The lysolipid sphingosine modulates pyrophosphatase activity in tonoplast vesicles and isolated vacuoles from a heterotrophic cell suspension culture of Chenopodium rubrum

The proton pumping activity of the tonoplast (vacuolar membrane) H⁺-ATPase and H⁺-pyrophosphatase (H⁺-PPase) has been studied on a tonoplast-enriched microsomal fraction and on intact vacuoles isolated from a heterotrophic cell suspension culture of Chenopodium rubrum L. in the presence of the lysosphingolipids D-sphingosine, psychosine (galactosylsphingosine) and lysosulfatide (sulfogalactosyl-sphingosine). Sphingosine strongly stimulates (K_a= 0.16 μ M ) the PPase activity, assayed both as ΔpH formation across the tonoplast vesicle membrane, and as reversible clamp current measured by the whole-vacuolar mode of the patch-clamp technique. Psychosine showed a minor, and lysosulfatide no stimulatory effect. No effect upon the ATPase activity has been observed. No sphingosine-induced change could be observed in the affinity of the PPase for its substrate (apparent K_m= 10 μ M MgPPi). We tentatively conclude that sphingosine, which is known as a potent inhibitor of the protein kinase C in animal cells, may be a regulator of the plant vacuolar PPase.     

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.     

Local Anesthetics Inhibit the Ca2+,Mg2+-ATPase Activity of Rat Brain Synaptosomes

Many biochemical effects of local anesthetics are expressed in Ca2+-dependent processes [Volpi M., Sha'afi R.I., Epstein P.M., Andrenyak P.M., and Feinstein M.B. (1981) Proc. Natl. Acad. Sci. USA 78, 795-799]. In this communication we report that local anesthetics (dibucaine, tetracaine, lidocaine, and procaine and the analogue quinacrine) inhibit the Ca2+-dependent and the Mg2+-dependent ATPase activity of rat brain synaptosomes and of membrane vesicles derived from them by osmotic shock. This inhibition is induced by concentrations of these drugs close to their pharmacological doses, and a good correlation between K0.5 of inhibition and their relative anesthetic potency is found. The Ca2+-dependent ATPase is more selectively inhibited at lower drug concentrations. The physiological relevance of these findings is discussed briefly.     

Na+/H+ antiport activity in tonoplast vesicles isolated from sunflower roots induced by NaCl stress

Na⁺ transport across the tonoplast and its accumulation in the vacuoles is of crucial importance for plant adaptation to salinity. Mild and severe salt stress increased both ATP- and PP_i-dependent H⁺ transport in tonoplast vesicles from sunflower seedling roots, suggesting the possibility that a Na⁺/H⁺ antiport system could be operating in such vesicles under salt conditions (E. Ballesteros et al. 1996. Physiol. Plant. 97: 259–268). During a mild salt stress, Na⁺ was mainly accumulated in the roots. Under a more severe salt treatment, Na⁺ was equally distributed in shoots and roots. In contrast to what was observed with Na⁺, all the salt treatments reduced the shoot K⁺ content. Dissipation by Na⁺ of the H⁺ gradient generated by the tonoplast H⁺-ATPase, monitored as fluorescence quenching of acridine orange, was used to measure Na⁺/H⁺ exchange across tonoplast-enriched vesicles isolated by sucrose gradient centrifugation from sunflower (Helianthus annuus L.) roots treated for 3 days with different NaCl regimes. Salt treatments induced a Na⁺/H⁺ exchange activity, which displayed saturation kinetics for Na⁺ added to the assay medium. This activity was partially inhibited by 125 μM amiloride, a competitive inhibitor of Na⁺/H⁺ antiports. No Na⁺/H⁺ exchange was detected in vesicles from control roots. The activity was specific for Na⁺. since K⁺ added to the assay medium slightly dissipated H⁺ gradients and displayed non-saturating kinetics for all salt treatments. Apparent K_m for Na⁺/H⁺ exchange in tonoplast vesicles from 150 mM NaCl-treated roots was lower than that of 75 mM NaCl-treated roots, V_max remaining unchanged. The results suggest that the existence of a specific Na⁺/H⁺ exchange activity in tonoplast-enriched vesicle fractions, induced by salt stress, could represent an adaptative response in sunflower plants, moderately tolerant to salinity.     

