Ca2+ pump and Ca2+/H+ antiporter in plasma membrane vesicles isolated by aqueous two-phase partitioning from corn leaves

Summary Plasma membrane vesicles, which are mostly right side-out, were isolated from corn leaves by aqueous two-phase partitioning method. Characteristics of Ca²⁺ transport were investigated after preparing inside-out vesicles by Triton X-100 treatment.⁴⁵Ca²⁺ transport was assayed by membrane filtration technique. Results showed that Ca²⁺ transport into the plasma membrane vesicles was Mg-ATP dependent. The active Ca²⁺ transport system had a high affinity for Ca²⁺(K _m (Ca²⁺)=0.4 m) and ATP(K _m (ATP)=3.9 m), and showed pH optimum at 7.5. ATP-dependent Ca²⁺ uptake in the plasma membrane vesicles was stimulated in the presence of Cl^– or NO ₃ ^– . Quenching of quinacrine fluorescence showed that these anions also induced H⁺ transport into the vesicles. The Ca²⁺ uptake stimulated by Cl^– was dependent on the activity of H⁺ transport into the vesicles. However, carbonylcyanidem-chlorophenylhydrazone (CCCP) and VO ₄ ^3– which is known to inhibit the H⁺ pump associated with the plasma membrane, canceled almost all of the Cl^–-stimulated Ca²⁺ uptake. Furthermore, artificially imposed pH gradient (acid inside) caused Ca²⁺ uptake into the vesicles. These results suggest that the Cl^–-stimulated Ca²⁺ uptake is caused by the efflux of H⁺ from the vesicles by the operation of Ca²⁺/H⁺ antiport system in the plasma membrane. In Cl^–-free medium, H⁺ transport into the vesicles scarcely occurred and the addition of CCCP caused only a slight inhibition of the active Ca²⁺ uptake into the vesicles. These results suggest that two Ca²⁺ transport systems are operating in the plasma membrane from corn leaves, i.e., one is an ATP-dependent active Ca²⁺ transport system (Ca²⁺ pump) and the other is a Ca²⁺/H⁺ antiport system. Little difference in characteristics of Ca²⁺ transport was observed between the plasma membranes isolated from etiolated and green corn leaves.     

Model of active transport of ions in cardiac cell

A model of the active transport of ions in a cardiac muscle cell, which takes into account the active transport of Na⁺, K⁺, Ca²⁺, Mg²⁺, HCO₃⁻ and Cl⁻ ions, has been constructed. The model allows independent calculations of the resting potential at the biomembrane and concentrations of basic ions (sodium, potassium, chlorine, magnesium and calcium) in a cell. For the analysis of transport processes in cardiac cell hierarchical algorithm “one ion-one transport system” was offered. The dependence of the resting potential on concentrations of the ions outside a cell has been established. It was shown, that ions of calcium and magnesium, despite their rather small concentration, play an essential role in maintenance of resting potential in cardiac cell. The calculated internal concentrations of ions are in good agreement with the corresponding experimental values.     

Activity of Plasma Membrane H<Superscript>+</Superscript>-ATPase in Coleoptile Cells during Development of Maize Seedlings

Hydrolytic activities of the H⁺-ATPase were compared for plasma membrane fractions isolated from coleoptile cells of 3-, 4-, and 5-day-old etiolated maize seedlings. The membrane preparations obtained by differential centrifugation were additionally purified in the gradient of sucrose density and in the polyethylene glycol-dextran system. The highest level of ATP-hydrolyzing activity was observed in the plasmalemma fraction obtained from 4-day-old seedlings. The pattern of age-dependent changes in H⁺-ATPase activity of the plasma membranes was clearly different from the monotonic deceleration of coleoptile cell elongation in the period examined. It is supposed that changes in ATPase activity reflect different regulatory roles of this principal ion-transporting enzyme of the plasma membrane at the stage of cell elongation and at a later developmental stage when the coleoptile has completed its physiological function.__________Translated from Fiziologiya Rastenii, Vol. 52, No. 4, 2005, pp. 566–572.Original Russian Text Copyright © 2005 by Rudashevskaya, Kirpichnikova, Shishova.     

