Inhibition of Rat Brain Microsomal Na+,K+-ATPase by S-Adenosylmethionine

Abstract: Rat brain microsomes were preincubated with S -adenosylmethionine (SAM), MgCl₂, and CaCl₂, then re-isolated, and the activity of Na⁺,K⁺-ATPase determined. SAM inhibited the Na⁺,K⁺-ATPase activity compared with microsomes subjected to similar treatment in the absence of SAM. A biphasic inhibitory effect was observed with a 50% decrease at a SAM concentration range of 0.4 μ M -3.2 μ M and a 70% reduction at a concentration range above 100 μ M . Inclusion of either S- adenosylhomocysteine or 3-deazaadenosine in the preincubations prevented the SAM inhibition of Na⁺,K⁺-ATPase activity. The inhibition by SAM appeared to be Mg²⁺- or Ca²⁺-dependent.     

Plasmalemma from the roots of cucumber: Isolation by two-phase partitioning and characterization

Plasmalemma was isolated from the roots of 2-week-old cucumber plants ( Cucumis sativus L. cv. Rhensk druv) by utilizing an aqueous polymer two-phase system with 6.5%:6.5% (w/w) Dextran T500 and polyethylene glycol (PEG) 3350 at pH 7.8. The plasmalemma fraction comprised ca 6% of the membrane proteins contained in the microsomal fraction. The specific activity of the plasma membrane marker enzyme (K⁺, Mg²⁺-ATPase) was 14- to 17-times higher in the upper (PEG-rich) than in the lower (Dextran-rich) phase, and the reverse was true for marker enzymes (cytochrome c oxidase, EC 1.9.3.1, and antimycin A-resistant NADPH cytochrome c reductase) of intracellular membranes. The ATPase was highly stimulated by the addition of detergent (Triton X-100), so that the isolated plasmalemma vesicles appear tightly sealed and in a right-side-out orientation. Further characterization of the ATPase activities showed a pH optimum at 6.0 in the presence of Mg²⁺. This optimum was shifted to pH 5.8 after addition of K⁺. K⁺ stimulated the ATPase activity below pH 6 and inhibited above pH 6. The ATPase activity was specific for ATP and sensitive to N,N-dicyclohexylcarbodiimide and sodium vanadate, with K⁺ enhancing the vanadate inhibition. The enzyme was insensitive to sodium molybdate, NO⁻₃, azide and oligomycin. No Ca²⁺-ATPase was detected, and even as little as 0.05 m M Ca²⁺ inhibited the Mg²⁺-ATPase activity.     

Probable Role of Allylisothiocyanate-Sensitive H+, K+-ATPase in Spicule Calcification in Embryos of the Sea Urchin, Hemicentrotus pulcherrimus

In embryos of the sea urchin, Hemicentrotus pulcherrimus , as well as in cultured cells derived from isolated micromeres, spicule formation was inhibited by allylisothiocyanate, an inhibitor of H⁺, K⁺-ATPase, at above 0.5 μM and was almost completely blocked at above 10 μM. Amiloride, an inhibitor of Na⁺, H⁺ antiporter, at above 100 μM exerted only slight inhibitory effect, if any, on spicule formation. Intravesicular acidification, determined using [ dimethylamine -¹⁴C]-aminopyrine as a pH probe, was observed in the presence of ATP and 200 mM KCl in microsome fraction obtained from embryos at the post gastrula stage, at which embryos underwent spicule calcification. Intravesicular acidification and K⁺-dependent ATPase activity were almost completely inhibited by allylisothiocyanate at 10 μM. Allylisothiocyanate-sensitive ATPase activity was found mainly in the mesenchyme cells with spicules isolated from prisms. H⁺, K⁺-ATPase, an H⁺ pump, probably mediates H⁺ release to accelerate CaCO₃ deposition from Ca²⁺, CO₂ and H₂O in the primary mesenchyme cells. Intravesicular acidification was stimulated by valinomycin at the late gastrula and the prism stages but not at the pluteus stage. K⁺ permeability probably increases after the prism stage to activate H⁺ release.     