Incubation of Tissue Slices in the Absence of Ca2+ and Mg2+ Can Cause Nonspecific Damage

Superfusion of striatal slices with a medium deficient in Ca2+ and Mg2+ caused a large and sustained increase in release of lactate dehydrogenase, a finding indicative of the disruption of plasma membranes. This was associated with an efflux of dopamine (DA) and the depletion of DA from the tissue. In addition, whereas DA efflux was stimulated by either D-amphetamine (10 microM) or L-glutamate (10 mM) in the absence of Ca2+, these effects were greatly reduced when Mg2+ also was withdrawn from the buffer. These results suggest that (a) incubation in a Ca2+/Mg2(+)-free buffer disrupts plasma membranes, (b) this disruption affects dopaminergic neurons as well as those of other striatal elements, and (c) the failure of a treatment to stimulate DA release in a Ca2+/Mg2(+)-free buffer cannot be used as a test of Ca2+ dependence.     

Intrasynaptosomal Free Mg2+ Concentration Measured with the Fluorescent Indicator Mag-Fura-2: Modulation by Na+ Gradient and by Extrasynaptosomal ATP

Abstract: Synaptosomes can be loaded with mag-fura-2 without significant perturbation of their ATP content by incubation for 10 min at 37°C with 10 µM mag-fura-2 acetoxymethyl ester in Hanks'-HEPES buffer (pH 7.45). The intrasynaptosomal free Mg²⁺ concentration ([Mg²⁺]_i) was found to be dependent on external Mg²⁺ concentration, increasing from 0.8 to 1.25 mM when the concentration of Mg²⁺ in the incubation medium increased from 1 to 8 mM. Dissipation of the Na⁺ gradient across the plasma membrane of synaptosomes by treatment with the Na⁺ ionophore monensin (0.2 mM) or with veratridine (0.2 mM) and ouabain (0.6 mM) produced a moderate increase of [Mg²⁺]_i, from 1.0 to 1.2–1.3 mM in an incubation medium containing 5 mM Mg²⁺. Plasma membrane depolarization by incubation of synaptosomes in a medium containing 68 mM KCl and 68 mM NaCl had no effect on [Mg²⁺]_i. Reversal of the Na⁺ gradient by incubation of synaptosomes in a medium in which external Na⁺ was replaced by choline increased [Mg²⁺]_i up to 1.6 and 2.2 mM for extrasynaptosomal Mg²⁺ concentrations of 1 and 8 mM, respectively. We conclude that a Na⁺/Mg²⁺ exchange operates in the plasma membrane of synaptosomes. In the presence of Mg²⁺ in the incubation medium, extrasynaptosomal ATP, but not ADP or adenosine, increased [Mg²⁺]_i from 1.1 ± 0.1 up to 1.6 ± 0.1 mM. The nonhydrolyzable ATP analogue adenosine 5′-(βγ-imido)triphosphate antagonized the effect of ATP, but had no effect by itself on [Mg²⁺]_i. It is concluded that Mg²⁺ transport across the plasma membrane of synaptosomes is modulated by the activity of an ecto-ATPase or an ecto-protein kinase.     