Mechanism of cAMP-induced H+-efflux ofDictyostelium cells: a role for fatty acids

AggregatingDictyostelium cells release protons when stimulated with cAMP. To find out whether the protons are generated by acidic vesicles or in the cytosol, we permeabilized the cells and found that this did not alter the cAMP-response. Proton efflux in intact cells was inhibited by preincubation with the V-type H⁺ ATPase inhibitor concanamycin A and with the plasma membrane H⁺ ATPase blocker miconazole. Surprisingly, miconazole also inhibited efflux in permeabilized cells, indicating that this type of H⁺ ATPase is present on intracellular vesicles as well. Vesicular acidification was inhibited by miconazole and by concanamycin A, suggesting that the acidic vesicles contain both V-type and P-type H⁺ ATPases. Moreover, concanamycin A and miconazole acted in concert, both in intact cells and in vesicles. The mechanism of cAMP-induced Ca²⁺-fluxes involves phospholipase A₂ activity. Fatty acids circumvent the plasma membrane and stimulate vesicular Ca²⁺-efflux. Here we show that arachidonic acid elicited H⁺-efflux not only from intact cells but also from acidic vesicles. The target of regulation by arachidonic acid seemed to be the vesicular Ca²⁺-relase channel.     

H+/Ca2+ exchange in rabbit renal cortical endosomes

Summary We have examined the effect of second messengers on ATP-driven H⁺ transport in an H⁺ ATPase-bearing endosomal fraction isolated from rabbit renal cortex. cAMP (0.1mm) had no effect on H⁺ transport. Acridine orange fluorescence in the presence of 0.5mm Ca²⁺ (+1mm EGTA) was 19±6% of control. Inhibition of ATP-driven H⁺ transport by Ca²⁺ was concentration dependent; 0.25 and 0.5mm Ca²⁺ (+1mm EGTA) inhibited acridine orange fluorescence by 50 and 80%, respectively. Ca²⁺ also produced a concentration-dependent increase in the rate of pH-gradient dissipation. Ca²⁺ did not affect ATP hydrolysis. ATP-dependent Br^– uptake was virtually unchanged in the presence of 0.5mm Ca²⁺ (+1mm EGTA). These vesicles were also shown to transport Ca²⁺ in an ATP-dependent mode. Inositol 1, 4, 5-trisphosphate had no effect on ATP-dependent Ca²⁺ uptake. These results are consistent with the co-existence of an H⁺ ATPase and an H⁺/Ca²⁺ exchanger on these endosomes, the latter transport system using the H⁺ gradient to energize Ca²⁺ uptake. Attempts to demonstrate an H⁺/Ca²⁺ antiporter in the absence of ATP have been unsuccessful. Yet, when a pH gradient was established by preincubation with ATP and residual ATP was subsequently removed by hexokinase + glucose, stimulation of Ca²⁺ uptake could be demonstrated. A Ca²⁺-dependent increase in H⁺ permeability and an ATP-dependent Ca²⁺ uptake might have important implications for the regulation of vacuolar H⁺ ATPase activity as well as the homeostasis of cytosolic Ca²⁺ concentration.     

Involvement of plasma membrane potential in the tolerance mechanism of plant roots to aluminium toxicity