Transport of Mg2+ and Ca2+, and Mg2+-stimulated adenosine triphosphatase activity in oat roots as affected by mineral nutrition

Mg²⁺- and Ca²⁺-uptake was measured in dark-grown oat seedlings ( Avena sativa L. cv. Brighton) cultivated at two levels of mineral nutrition. In addition the stimulation of the ATPase activity of the microsomal fraction of the roots by Mg²⁺ was measured. Ca²⁺-uptake by the roots was mainly passive. Mg²⁺-uptake mainly active; the passive component of Mg²⁺-uptake was accompanied by Ca²⁺-efflux up to 60% of the Ca²⁺ present in the roots.
In general Mg²⁺ -uptake of oat roots was biphasic. The affinity of the second phase correspond well with that of the Mg²⁺-stimulation of the ATPase activity, in low-salt roots as well as in high-salt roots and in roots of plants switched to the other nutritional condition. Linear relationships were observed when [phase 2] Mg²⁺-uptake was plotted against Mg²⁺-stimulation of the ATPase activity of the microsomal fraction of the roots. In 5 days old high-salt plants 1 ATP (hydrolysed in the presence of Mg²⁺ J corresponded with active uptake of a single Mg²⁺ ion, but in older high-salt roots and in low-salt roots more ATP was hydrolysed per net uptake of a Mg²⁺ ion. The results are discussed against the background of regulation of the Mg²⁺-level of the cytoplasm of root cells by transport of Mg²⁺ by a Mg²⁺-ATPase to the vacuole, to the xylem vessels, and possibly outwards.     

Long-term effects of abscisic acid on K+ transport in young wheat plants of different K+ status

The effects of abscisic acid (ABA) on growth, uptake and translocation of potassium ions, K⁺,Mg²⁺-ATPase activity and transpiration were investigated in young wheat ( Triticum aestivum L. cv. Martonvásári-8) plants grown at different K⁺ supplies. Long-term treatment with ABA (10 μ M ) reduced growth in high-K⁺ plants, but had less effect under low-K⁺ conditions. K⁺(⁸⁶Rb) uptake was inhibited by about 70 and 40% in low- and high-K⁺ plants, respectively. The stimulation by K⁺ of the Mg²⁺-ATPase activity in the root microsomal fraction was lost with ABA treatment. It is suggested that the inhibitory effect of ABA on K⁺ uptake may be related to this effects on the K⁺,Mg²⁺-ATPase. Translocation of K⁺ to the shoot was inhibited in low-K⁺ plants only, and it was not affected in high-K⁺ plants. In parallel to this, ABA treatment reduced transpiration by about 50% in low-K⁺ plants, whereas a much smaller effect was seen in high-K⁺ plants. These observations suggest that the regulation by ABA of the stomatal movements is strongly counteracted by high-K⁺ status.     

DEFICIENCY OF A Ca2+-ATPase IN BRAINS OF SEIZURE PRONE MICE

Abstract– Ca²⁺-stimulated ATPase activity was studied in membrane enriched preparations from the brains of audiogenic seizure-prone (DBA) and control (C57 and C3H) mice. The animals ranged in age from 7 to 60 days. Na⁺, K ⁺ -ATPase, 5'-nucleotidase and p -nitrophenylphosphatase were assayed to evaluate membrane integrity.
Ca²⁺-ATPase was significantly lower in DBA mice; notably during the period of maximal seizure sensitivity. Mg²⁺ -ATPase somewhat followed the pattern shown by Ca²⁺ -ATPase. Na⁺, K⁺ -ATPase in DBA did not differ significantly from controls and there were no differences in either 5'-nucleotidase or p-nitrophenylphosphatase activities.
Ca²⁺-ATPase kinetics experiments showed even more clearly the difference between DBA and control preparations. V_max was consistently lower in DBA than in controls. The K_m values appeared to fall into groupings suggestive of sequential synthesis of isozymes. Differences in the patterns of DBA and C57 just prior to the time of maximal seizure sensitivity are interpreted as reflecting failure to synthesize an isozyme or delay of its synthesis. The genesis of seizures through such an enzymatic defect may be related to the action of translocated ATP on the plasma membrane.     

Effects of 2-iodobenzanilide on potassium uptake, H+ extrusion and K+-stimulated ATPase of corn roots