Effects of Cations on (Ca2++ Mg2+)-Activated ATPase from Rat Brain

Abstract: With a partially purified, membrane-bound (Ca + Mg)-activated ATPase preparation from rat brain, the K_0.5 for activation by Ca²⁺ was 0.8 p μm in the presence of 3 mm -ATP, 6 mm -MgCl₂, 100 m_M-KCI, and a calcium EGTA buffer system. Optimal ATPase activity under these circumstances was with 6-100 μm -Ca²⁺, but marked inhibition occurred at higher concentrations. Free Mg²⁺ increased ATPase activity, with an estimated K_0.5, in the presence of 100 μm -CaCl₂, of 2.5 mm ; raising the MgCl₂ concentration diminished the inhibition due to millimolar concentrations of CaCl₂, but antagonized activation by submicromolar concentrations of Ca²⁺. Dimethylsulfoxide (10%, v/v) had no effect on the K_0.5 for activation by Ca²⁺, but decreased activation by free Mg²⁺ and increased the inhibition by millimolar CaCl₂. The monovalent cations K⁺, Na⁺, and TI⁺ stimulated ATPase activity; for K⁺ the K_0.5 was 8 mm , which was increased to 15 mm in the presence of dimethylsulfoxide. KCI did not affect the apparent affinity for Ca²⁺ as either activator or inhibitor. The preparation can be phosphorylated at 0°C by [γ-³²P]-ATP; on subsequent addition of a large excess of unlabeled ATP the calcium dependent level of phosphorylation declined, with a first-order rate constant of 0.12 s^?1. Adding 10 mm -KCI with the unlabeled ATP increased the rate constant to 0.20 s^?1, whereas adding 10 mm -NaCl did not affect it measurably. On the other hand, adding dimethyl-sulfoxide slowed the rate of loss, the constant decreasing to 0.06 s^?1. Orthovanadate was a potent inhibitor of this enzyme, and inhibition with 1 μm -vanadate was increased by both KCI and dimethylsulfoxide. Properties of the enzyme are thus reminiscent of the plasma membrane (Na + K)-ATPase and the sarcoplasmic reticulum (Ca + Mg)-ATPase, most notably in the K⁺ stimulation of both dephosphorylation and inhibition by vanadate.     

Cyclothiazide Modulates AMPA Receptor-Mediated Increases in Intracellular Free Ca2+ and Mg2+ in Cultured Neurons from Rat Brain

Abstract: We investigated the modulation of (±)-α-amino-3-hydroxy-5-methylisoxazole-4-propionic acid (AMPA)-induced increases in intracellular free Ca²⁺ ([Ca²⁺]_i) and intracellular free Mg²⁺ ([Mg²⁺]_i) by cyclothiazide and GYKI 52466 using microspectrofluorimetry in single cultured rat brain neurons. AMPA-induced changes in [Ca²⁺]_i were increased by 0.3–100 µ M cyclothiazide, with an EC₅₀ value of 2.40 µ M and a maximum potentiation of 428% of control values. [Ca²⁺]_i responses to glutamate in the presence of N -methyl- d -aspartate (NMDA) receptor antagonists were also potentiated by 10 µ M cyclothiazide. The response to NMDA was not affected, demonstrating specificity of cyclothiazide for non-NMDA receptors. Almost all neurons responded with an increase in [Ca²⁺]_i to both kainate and AMPA in the absence of extracellular Na⁺, and these Na⁺-free responses were also potentiated by cyclothiazide. GYKI 52466 inhibited responses to AMPA with an IC₅₀ value of 12.0 µ M . Ten micromolar cyclothiazide significantly decreased the potency of GYKI 52466. However, the magnitude of this decrease in potency was not consistent with a competitive interaction between the two ligands. Cyclothiazide also potentiated AMPA- and glutamate-induced increases in [Mg²⁺]_i. These results are consistent with the ability of cyclothiazide to decrease desensitization of non-NMDA glutamate receptors and may provide the basis for the increase in non-NMDA receptor-mediated excitotoxicity produced by cyclothiazide.     