H⁺-ATPase activity of a plasma membrane-enriched fraction decreased after the treatment of barley (Hordeum vulgare) seedlings with Al for 5 days. A remarkably high level of Al was found in the membrane fraction of Al-treated roots. A long-term effect of Al was identified as the repression of the H⁺-ATPase of plasma membranes isolated from the roots of barley and wheat (Triticum aestivum) cultivars, Atlas 66 (Al-tolerant) and Scout 66 (Al-sensitive). To monitor short-term effects of Al, the electrical membrane potentials across plasma membranes of both wheat cultivars were compared indirectly by measuring the efflux of K⁺ for 40 min under various conditions. The rate of efflux of K⁺ in Scout was twice that in Atlas at low pH values such as 4.2. Vanadate, an inhibitor of the H⁺-ATPase of the plasma membrane, increased the efflux of K⁺. Al repressed this efflux at low pH, probably through an effect on K⁺ channels, and repression was more pronounced in Scout. Al strongly repressed the efflux of K⁺ irrespective of the presence of vanadate. Ca²⁺ also had a repressive effect on the efflux of K⁺ at low pH. The effect of Ca²⁺, greater in Scout, might be related to the regulation of the net influx of H⁺, since the effect was negated by vanadate. The results suggest that extracellular low pH may cause an increase in the influx of H⁺, which in turn is counteracted by the efflux of K⁺ and H⁺. These results suggest that the ability to maintain the integrity of the plasma membrane and the ability to recover the electrical balance at the plasma membrane through a net influx of H⁺ and the efflux of K⁺ seem to participate in the mechanism of tolerance to Al stress under acidic conditions.     

Electrogenic behavior of the human red cell Ca2+ pump revealed by disulfonic stilbenes

Summary A systematic study was made of the action of 4-acetamido-4-isothiocyanostilbene-2,2-disulfonic acid (SITS) and 4,4-diisothiocyanostilbene-2,2-disulfonic acid (DIDS) on active Ca²⁺ transport of human erythrocytes. Pumping activity was estimated in inside-out vesicles (IOV's) by means of Ca²⁺-selective electrodes or use of tracer⁴⁵Ca²⁺. The stilbenes exhibited an approximately equal inhibitory potency and their action could be overcome by carbonyl cyanidep-trifluoromethoxyphenylhydrazone (FCCP) at low but not at high stilbene concentrations. In the absence of DIDS. Ca²⁺ transport was not affected upon addition of valinomycin, but it was appreciably reduced when vesicles were preincubated with low DIDS concentrations. Such an effect was strictly dependent on the external K⁺ concentration and it was abolished when valinomycin was added together with FCCP. Similar results were obtained using IOV's prepared from intact cells which had been previously exposed to the stilbene. The findings clearly demonstrate the presence in human red cells of a partially electrogenic Ca²⁺ pump, exchanging one Ca²⁺ ion for one proton.     

Active transport of Ca in membrane vesicles from pea: Evidence for a H/Ca antiport

Two non mitochondrial systems involved in ATP-dependent Ca²⁺ accumulation have been described and characterized in two membrane fractions from pea internodes purified on a metrizamide-sucrose discontinuous gradient. In the lighter membrane fraction an ATP-dependent Ca²⁺ accumulation system, which shows the characteristics of an ATP-dependent H⁺/Ca²⁺ antiport, predominates. This system is inhibited by FCCP and nigericin and stimulated by 50 mM KCl. It is saturated by 0.8–1.0 mM MgSO₄-ATP, strictly requires ATP and is severely inhibited by an excess of free Mg²⁺ or Mn²⁺. A second system of ATP-dependent Ca²⁺ accumulation, recovered mainly in the heavier membrane fraction, is insensitive to FCCP, is saturated by 8–10 mM MgSO₄-ATP, can utilize also ITP or other nucleoside triphosphates although at lower rate than ATP and is only scarcely affected by an excess of free Mg²⁺ or Mn²⁺. This system is interpreted as corresponding to the (Ca²⁺ + Mg²⁺)-ATPase described by Dieter, P. and Marmé, D. ((1980) Planta 150, 1–8).     

Calcium efflux from platelet vesicles of the dense tubular system. Analysis of the possible contribution of the Ca2+ pump