The carboxanilide systemic fungicide 2-iodobenzanilide (2-IB) after 2 h pretreatment at 0.25 m M inhibited K⁺ and SO₄^2- uptake by excised corn roots ( Zea mays L., cv. Dekalb 342) up to ca 70 and 40%, respectively. Proton extrusion from corn roots was also reduced by ca 50% after 1 h contact, and the microsomal K⁺-stimulated ATPase activity from corn roots and pea stems ( Pisum sativum L., cv. Alaska) inhibited by 50 and 72%, respectively. In contrast, the Mg²⁺-ATPase activities of microsomes and mitochondria at pH 6.0 and 8.7, respectively, were unaffected. After 2 h of preincubation with 0.25 m M 2-IB, O₂ consumption by corn roots and pea stems was inhibited by 12 and 18%, respectively. ATP content of corn roots was not altered by 2-IB treatment. Therefore, energy availability "in vivo" was unaffected and the primary effect on corn roots is suggested to be at the plasmalemma ATPase which forms the proton gradient.
With isolated pea stem mitochondria, 0.25 m M 2-IB inhibited O₂ consumption by ca 60% when NADH or malate plus pyruvate were added as substrates; when succinate was used O₂ consumption was unaffected. The mode of action on isolated mitochondria was different from that shown for carboxin and also formerly attributed to the whole class of carboxanilide fungicides.     

Temperature dependence of the barley root plasma membrane-bound Ca2+- and Mg2+-dependent ATPase

Arrhenius plots of the maximal velocities for the Ca²⁺-and Mg²⁺-dependent ATPase activities found in a plasma membrane-rich microsome fraction isolated from the roots of barley ( Hordeum vulgare L. cv. Conquest) were nonlinear. Arrhenius plot analyses using a relation which produced curvilinear Arrhenius plots accurately fit the data and allowed the calculation of the activation enthalpies and molar heat capacities of activation. The temperature dependence of the computed Km values for the Ca²⁺- and Mg²⁺-dependent ATPase activities was complex, with the highest enzyme-substrate affinities being obtained near the barley seedling growth temperature (16°C). Using electron paramagnetic resonance spectroscopy with amphiphilic cationic and anionic spin probes, it was possible to demonstrate that temperature changes and increasing Ca²⁺ concentrations could alter the mobility of the membrane lipid polar head groups. Inhibition of the ATPase activities by high levels of Ca²⁺ may result from a Ca2+-induced reduction in the lipid polar head group mobility. The possible role of lipid polar head group-protein interactions in the complex temperature dependence of the barley root ATPase kinetic constants is discussed.     

NMDA Receptor-Mediated Neurotoxicity: A Paradoxical Requirement for Extracellular Mg2+ in Na+/Ca2+-Free Solutions in Rat Cortical Neurons In Vitro

Abstract: Accumulation of intracellular Ca²⁺ is known to be critically important for the expression of NMDA receptor-mediated glutamate neurotoxicity. We have observed, however, that glutamate can also increase the neuronal intracellular Mg²⁺ concentration on activation of NMDA receptors. Here, we used conditions that elevate intracellular Mg²⁺ content independently of Ca²⁺ to investigate the potential role of Mg²⁺ in excitotoxicity in rat cortical neurons in vitro. In Ca²⁺-free solutions in which the Na⁺ was replaced by N -methyl- d -glucamine or Tris (but not choline), which also contained 9 m M Mg²⁺, exposure to 100 µ M glutamate or 200 µ M NMDA for 20 min produced delayed neuronal cell death. Neurotoxicity was correlated to the extracellular Mg²⁺ concentration and could be blocked by addition of NMDA receptor antagonists during, but not immediately following, agonist exposure. Finally, we observed that rat cortical neurons grown under different serum conditions develop an altered sensitivity to Mg²⁺-dependent NMDA receptor-mediated toxicity. Thus, the increase in intracellular Mg²⁺ concentration following NMDA receptor stimulation may be an underestimated component critical for the expression of certain forms of excitotoxic injury.     

Glutamate Induces a Calcineurin-Mediated Dephosphorylation of Na+,K+-ATPase that Results in Its Activation in Cerebellar Neurons in Culture

Abstract: In primary cultures of cerebellar neurons glutamate neurotoxicity is mainly mediated by activation of the NMDA receptor, which allows the entry of Ca²⁺ and Na⁺ into the neuron. To maintain Na⁺ homeostasis, the excess Na⁺ entering through the ion channel should be removed by Na⁺,K⁺-ATPase. It is shown that incubation of primary cultured cerebellar neurons with glutamate resulted in activation of the Na⁺,K⁺-ATPase. The effect was rapid, peaking between 5 and 15 min (85% activation), and was maintained for at least 2 h. Glutamate-induced activation of Na⁺,K⁺-ATPase was dose dependent: It was appreciable (37%) at 0.1 µ M and peaked (85%) at 100 µ M . The increase in Na⁺,K⁺-ATPase activity by glutamate was prevented by MK-801, indicating that it is mediated by activation of the NMDA receptor. Activation of the ATPase was reversed by phorbol 12-myristate 13-acetate, an activator of protein kinase C, indicating that activation of Na⁺,K⁺-ATPase is due to decreased phosphorylation by protein kinase C. W-7 or cyclosporin, both inhibitors of calcineurin, prevented the activation of Na⁺,K⁺-ATPase by glutamate. These results suggest that activation of NMDA receptors leads to activation of calcineurin, which dephosphorylates an amino acid residue of the Na⁺,K⁺-ATPase that was previously phosphorylated by protein kinase C. This dephosphorylation leads to activation of Na⁺,K⁺-ATPase.     

