel and L-type Ca²⁺ channel, Na⁺-K⁺-ATPase, and sarcolemmal and sarcoplasmic Ca²⁺-ATPases, and equations describing the basic pathways (creatine and adenylate kinase reactions) known to communicate the flux changes generated by intracellular ATPases. Under normal conditions and during 20 min of ischemia, the three regions were characterized by different I_Na, I_to, I_Kr, I_Ks, and I_Kp channel properties. The results indicate that the ATP-sensitive K⁺ channel is activated by the smallest reduction in ATP in epicardial cells and largest in endocardial cells when cytosolic ADP, AMP, PCr, Cr, P_i, total Mg²⁺, Na⁺, K⁺, Ca²⁺, and pH diastolic levels are normal. The model predicts that only K_ATP ionophore (Kir6.2 subunit) and not the regulatory subunit (SUR2A) might differ from endocardium to epicardium. The analysis suggests that during ischemia, the inhomogeneous accumulation of the metabolites in the tissue sublayers may alter in a very irregular manner the K_ATP channel opening through metabolic interactions with the endogenous PI cascade (PIP₂, PIP) that in turn may cause differential action potential shortening among the ventricular myocyte subtypes. The model predictions are in qualitative agreement with experimental data measured under normal and ischemic conditions in rabbit ventricular myocytes. ATP-sensitive K⁺ channel; creatine and adenylate kinase reactions; phosphatidylinositol phosphates; heart; mathematical model     

Cyclic AMP and calcium modulated ATPase activity in the salivary glands of the lone star tick Amblyomma americanum (L.)

Na⁺,K⁺-activated ATPase activity in tick salivary glands increases during the rapid stage of tick feeding paralleling similar increases in dopamine and cAMP-stimulated fluid secretion. High concentrations of cyclic AMP increase Na⁺,K⁺-ATPase activity in a plasma membrane-enriched fraction from the salivary glands of rapidly feeding ticks. Cyclic AMP-dependent protein kinase inhibitor protein blocks activation of Na⁺,K⁺-ATPase activity at low but not high concentrations of cAMP indicating that both activator and inhibitor modulator phosphoproteins of Na⁺,K⁺-ATPase activity exist in the plasma membrane-enriched fraction.ATPase activity in the plasma membrane-enriched fraction is not measurable in the absence of Mg²⁺, Ca²⁺ and Na⁺. Ca-stimulated nucleotidase activity is highest with ATP serving as the preferred substrate in a series including CTP, UTP, GTP and ADP. Calcium, Mg²⁺ stimulated ATPase activity is activated further by calmodulin and partially inhibited by low concentration of vanadate, trifluoperazine and oligomycin. Results suggest that the plasma membrane-enriched fraction of tick salivary glands contains both Ca²⁺-ATPase activity and oligomycin-sensitive Ca²⁺, Mg²⁺-ATPase activities, the latter likely from a small amount of mitochondria in the partially purified organelle fraction.     

Plasma Membrane H+-ATPase in Guard-Cell Protoplasts from Vicia faba L.

The activity of plasma membrane H⁺-ATPase was measured withmembrane fragments of guard-cell protoplasts isolated from Viciafaba L. ATP hydrolytic activity was slightly inhibited by oligomycinand ammonium molybdate, and markedly inhibited by NO₃^–and vanadate. In the presence of oligomycin, ammonium molybdateand NO₃^–, the ATP-hydrolyzing activity was strongly inhibitedby vanadate. It was also inhibited by diethylstilbestrol (DES),p-chloromercuribenzoic acid (PCMB) and Ca²⁺, but slightly stimulatedby carbonyl cyanide m-chlorophenylhydrazone (CCCP). The acitivityhad higher specificity for ATP as a substrate than other phosphoricesters such as ADP, AMP, GTP and p-nitrophenylphosphate; theK_m was 0.5 mM for ATP. The activity required Mg²⁺ but was notaffected by K⁺, and it was maximal around pH 6.8. When guard-cellprotoplasts were used instead of membrane fragments, the ATPaseactivity reached up to 800µmol Pi.(mg Chl)^–1.h^–1in the presence of lysolecithin. These results indicate thatthe guard cell has a high plasma membrane H⁺-ATPase activity. (Received December 23, 1986; Accepted April 28, 1987)     