The ATP dependent Ca²⁺ uptake of platelet vesicles was inhibited by the two hydrophobic drugs trifluoperazine (TFP) and propranolol (PROP). Inhibition was significantly lowered when Pi was used instead of oxalate as a precipitant agent. When the ATPase ligands substrate (Mg²⁺ and Pi) were absent of the efflux medium, a slow release of Ca²⁺ which did not couple with ATP synthesis (passive Ca²⁺ efflux) was observed. Both, TFP and PROP enhanced the passive Ca²⁺ efflux. This enhanced efflux was partially inhibited only when Mg²⁺ and Pi were added together to the efflux reaction media, but it was not affected by spermidine, ruthenium red or thapsigargin (TG). The Ca²⁺ ionophores A23187 and ionomycin, also enhanced passive Ca²⁺ efflux. However, in this case, Ca²⁺ efflux was inhibited just by inclusion of Mg²⁺ to the medium. Ca²⁺ efflux promoted by Triton X-100 was not affected by either Mg²⁺ or Pi, included together or separately into the efflux medium. The ATP Pi measured in the presence of Triton X-100 and millimolar Ca²⁺ concentrations was inhibited by both TFP and PROP, but not by Ca²⁺ ionophores up to 4 M. The data suggest that the observed enhancement of passive Ca²⁺ efflux promoted by TFP and PROP could be attributed to a direct effect of these drugs over the platelet Ca²⁺ pump isoforms (Sarco Endoplasmic Reticulum Calcium ATPase, SERCA_2b and SERCA₃) themselves, as it was reported for the sarcoplasmic reticulum Ca²⁺ ATPase (SERCA₁).     

Specificity of ligand binding to transport sites: Ca2+ binding to the Ca2+ transport ATPase and its dependence on H+ and Mg2+

Ligand binding to transport sites constitutes the initial step in the catalytic cycle of transport ATPases. Here, we consider the well characterized Ca²⁺ ATPase of sarcoplasmic reticulum (SERCA) and describe a series of Ca²⁺ binding isotherms obtained by equilibrium measurements in the presence of various H⁺ and Mg²⁺ concentrations. We subject the isotherms to statistical mechanics analysis, using a model based on a minimal number of mechanistic steps. The analysis allows satisfactory fits and yields information on occupancy of the specific Ca²⁺ sites under various conditions. It also provides a fundamental method for analysis of binding specificity to transport sites under equilibrium conditions that lead to tightly coupled catalytic activation.     

Maize tonoplast PP(i)-dependent H(+)/Ca(2+) exchange: two K(s) for Ca(2+) and inhibition by thapsigargin

Maize root tonoplasts are able to accumulate Ca(2+) using the energy derived from the H(+) gradient formed during PP(i) hydrolysis. Oxalate increases 6- to 10-fold the amount of Ca(2+) accumulated by tonoplast. Two apparently different K(s) values for Ca(2+) with values of 0.36 and 4.70 microM were detected when oxalate was included in the medium and the free Ca(2+) concentration in the medium was buffered with the use of EGTA. Binding of Ca(2+) to the outer surface of tonoplasts inhibits the outflow of Ca(2+) previously accumulated by the tonoplast, half-maximal inhibition being observed in presence of 1 microM Ca(2+). Thapsigargin, a specific inhibitor of Ca(2+)-ATPase, inhibits the Ca(2+) uptake driven by H(+) gradient but does not inhibit the hydrolysis of PP(i) nor the formation of a H(+) gradient.     

Reversible changes of extracellular pH during action potential generation in a higher plant Cucurbita pepo

A pH-sensitive electrode was applied to measure activity of H⁺ ions in the medium surrounding excitable cells of pumpkin (Cucurbita pepo L.) seedlings during cooling-induced generation of action potential (AP). Reversible alkalization shifts were found to occur synchronously with AP, which could be due to the influx of H⁺ ions from external medium into excitable cells. Ethacrynic acid (an anion channel blocker) reduced the AP amplitude but had no effect on the transient alkalization of the medium. An inhibitor of plasma membrane H⁺-ATPase, N,N’-dicyclohexylcarbodiimide suppressed both the AP amplitude and the extent of alkalization. In experiments with plasma membrane vesicles, the hydrolytic H⁺-ATPase activity was subjected to inhibition by Ca²⁺ concentrations in the range characteristic of cytosolic changes during AP generation. The addition of a calcium channel blocker verapamil and a chelating agent EGTA to inhibit Ca²⁺ influx from the medium eliminated the AP spike and diminished reversible alkalization of the external solution. An inhibitor of protein kinase, H-7 alleviated the inhibitory effect of Ca²⁺ on hydrolytic H⁺-ATPase activity in plasma membrane vesicles and suppressed the reversible alkalization of the medium during AP generation. The results provide evidence that the depolarization phase of AP is associated not only with activation of chloride channels and Cl^? efflux but also with temporary suppression of plasma membrane H⁺-ATPase manifested as H⁺ influx. The Ca²⁺-induced inhibition of the plasma membrane H⁺-ATPase is supposedly mediated by protein kinases.     