Glutamate Uptake Stimulates Na+,K+-ATPase Activity in Astrocytes via Activation of a Distinct Subunit Highly Sensitive to Ouabain

Abstract: The excitatory amino acid glutamate was previously shown to stimulate aerobic glycolysis in astrocytes by a mechanism involving its uptake through an Na⁺-dependent transporter. Evidence had been provided that Na⁺,K⁺-ATPase might be involved in this process. We have now measured the activity of Na⁺,K⁺-ATPase in cultured astrocytes, using ouabain-sensitive ⁸⁶Rb uptake as an index. l -Glutamate increases glial Na⁺,K⁺-ATPase activity in a concentration-dependent manner with an EC₅₀ = 67 µ M . Both l - and d -aspartate, but not d -glutamate, produce a similar response, an observation that is consistent with an uptake-related effect rather than a receptor-mediated one. Under basal conditions, concentration-dependent inhibition of Na⁺,K⁺-ATPase activity in astrocytes by ouabain indicates the presence of a single catalytic site with a low affinity for ouabain ( K _0.5 = 113 µ M ), compatible with the presence of an α₁ isozyme. On stimulation with glutamate, however, most of the increased activity is inhibited by low concentrations of ouabain ( K _0.5 = 20 n M ), thus revealing a high-affinity site akin to the α₂ isozyme. These results suggest that astrocytes possess a glutamate-sensitive isoform of Na⁺,K⁺-ATPase that can be mobilized in response to increased neuronal activity.     

Ca2+ dependence and La3+ interference of ultraviolet radiationinduced K+ efflux from rose cells

A low fluence of ultraviolet radiation (UV) causes cultured cells of Rosa damascena Mill cv. Gloire de Guilan to lose intracellular K⁺. This effect required the presence of Ca²⁺ in the medium. A reduction in the concentration of free Ca²⁺ to 10⁻⁵ M with ethyleneglycol-bis-(β-aminoethyl-ether)-N.N.N',N'-tetraacetic acid (EGTA) buffer inhibited the UV-stimulated efflux; this was correlated with a discharge of the membrane potential and a stimulation of the leakage of K⁺ from unirradiated cells. All the same effects were seen with La³⁺ at 0.2 m M. At 0.02 m M La³⁺, the UV-stimulated efflux of K⁺ was blocked without concomitant effects on the membrane potential or K⁺ efflux from control cells. It is suggested that removal of Ca²⁺ blocks or masks the UV-induced leakage of K⁺ by destabilizing the plasma membrane. In addition, La³⁺ may specifically inhibit the UV-stimulated opening of K⁺ or anion channels.     

Nitroprusside and Cyclic GMP Stimulate Na+-Ca2+ Exchange Activity in Neuronal Preparations and Cultured Rat Astrocytes

Abstract: The effects of nitric oxide (NO)-generating agents on ⁴⁵Ca²⁺ uptake in rat brain slices and cultured rat astrocytes were studied in the presence of monensin, which is considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Sodium nitroprusside (SNP) at >10 µ M increased monensin-stimulated Ca²⁺ uptake in the slices, although it did not affect high K⁺-stimulated Ca²⁺ uptake. Another NO donor, 3-morpholinosydnonimine, was effective. The effect of SNP was antagonized by hemoglobin (50 µ M ), a NO scavenger, and mimicked by 8-bromo-cyclic GMP (100 µ M ). In rat brain synaptosomes, SNP increased monensin-stimulated Ca²⁺ uptake, but it did not affect high K⁺-stimulated Ca²⁺ uptake. 8-Bromocyclic GMP, but not SNP, increased Na⁺-dependent Ca²⁺ uptake significantly in synaptic membrane vesicles in the absence of monensin. In cultured rat astrocytes, SNP and 8-bromo-cyclic GMP increased Ca²⁺ uptake in the presence of ouabain and monensin, which were required for the Ca²⁺ uptake in the cells. These findings suggest that NO stimulates the Na⁺-Ca²⁺ exchanger in neuronal preparations and astrocytes in a cyclic GMP-dependent mechanism.     