Caloxin: a novel plasma membrane Ca2+ pump inhibitor

Plasma membrane (PM) Ca²⁺ pump is aCa²⁺-Mg²⁺-ATPase that expels Ca²⁺from cells to help them maintain low concentrations of cytosolic Ca²⁺. There are no known extracellularly acting PMCa²⁺ pump inhibitors, as digoxin and ouabain are forNa⁺ pump. In analogy with digoxin, we define caloxins asextracellular PM Ca²⁺ pump inhibitors and describe caloxin2A1. Caloxin 2A1 is a peptide obtained by screening a random peptidephage display library for binding to the second extracellular domain(residues 401-413) sequence of PM Ca²⁺ pump isoform1b. Caloxin 2A1 inhibits Ca²⁺-Mg²⁺-ATPase inhuman erythrocyte leaky ghosts, but it does not affect basalMg²⁺-ATPase or Na⁺-K⁺-ATPase in theghosts or Ca²⁺-Mg²⁺-ATPase in the skeletalmuscle sarcoplasmic reticulum. Caloxin 2A1 also inhibitsCa²⁺-dependent formation of the 140-kDa acid-stableacylphosphate, which is a partial reaction of this enzyme. Consistentwith inhibition of the PM Ca²⁺ pump in vascularendothelium, caloxin 2A1 produces an endothelium-dependent relaxationthat is reversed byN^G-nitro-L-arginine methyl ester.Thus caloxin 2A1 is a novel PM Ca²⁺ pump inhibitor selectedfor binding to an extracellular domain.

     

Ca2+ Regulation of Outward Rectifying K+ Channel in the Plasma Membrane of Tobacco Cultured Cells in Suspension: a Role of the K +Channel in Mitigation of Salt-Stress Effects by External Ca2+

The patch-clamp technique was used to study effect of the Ca²⁺on K⁺ channels in the plasma membrane of protoplasts isolatedfrom tobacco (Nicotiana tabacum L., cv. Bright Yellow) culturedcells in suspension. The outward rectifying whole-cell K⁺ currentswere not affected by in-tracellular Ca²⁺, but they were reducedwith increasing extracellular Ca²⁺. Neither extracellular norintracellular Ca²⁺ affected the permeability ratios (p_K+/P_Na+)of the plasma membrane. These results suggest that the inhibitionof outward-rectifying K⁺ channels by extracellular Ca²⁺may partiallycontribute towards the mitigation of detrimental effects ofsalinity on growth by extracellular Ca²⁺. (Received January 19, 1998; Accepted July 30, 1998)     

Stimulation of the Extrusion of Protons and H+-ATPase Activities with the Decline in Pyrophosphatase Activity of the Tonoplast in Intact Mung Bean Roots under High-NaCl Stress and Its Relation to External Levels of Ca2+ Ions

Extrusion of protons as a response to high-NaCl stress in intactmung bean roots was investigated at different external concentrationsof Ca²⁺ ions ([Ca²⁺]_ex). The extrusion of protons was graduallyenhanced in the roots exposed to 100 mM NaCl, and high [Ca²⁺]_exdiminished this enhancement of the extrusion. Vesicles of plasmalemmaand tonoplast were prepared from the roots and the H⁺-translocatingATPase (H⁺-ATPase) activities associated with the two typesof membrane and the H⁺-pyrophosphatase (H⁺-PPase) activity ofthe tonoplast were assayed. The plasmalemma ATPase was stimulatedin parallel with dramatic increases in the intracellular concentrationof Na⁺([Na⁺]_in). High [Ca²⁺]ex prevented the increase in [Na⁺]inand diminished the stimulation of ATPase activity. The tonoplastATPase showed a rapid response to salt stress and was similarlystimulated even at high [Ca²⁺]M. The activities of both ATPaseswere, however, insensitive to concentrations of Na⁺ ions upto 100 HIM. By contrast, H⁺-PPase activity of the tonoplastwas severely inhibited with increasing [Na⁺]in under salt stressand recovered with high [Ca²⁺]ex. These findings suggest thathigh-NaCl stress increases the intracellular concentration ofNa⁺ ions in mung bean roots, which inhibits the tonoplast H⁺-PPase,and the activity of the plasmalemma H⁺-ATPase is thereby stimulatedand regulates the cytoplasmic pH. (Received March 26, 1991; Accepted December 13, 1991)     

Effect of Divalent Cations on Na+ Permeability of Chara corallina and Freshwater Grown Chara buckellii