Vanadate inhibition of the Ca-ATPase from human red cell membranes

1. (1) VO₃⁻ combines with high affinity to the Ca²⁺-ATPase and fully inhibits Ca²⁺-ATPase and Ca²⁺-phosphatase activities. Inhibition is associated with a parallel decrease in the steady-state level of the Ca²⁺-dependent phosphoenzyme.

2. (2) VO₃⁻ blocks hydrolysis of ATP at the catalytic site. The sites for VO₃⁻ also exhibit negative interactions in affinity with the regulatory sites for ATP of the Ca²⁺-ATPase.

3. (3) The sites for VO₃⁻ show positive interactions in affinity with sites for Mg²⁺ and K⁺. This accounts for the dependence on Mg²⁺ and K⁺ of the inhibition by VO₃⁻. Although, with less effectiveness, Na⁺ substitutes for K⁺ whereas Li⁺ does not. The apparent affinities for Mg²⁺ and K⁺ for inhibition by VO₃⁻ seem to be less than those for activation of the Ca²⁺-ATPase.

4. (4) Inhibition by VO₃⁻ is independent of Ca²⁺ at concentrations up to 50 μM. Higher concentrations of Ca²⁺ lead to a progressive release of the inhibitory effect of VO₃⁻.

Action of mercurials on the active and passive transport properties of sarcoplasmic reticulum

The effect of Hg²⁺ and Ch₃-Hg⁺ on the passive and active transport properties of the Ca²⁺-Mg²⁺-ATPase-rich fraction of skeletal sarcoplasmic reticulum (SR) is reported. The agents abolish active transport, at 10^–5 and 10^–4 M concentrations, respectively. Addition of the mercurials was also shown to release actively accumulated Ca²⁺. The mercurials increase the passive Ca²⁺ and Mg²⁺ permeability in the absence of ATP at the same concentrations at which they inhibit transport. It is proposed that both effects are the result of direct binding of the mercurials to the SH groups of the Ca²⁺-Mg²⁺-ATPase pump, altering the conformational equilibria of the pump. The agents were also shown to increase the passive KCl permeability. The SR preparation consists of two vesicle populations with respect to K⁺ permeability, one with rapid KCl equilibration faciliated by a monovalent cation channel function and one with slow KCl equilibration. The mercurials increase the rates of KCl equilibration in both fractions, but produce higher rates in the fraction containing the channel function. The results are discussed in terms of pump and channel function and are compared with results for the electrical behavior of the Ca²⁺-Mg²⁺-ATPase and other SR proteins in black lipid membranes, as presented by others.     