Effect of K+ and H+ stress and role of Ca2+ in the regulation of intracellular K+ concentration in mung bean roots

The effects of external K⁺, H⁺ and Ca2⁺ concentrations on the intracellular K+ concentration, [K⁺]i, and the K⁺-ATPase activity in 2-day-old mung bean roots [ Vigna mungo (L.) Hepper] were investigated. [K⁺]i, in mung bean roots was markedly decreased by external K⁺ or H⁺ stress and did not recover the initial value even after the stress was removed. This decrease in [K⁺]i, gradually disappeared with the addition of (Ca2⁺. Ca2⁺ may offset the harmful effects of ion stress. Ca2⁺ seems to have two effects on K+ transport; control of K⁺ permeability and activation of K⁺ uptake, although K⁺-ATPase activity was inhibited by Ca2⁺ concentrations higher than 10–4 M. We suggest that Ca2⁺ activates K⁺ uptake indirectly through the acidification of the cytoplasm.     

Several compartments of Saccharomyces cerevisiae are equipped with Ca2+-ATPase(s)

Abstract Sucrose density fractionation of yeast membranes revealed two major and two minor peaks of ⁴⁵Ca²⁺ transport activity which all co-migrate with marker enzymes of the endoplasmic reticulum, Golgi and membranes associated with these compartments as well as with ATPase activity measured when all other known ATPase are inhibited. Co-migration of ⁴⁵Ca²⁺ transport and ATPase activities was also found after removal of plasma membranes by concanavalin A treatment. SDS-PAGE at pH 6.3 shows the Ca²⁺-dependent formation of acyl phosphate polypeptides of about 110 and 200 kDa. It is concluded that several compartments or sub-compartments of yeast are equipped with Ca²⁺-ATPase(s). It is proposed that these compartments are derived from the protein secretory apparatus of yeast.     

ATP-dependent H+-pumping of a low-density fraction of pea stem microsomes

A low-density fraction of pea ( Pisum sativum L. cv. Alaska) stem microsomes, obtained from a discontinuous sucrose gradient, possessed an H⁺-ATPase able to generate a proton gradient and an electrical potential. The proton pumping was insensitive to monovalent cations, to vanadate and oligomycin, required a permeant anion and was inhibited by nitrate, N, N'-dicyclohexylcarbodiimide and diethylstilbestrol. The H⁺-ATPase had a pH optimum around 6.0–6.5 and was saturable with respect to the substrate Tris-ATP (K_m≅ 0.4 m M ). Ca²⁺ (0.05–1 m M ) induced a dissipation of the ATP-generated δpH without affecting ATPase activity. At physiological concentrations (1–5 m M ), nitrate caused an initial slight increase of the ATP-generated proton gradient followed by a complete dissipation after 2–3 min. The dissipating effect was not caused by inhibition of ATPase activity, since ATP prevented the nitrate-induced collapse of δpH. On the other hand, ATPase activity, evaluated as release of P_i, was not inhibited by concentrations lower than 20 m M KNO₃. These results indicate that nitrate entered the vesicles in response to an electrical potential and then could exit in symport with protons, while Ca²⁺ entered in exchange for protons (antiport).     

Intracellular Ascorbic Acid Inhibits the Na+-Ca2+ Exchanger in Cultured Rat Astrocytes

Abstract: The effect of ascorbic acid on Ca²⁺ uptake in cultured rat astrocytes was examined in the presence of ouabain and monensin, which are considered to drive the Na⁺-Ca²⁺ exchanger in the reverse mode. Ascorbic acid at 0.1–1 m M inhibited Na⁺-dependent Ca²⁺ uptake significantly but not Na⁺-dependent glutamate uptake in the cells, although the inhibition required pretreatment for more than 30 min. The effect of ascorbic acid on the Ca²⁺ uptake was blocked by simultaneous addition of ascorbate oxidase (10 U/ml). Na⁺-dependent Ca²⁺ uptake was also inhibited by isoascorbate at 1 m M but not by ascorbate 2-sulfate, dehydroascorbate, and sulfhydryl-reducing reagents such as glutathione and 2-mercaptoethanol. The inhibitory effect of ascorbic acid was observed even in the presence of an inhibitor of lipid peroxidation, o -phenanthroline, or a radical scavenger, mannitol, and the degrading enzymes such as catalase and superoxide dismutase. On the other hand, the inhibitory effect was not observed under the Na⁺-free conditions that inhibited the uptake of ascorbic acid in astrocytes. When astrocytes were cultured for 2 weeks in a medium containing ascorbic acid, the content of ascorbic acid in the cells was increased and conversely Na⁺-dependent Ca²⁺ uptake was decreased. These results suggest that an increase in intracellular ascorbic acid results in a decrease of Na⁺-Ca²⁺ exchange activity in cultured astrocytes and the mechanism is not related to lipid peroxidation.     