The effect of elevated Na⁺ concentration on Na⁺ permeability(P_Na) and Na⁺ influx in the presence of two levels of externaldivalent cations was determined in Chara corallina and freshwater-culturedChara buckellii. When Na⁺ in the medium was increased from 1.0to 70 mol m^–3, Na⁺ influx increased in both species ifCa²⁺ was low (0.1 mol m–3). If Ca²⁺ was increased to 7.0mol m^–3 when Na⁺ was increased, Na⁺ influx remained atthe low control level in C. corallina, and showed only a temporaryincrease in C. buckellii. Mg²⁺ was a better substitute for Ca²⁺in C. buckellii than in C. corallina. Na⁺ permeability data suggest that when the external Ca²⁺ concentrationis low, P_Na does not increase in the presence of elevated NaCl;the increase in Na⁺ influx appears to be due to the increasein external Na⁺ concentration alone. Ca^{2 +} supplementation appearsto decrease P_Na whereas supplemental Mg²⁺ has no effect. Na⁺ effluxes were computed from previously determined net fluxesand the influxes. It was found that for both species, fluxesin both directions were stimulated in response to all experimentaltreatments, but Na⁺ influx always exceeded efflux. This resultedin net Na⁺ accumulation in the vacuoles of both species. The results are discussed with reference to net flux and electrophysiologicaldata obtained previously under identical conditions, as wellas the comparative salinity tolerance of both species and theNa⁺/divalent cation ratio. Key words: Na⁺ influx, Na⁺ tolerance, membrane potential, permeability, Chara     

Role of caveolae in signal-transducing function of cardiac Na+/K+-ATPase

Ouabain binding toNa⁺/K⁺-ATPase activates Src/epidermal growthfactor receptor (EGFR) to initiate multiple signal pathways thatregulate growth. In cardiac myocytes and the intact heart, the earlyouabain-induced pathways that cause rapid activations of ERK1/2 alsoregulate intracellular Ca²⁺ concentration([Ca²⁺]_i) and contractility. The goal of thisstudy was to explore the role of caveolae in these early signalingevents. Subunits of Na⁺/K⁺-ATPase were detectedby immunoblot analysis in caveolae isolated from cardiac myocytes,cardiac ventricles, kidney cell lines, and kidney outer medulla byestablished detergent-free procedures. Isolated rat cardiac caveolaecontained Src, EGFR, ERK1/2, and 20-30% of cellular contents of₁- and ₂-isoforms ofNa⁺/K⁺-ATPase, along with nearly all ofcellular caveolin-3. Immunofluorescence microscopy of adult cardiacmyocytes showed the presence of caveolin-3 and -isoforms inperipheral sarcolemma and T tubules and suggested their partialcolocalization. Exposure of contracting isolated rat hearts to apositive inotropic dose of ouabain and analysis of isolated cardiaccaveolae showed that ouabain caused 1) no change in totalcaveolar ERK1/2, but a two- to threefold increase in caveolarphosphorylated/activated ERK1/2; 2) no change in caveolar ₁-isoform and caveolin-3; and 3) 50-60%increases in caveolar Src and ₂-isoform. These findings,in conjunction with previous observations, show that components of thepathways that link Na⁺/K⁺-ATPase to ERK1/2 and[Ca²⁺]_i are organized within cardiac caveolaemicrodomains. They also suggest that ouabain-induced recruitments ofSrc and ₂-isoform to caveolae are involved in themanifestation of the positive inotropic effect of ouabain.

     

Ontogenetic Changes in the Chemical Composition of Ricinus communis Grown with NO-3 or NH+4 as N Source

Ricinus communis L. var. Gibsonii was grown in Long Ashton nutrientmedium with either 12mol m^–3 NO^–₃ or 8.0 mol m^–3NH⁺₄ as N source. Two plants from each N treatment were harvestedtwice a week and analysed for C, N, P, S, NO^–₃, SO^2–₄Cl^–K⁺Na⁺, Ca²⁺ Mg²⁺ and ash alkalinity. Statistical analysis of thedata showed that the effect of age and N source was differentfor the chemical variables analysed. Thus [Na⁺] was unaffectedby age or N source, and for both N sources [Mg²⁺] started atthe same level and decreased at the same rate as the plantsmatured. With NH⁺₄ as N source, [SO^2–₄] was higher thanwith NO^–₃, but did not alter with age. The concentrations,in mmol g^–1 dry wt, of C, organic N, K⁺ and Ca²⁺ weredifferent for the two N sources, but the levels of these variablesaltered with age in the same way for both N sources; i.e. therewas no age x N interaction. In the case of P, NO^–₃, Cl^– and COO^–, however,age-related variations were different for the two N sources.It is concluded, inter alia, that [Na⁺] is determined by external[Na⁺] alone, and that K⁺, Ca²⁺ and Cl^– are the inorganicions actively involved in charge balance during ion uptake bythe roots. Key words: Ontogeny, Chemical composition, Plant nutrition     