Activation of the human red cell calcium ATPase by calcium pretreatment

Some kinetic parameters of the human red cell Ca²⁺-ATPase were studied on calmodulin-free membrane fragments following preincubation at 37°C. After 30 min treatment with EGTA(1 mm) plus dithioerythritol (1 mm), a V _max of about 0.4 μmol P_i/mg × hr and a K _s of 0.3 μm Ca²⁺ were found. When Mg²⁺ (10 mm) or Ca²⁺(10 μm) were also added during preincubation, V _maxbut not Kwas altered. Ca²⁺ was more effective than Mg²⁺, thus increasing V _max to about 1.3 μmol P_i/mg × hr. The presence of both Ca²⁺ and Mg²⁺ during pretreatment decreasedKto 0.15 μm, while having no apparent effect on V _max. Conversely, addition of ATP (2 mm) with either Ca²⁺ or Ca²⁺ plus Mg²⁺increased V_max without affecting K. Preincubation with Ca²⁺ for periods longer than 30 min further increased V_maxand reduced Kto levels as low as found with calmodulin treatment. The Ca²⁺ activation was not prevented by adding proteinase inhibitors (iodoacetamide, 10 mm; leupeptin, 200 μm; pepstatinA, 100 μm; phenylmethanesulfonyl fluoride, 100 μm). The electrophoretic pattern of membranes preincubated with or without Mg²⁺, Ca²⁺ or Ca²⁺ plus Mg²⁺ did not differ significantly from each other. Moreover, immunodetection of Ca²⁺-ATPase by means of polyclonal antibodiesrevealed no mobility change after the various treatments. The above stimulation was not altered by neomycin (200 μm), washing with EGTA (5 mm) or by both incubating and washing with delipidized serum albumin (1 mg/ml), or omitting dithioerythritol from the preincubation medium. On the other hand, the activation elicited by Ca²⁺ plus ATP in the presence of Mg²⁺ was reduced 25–30% by acridine orange (100 μm), compound 48/80 (100 μm) or leupeptin (200 μm) but not by dithio-bis-nitrobenzoic acid (1 mm). The fluorescence depolarization of 1,6-diphenyl-and l-(4-trimethylammonium phenyl)-6-phenyl 1,3,5-hexatriene incorporated into membrane fragments was not affected after preincubating under the different conditions. The results show that proteolysis, fatty acid production, an increased phospholipid metabolism or alteration of membrane fluidity are not involved in the Ca²⁺ effect. Ca²⁺ preincubation may stimulate the Ca²⁺-ATPase activity by stabilizing or promoting the E₁ conformation.     

Kinetic properties of the ATP-dependent Ca2+ pump and the Na+/Ca2+ exchange system in basolateral membranes from rat kidney cortex

Summary Basolateral plasma membranes from rat kidney cortex have been purified 40-fold by a combination of differential centrifugation, centrifugation in a discontinuous sucrose gradient followed by centrifugation in 8% percoll. The ratio of leaky membrane vesicles (L) versus right-side-out (RO) and inside-out (IO) resealed vesicles appeared to be LROIO=431. High-affinity Ca²⁺-ATPase, ATP-dependent Ca²⁺ transport and Na⁺/Ca²⁺ exchange have been studied with special emphasis on the relative transport capacities of the two Ca²⁺ transport systems. The kinetic parameters of Ca²⁺-ATPase activity in digitonin-treated membranes are:K _m =0.11 m Ca²⁺ andV _max=81±4 nmol P_i/min·mg protein at 37°C. ATP-dependent Ca²⁺ transport amounts to 4.3±0.2 and 7.4±0.3 nmol Ca²⁺/min·mg protein at 25 and 37°C, respectively, with an affinity for Ca²⁺ of 0.13 and 0.07 m at 25 and 37°C. After correction for the percentage of IO-resealed vesicles involved in ATP-dependent Ca²⁺ transport, a stoichiometry of 0.7 mol Ca²⁺ transported per mol ATP is found for the Ca²⁺-ATPase. In the presence of 75mm Na⁺ in the incubation medium ATP-dependent Ca²⁺ uptake is inhibited 22%. When Na⁺ is present at 5mm an extra Ca²⁺ accumulation is observed which amounts to 15% of the ATP-dependent Ca²⁺ transport rate. This extra Ca²⁺ accumulation induced by low Na⁺ is fully inhibited by preincubation of the vesicles with 1mm ouabain, which indicates that (Na⁺–K⁺)-ATPase generates a Na⁺ gradient favorable for Ca²⁺ accumulation via the Na⁺/Ca²⁺ exchanger. In the absence of ATP, a Na⁺ gradient-dependent Ca²⁺ uptake is measured which rate amounts to 5% of the ATP-dependent Ca²⁺ transport capacity. The Na⁺ gradient-dependent Ca²⁺ uptake is abolished by the ionophore monensin but not influenced by the presence of valinomycin. The affinity of the Na⁺/Ca²⁺ exchange system for Ca²⁺ is between 0.1 and 0.2 m Ca²⁺, in the presence as well as in the absence of ATP. This affinity is surprisingly close to the affinity measured for the ATP-dependent Ca²⁺ pump. Based on these observations it is concluded that in isolated basolateral membranes from rat kidney cortex the Ca²⁺-ATPase system exceeds the capacity of the Na⁺/Ca²⁺ exchanger four- to fivefold and it is therefore unlikely that the latter system plays a primary role in the Ca²⁺ homeostasis of rat kidney cortex cells.     