Ischemia-Induced Inhibition of Calcium Uptake into Rat Brain Microsomes Mediated by Mg2+/Ca2+ ATPase

Abstract: It is well established that ischemia is associated with prolonged increases in neuronal intracellular free calcium levels. Recent data suggest that regulation of calcium uptake and release from the endoplasmic reticulum is important in maintaining calcium homeostasis. The endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase is the major mechanism for sequestering calcium in this organelle. Inhibition of this enzyme may play a causal role in the loss of calcium homeostasis. In order to investigate the effect of ischemia on calcium sequestration into the endoplasmic reticulum, microsomes were isolated from control and ischemic whole brain homogenates by differential centrifugation. Calcium uptake was measured by radioactive calcium (⁴⁵Ca²⁺) accumulation in the microsomes mediated by Mg²⁺/Ca²⁺ ATPase. Ischemia caused a statistically significant inhibition of presteady-state and steady-state calcium uptake. Duration of ischemia was directly proportional to the degree of inhibition. Decreased calcium uptake was shown not to be the result of increased calcium release from ischemic compared with control microsomes nor the result of selective isolation of ischemic microsomes from the homogenate with a decreased capacity for calcium uptake. The data demonstrate that ischemia inhibits the ability of brain microsomes to sequester calcium and suggest that loss of calcium homeostasis is due, in part, to ischemia-induced inhibition of endoplasmic reticulum Mg²⁺/Ca²⁺ ATPase.     

Interaction between poly(L-lysine) and membranes inhibits proton pumping by corn root tonoplast H+-ATPase

The influence of poly(L-lysine) binding on the coupled activities of nitrate-sensitive H⁺-ATPase in isolated corn ( Zea mays L. cv. FRB73) root tonoplast vesicles was investigated. The addition of membrane-impermeable poly(L-lysine) caused a slow increase in light scattering of the tonoplast suspension. Electron microscopy showed that the increase was the result of an aggregation of the vesicles. In the presence of 75 m M KCl, a concentration sufficient to sustain near optimal ATP hydrolysis, poly(L-lysine) slightly enhanced the hydrolysis activity but significantly inhibited proton pumping of the H⁺-ATPase. Inhibition increased with the average molecular mass of poly(L-lysine) and reached a maximum at 58 kDa. When total osmolarity was kept constant, the replacement of sucrose by KCl enhanced both ATP hydrolysis and proton pumping activities. However, enhancement of proton pumping was significantly greater than that of ATP hydrolysis. An increase in KCl, but not K₂SO₄, significantly relieved poly(L-lysine)-induced inhibition of proton pumping. Kinetic analysis indicated that poly(L-lysine) did not significantly affect the proton leakage of the tonoplast membranes under different energetic conditions. These results suggest that the electrostatic interaction between poly(L-lysine) and the negative charges on the exterior surface of tonoplast vesicles could change the coupling ratio of ATP hydrolysis to proton pumping. Thus, the surface charge of the tonoplast membrane may be involved in the regulation of these two activities.     

Ca2+- or Mg2+-Stimulated ATPase Activity in Bullfrog Spinal Nerve: Relation to Ca2+ Requirements for Fast Axonal Transport

Adenosine triphosphatase (ATPase) activity stimulated by Ca2+ or Mg2+ was characterized in spinal nerve and spinal sensory ganglion of bullfrog. Enzyme activity of homogenates from both sources reached a maximum at a 1-2 mM concentration of either cation, although the level of maximal activity in nerve trunks was approximately twice that in ganglia. Enzyme activation was not observed with 2 mM-Sr2+ or Ba2+. Co2+ or Mn2+, at 2 mM, depressed Ca2+ activation of the enzyme by 50-60% in nerve but had no inhibitory effect on ganglia activity. In intact spinal ganglion/spinal nerve preparations, incubated for 20 h in medium containing 0.2 mM-Co2+, no effect was detected on Ca2+/Mg2+ ATPase activity in ganglia or nerve trunks whereas fast axonal transport was inhibited by 80%. Incubation in medium containing 0.02 mM-Hg2+ depressed enzyme activity in ganglia by 64% and in nerve trunks by 44%, whereas fast transport was again inhibited by 80%. When only nerve trunks were exposed to these ions, Hg2+ but not Co2+ was observed to slow the rate of fast axonal transport. The divalent cation specificity of the Ca2+/Mg2+ ATPase activity is distinct from the ion specificities, determined in previous work, of the Ca2+ requirement during initiation of fast axonal transport in the soma, and of the Ca2+ requirement during translocation in the axon. Thus, previous observations of Ca2+-dependent events in fast axonal transport cannot be taken per se to suggest the involvement of Ca2+/Mg+ ATPase in the transport process.     