Isolation and properties of ion-stimulated ATPase activity associated with cauliflower plasma membranes

The association of K⁺-stimulated, Mg²⁺-dependent ATPase activity with plasma membranes from higher plants has been used as a marker for the isolation and purification of a plasma membrane-enriched fraction from cauliflower (Brassica oleraceae L.) buds. Plasma membranes were isolated by differential centrifugation followed by density gradient centrifugation of the microsomal fraction. The degree of purity of plasma membranes was determined by increased sensitivity of Mg²⁺-ATPase activity to stimulation by K⁺ and by assay of approximate marker enzymes. In the purified plasma membrane fraction, Mg²⁺-ATPase activity was stimulated up to 700% by addition of K⁺. Other monovalent cations also markedly stimulated the enzyme, but only in the presence of the divalent cation Mg²⁺. Ca²⁺ was inhibitory to enzyme activity. ATPase was the preferred substrate for hydrolysis, there being little hydrolysis in the presence of ADP, GTP, or p-nitrophenylphosphate. Monovalent cation-stimulated activity was optimum at alkaline pH. Enzyme activity was inhibited nearly 100% by AgNO₃ and about 40% by diethylstilbestrol.     

Taste responses to divalent cations in the frog glossopharyngeal nerve: competitive inhibition of the Mg2+ response by Ca2+

In the frog glossopharyngeal nerve, single water fibers respondto low CaCl₂ (1–2 mM) and relatively high MgCl₂ (100 mM).In the present study, it was found that stimulation by a mixtureof low CaCl₂ and relatively high MgCl₂ led to a small response.This suggests that the Ca⁺ response is inhibited by the presenceof Mg²⁺ and the Mg²⁺ response is inhibited by the presence ofCa²⁺. Hence, it is suggested that there are different receptorsites for divalent cations in single water fibers of the frogglossopharyngeal nerve, a calcium receptor site (X_Ca) responsiblefor the Ca²⁺ response and a magnesium receptor site (X_Mg) responsiblefor the Mg²⁺ response. It has been reported that Mg²⁺ inhibitsthe Ca²⁺ response by competing with Ca²⁺ for X_Ca (Kitada andShimada, 1980). In the present study, the inhibition of theMg²⁺ response by Ca²⁺ was examined quantitatively under theassumption that the magnitude of the neural response is proportionalto the amount of MgX_Mg complex minus a constant (the thresholdconcentration of the MgX_Mg complex). The results obtained indicatethat Ca²⁺ competes with Mg²⁺ for X^Mg. The apparent dissociationconstants for MgX_Mg complex and CaX_Mg complex, which were obtainedfrom the present study, were 8.0 x 10^–2 M and 7.2 x 10^–4M, respectively. Thus, competition between Ca⁺ and Mg²⁺ forthe distinct receptor sites involved in taste reception wasdemonstrated by the results described in this paper. Since thedivalent cations do not always bring about activation of tastereceptors, the responses to salts in the frog glossopharyngealnerve cannot be explained in terms of changes in the surfacepotential outside the taste cells. The present results suggestthat there exist multiple specific receptor sites for cationsinvolved in salt taste responses, and only the binding of eachseparate cation to its appropriate receptor sites leads to activationof the receptor and the initiation of impulses in sensory nerveendings.     

Significance of Ca2+ and K+ in Micrasterias Growth and Morphogenesis

Significance of Ca²⁺ and K⁺ for the complex morphogenesis ofMicrasterias, which takes place through multipolar tip growth,was investigated. Studies with different external Ca²⁺ concentrationsand Ca²⁺ channel inhibitors LaCl₃ and verapamil indicate thatCa²⁺ and Ca²⁺ channels are essential in the development, whiletreatments with different K⁺ concentrations and K⁺ channel inhibitorTEA demonstrate that potassium or K⁺ channels are not neededin the process, albeit the existence of K⁺ channels. K⁺ is notneeded even for the regulation of turgor pressure, which wasfound to decrease clearly during cell development. The plasmamembrane ATPase inhibitors diethylstilbesterol (DES) and Na-orthovanadatestop morphogenesis and indicate the importance of ion pumpsin the developmental process. Both supraoptimal, external K⁺and Ca²⁺ cause abundant Ca²⁺ precipitate formation in chloroplasts,which shows that chloroplasts are important in regulation ofcytoplasmic Ca²⁺ metabolism and that K⁺ activates the uptakeof Ca²⁺ through Ca²⁺ channels. (Received June 13, 1995; Accepted September 13, 1996)