Direct inhibition of epithelial Na+ channels by a pH-dependent interaction with calcium,and by other divalent ions

Summary Direct inhibitory effects of Ca²⁺ and other ions on the epithelial Na⁺ channels were investigated by measuring the amiloride-blockable²²Na⁺ fluxes in toad bladder vesicles containing defined amounts of mono- and divalent ions. In agreement with a previous report (H.S. Chase, Jr., and Q. Al-Awqati,J. Gen. Physiol. 81:643–666, 1983) we found that the presence of micromolar concentrations of Ca²⁺ in the internal (cytoplasmic) compartment of the vesicles substantially lowered the channel-mediated fluxes. This inhibition, however, was incomplete and at least 30% of the amiloride-sensitive²²Na⁺ uptake could not be blocked by Ca²⁺ (up to 1mm). Inhibition of channels could also be induced by millimolar concentrations of Ba²⁺, Sr²⁺, or VO²⁺, but not by Mg²⁺. The Ca²⁺ inhibition constant was a strong function of pH, and varied from 0.04 m at pH 7.8 to >10 m at pH 7.0 Strong pH effects were also demonstrated by measuring the pH dependence of²²Na⁺ uptake in vesicles that contained 0.5 m Ca²⁺. This Ca²⁺ activity produced a maximal inhibition of²²Na⁺ uptake at pH7.4 but had no effect at pH7.0. The tracer fluxes measured in the absence of Ca²⁺ were pH independent over this range. The data is compatible with the model that Ca²⁺ blocks channels by binding to a site composed of several deprotonated groups. The protonation of any one of these groups prevents Ca²⁺ from binding to this site but does not by itself inhibit transport. The fact that the apical Na⁺ conductance in vesicles, can effectively be modulated by minor variations of the internal pH near the physiological value, raises the possibility that channels are being regulated by pH changes which alter their apparent affinity to cytoplasmic Ca²⁺, rather than, or in addition to changes in the cytoplasmic level of free Ca²⁺.     

The changing sensitivity to auxin of the plasma-membrane H+-ATPase: Relationship between plant development and ATPase content of membranes

The auxin sensitivity of the plasma-membrane H⁺-ATPase from tobacco leaves (Nicotiana tabacum L. cv. Xanthi) depends on the physiological state of the plant (Santoni et al., 1990, Plant Sci. 68, 33–38). Results based on the study of auxin sensitivity according to culture conditions which accelerate or delay tobacco development demonstrate that the highest auxin sensitivity is always associated with the end of the period of induction to flowering. Auxin stimulation of H⁺-translocation activity corresponds to an increase of the apparent ATPase affinity for ATP. The plasma-membrane H⁺-ATPase content, measured with an enzyme-linked immunosorbent assay using a specific anti-H⁺-ATPase antibody, varies according to plant development, and was found to increase by 100% during floral induction. The specific molecular ATPase activity also changes according to plant development; more particularly, the decrease in molecular ATPase activity upto and during the floral-induction period parallels the increase of sensitivity to indole-3-acetic acid.Abbreviations ELISA enzyme-linked immunosorbent assay - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate Authors are grateful to Mrs. Grosclaude (Lab. Virologie, INRA, Jouy-en-Josas, France) and Mrs. Boudon (Lab. Mycoplasmes, INRA, Dijon, France) for support and advice in the preparation of antibodies. This work was supported by grants No. 89/512/6 from the E.P.R of Bourgogne and No. 89 C 0662 from M.R.T.