Effects of Opiates on High-Affinity Ca2+,Mg2+-ATPase in Brain Membrane Subfractions

The effect of a single administration of morphine sulfate (15 mg/kg, s.c. or 30 mg/kg, i.p., 30 min) on Ca2+-stimulated Mg2+-dependent ATPase activity was investigated in synaptosomal plasma membranes (SPM) prepared from rat cortex. Morphine produced a significant decrease in Ca2+,Mg2+-ATPase activity in synaptosomal fractions (SPM 1 + 2) known to contain a high density of opiate receptors and calmodulin-dependent Ca2+,Mg2+-ATPase. However, in another subpopulation (SPM 3) that contains fewer opiate receptors and less enzyme activity, no such decrease in the enzyme activity was observed after the opiate administration. The decrease in Ca2+,Mg2+-ATPase activity seen in SPM 1 + 2 was specifically antagonized by the opiate antagonist naloxone hydrochloride (2 mg/kg, s.c.) when given 15 min before morphine administration. Mg2+-ATPase was not altered either by morphine or by a naloxone-morphine combination. These findings give further evidence for the role of intracellular Ca2+ in mediating many of the acute effects of opiates.     

Anion-sensitive ATPase activity and proton transport in isolated vacuoles of species of the CAM genus Kalanchoë

Vacuoles were isolated from leaves of Kalanchoë daigremontiana Hamet et Perrier de la Bathie, and the ionic sensitivity of the vacuolar ATPase was studied in vacuole homogenates desalted on Sephadex G-25. The ATPase activity was dependent on the presence of divalent cations (Mg²⁺≥ Mn²⁺≥ Ca²⁺, Co²⁺; Zn²⁺ had no effect). Mg²⁺-dependent ATPase activity was stimulated by anions (Cl^? > malate²⁺, HCO^?₃), with maximal stimulation at concentrations above 50 mM. Mg²⁺-Dependent activity was inhibited by NO^?₃ above 2 mM, but no saturation was observed up to 100 mM. No stimulation by K⁺ or Na⁺ was detected; stimulation by NH⁺₄ was abolished by 0.01% (w/v) Triton X-100, suggesting that the NH⁺₄ effect was due to the permeability of vacuolar membrane vesicles to NH₃. Trans-tonoplast electrical potentials (Δψ) and intra-vacuolar pH were measured with glass microelectrodes and antimony covered glass micro-pH-electrodes, respectively. Free vacuofes isolated from Kalanchoë tubiflora (Harv.) Hamet were slightly positive with respect to the suspension medium. This Δψ was insensitive to the protonophore FCCP and depolarized by about 4 mV on addition of 50 mM KCl, still remaining about +5 mV. Upon addition of 7 mM Mg-ATP, vacuoles showed an FCCP-sensitive increase of Δψ from +9.2 ± 2.8 (13) to +17.8 ± 3.7 (12) mV [given as x?± sd (n)] and an internal acidification from pH 5.4 ± 0.2 (11) to pH 4.3 ± 0.4 (12). Mg-ADP and ATP without Mg²⁺ had no effect on Δψ. It is concluded that the H4 pumping at the tonoplast is due to the functioning of the anion-sensitive vacuolar ATPase and that this is an essential part of the mechanism of nocturnal acid accumulation in CAM.     