Barium Evokes Glutamate Release from Rat Brain Synaptosomes by Membrane Depolarization: Involvement of K+, Na+, and Ca2+ Channels

Abstract: During K⁺ -induced depolarization of isolated rat brain nerve terminals (synaptosomes), 1 m M Ba²⁺ could substitute for 1 m M Ca²⁺ in evoking the release of endogenous glutamate. In addition, Ba²⁺ was found to evoke glutamate release in the absence of K⁺-induced depolarization. Ba²⁺ (1–10 m M ) depolarized synaptosomes, as measured by voltage-sensitive dye fluorescence and [³H]-tetraphenylphosphonium cation distribution. Ba²⁺ partially inhibited the increase in synaptosomal K⁺ efflux produced by depolarization, as reflected by the redistribution of radiolabeled ⁸⁶Rb⁺. The release evoked by Ba²⁺ was inhibited by tetrodotoxin (TTX). Using the divalent cation indicator fura-2, cytosolic [Ca²⁺] increased during stimulation by approximately 200 n M , but cytosolic [Ba²⁺] increased by more than 1 μ M . Taken together, our results indicate that Ba²⁺ initially depolarizes synaptosomes most likely by blocking a K⁺ channel, which then activates TTX-sensitive Na⁺ channels, causing further depolarization, and finally enters synaptosomes through voltage-sensitive Ca²⁺channels to evoke neurotransmitter release directly. Though Ba²⁺-evoked glutamate release was comparable in level to that obtained with K⁺-induced depolarization in the presence of Ca²⁺, the apparent intrasynaptosomal level of Ba²⁺ required for a given amount of glutamate release was found to be several-fold higher than that required of Ca²⁺.     

ATPase activity of isolated plasma membrane vesicles of leaves of Elodea as affected by thiol reagents and NADH/NAD+ ratio

The goal of this study was to test the hypothesis that the plasma membrane-bound ATPase activity is influenced by the redox poise of the cytoplasm. Purified plasma membrane vesicles from leaves of Elodea canadensis Michx. and E. nuttallii (Planch.) St. John were isolated using an aqueous polymer two-phase batch procedure. The distribution of marker enzyme activities confirmed the plasma membrane origin of the vesicles. The vesicles exhibited NADH-ferricyanide reductase activity, indicating the presence of a redox chain in the plasma membrane. The K⁺, Mg²⁺-ATPase activity associated with these vesicles was inhibited by the sulfhydryl reagents N-ethylmaleimide and glutathione (GSSG). Furthermore the activity was inhibited by NAD⁺. This inhibition by NAD⁺ was relieved by increasing the NADH/NAD⁺ ratio. The possibility that the ATPase activity is regulated by the cytoplasmic NAD(P)H/ NAD(P)⁺ ratio is discussed, as well as the role of a plasma membrane-bound redox chain.     

Characterization of the plasmalemma ATPase activity from roots of Plantago major ssp. pleiosperma, purified by the two-phase partitioning method

A purified plasmalemma preparation from roots of Plantago major L. ssp. pleiosperma (Pilger) was obtained by the two-phase partitioning method, using 6.5% (w/w) of Dextran T-500 and polyethylene glycol 3350, respectively. The distribution of murker enzymes proved the purity of the plasmalemma fraction. The ATPase activity was characterized by determining its sensitivity to anions, cations and inhibitors. The Mg²⁺-dependent ATPase activity peaked at pH 7.25, K⁺-stimulation at pH 6.75, and the Cl⁻ -stimulation both at pH 6.75 and 7.5 (all in the presence of 3 m M MgSO₄). The plasmalemma preparations hydrolyzed preferentially ATP (in the presence of Mg²⁺), although they were less specific for ATP at pH 7.5 than at pH 6.75. The Cl⁻ - stimulated ATPase is probably associated with and located on the plasmalemma. The question if the Cl⁻ -stimulated activity is due to an ATPase distinct from the classical K⁺-stimulated ATPase is considered.