Purification and Characterization of Posterior Pituitary Calmodulin and Its Activation of Neurosecretosome Ca2++ Mg2+-ATPase Activities

Abstract: Calmodulin was isolated as an electrophoretically homogeneous protein from bovine posterior pituitary glands. The yield indicated that this gland is a particularly rich source. Purified bovine posterior pituitary calmodulin and bovine brain calmodulin had identical electrophoretic mobilities on 10% and 12% polyacrylamide gels. The protein was further identified by molecular weight determination and by amino acid analysis which showed that it contained trimethyllysine, one residue per molecule. Bovine posterior pituitary calmodulin was found to activate a preparation of calmodulin-deficient phosphodiesterase from bovine heart. In addition, pituitary calmodulin stimulated Ca2⁺+ Mg²⁺-ATPase activity associated with a purified nerve ending plasma membrane fraction. This dependence could only be demonstrated after successive washing of the membranes with EGTA buffers, a procedure designed to remove endogenous calmodulin.     

Mg2+- or Ca2+-Activated ATPase in Squid Giant Fiber Axoplasm

A divalent cation-activated ATPase in axoplasm from the squid giant axon is described. The enzyme requires Mg2+ or Ca2+, has a K+ optimum of 60 mM, and has a pH optimum of 7.5. Several nucleotide triphosphates other than ATP can serve as substrates. The enzyme is inhibited by excess ATP or Mg2+. The enzyme is enriched in a rapidly sedimenting fraction of the axoplasm, and is eluted in the exclusion volume of a Sepharose 4B column, suggesting that it is associated with a highly aggregated structure. Comparison of the properties of enzyme with those of myosin and Na+-K+-ATPase suggests that differs from both of these enzymes. The enzyme has many similarities to vertebrate nerve ATPases previously described. The demonstration of the presence of this ATPase in squid axoplasm proves the neuronal localization of the enzyme.     

Modulation of the Ca2+,Mg2+-ATPase Activity of Synaptosomal Plasma Membrane by the Local Anesthetics Dibucaine and Lidocaine

It has been previously shown that local anesthetics inhibit the total Ca2+, Mg2(+)-ATPase activity of synaptosomal plasma membranes. We have carried out kinetic studies to quantify the effects of these drugs on the different Ca2(+)-dependent and Mg2(+)-dependent ATPase activities of these membranes. As a result we have found that this inhibition is not altered by washing the membranes with EDTA or EGTA. We have also found that the Ca2(+)-dependent ATPase activity is not significantly inhibited in the concentration range of these local anesthetics and under the experimental conditions used in this study. The inhibition of the Mg2(+)-dependent ATPase activities of these membranes was found to be of a noncompetitive type with respect to the substrate ATP-Mg2+, did not significantly shift the Ca2+ dependence of the Ca2+, Mg2(+)-ATPase activity, and occurred in a concentration range of local anesthetics that does not significantly alter the order parameter (fluidity) of these membranes. Modulation of this activity by the changes of the membrane potential that are associated with the adsorption of local anesthetics on the synaptosomal plasma membrane is unlikely, on the basis of the weak effect of membrane potential changes on the Ca2+,Mg2(+)-ATPase activity. It is suggested that the local anesthetics lidocaine and dibucaine inhibit the Ca2+, Mg2(+)-ATPase of the synaptosomal plasma membrane by disruption of the lipid annulus.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

Changes of membrane-associated Mg2+-activated ATPase of cucumber roots during calcium starvation

The properties of membrane-associated ATPase of cucumber (Cucumis sativus cv. Seiriki No. 2) roots cultured in a complete medium (complete enzyme) and in a medium lacking Ca²⁺ (Ca²⁺-deficient enzyme) were investigated. The basal activity of membrane-associated ATPase increased during Ca²⁺ starvation, while Mg²⁺-activation of the enzyme decreased and even resulted in inhibition by high Mg²⁺ concentration at the late stage of the Ca²⁺ starvation. The complete enzyme had low basal activity and showed a Mg²⁺-activated hyperbolic reaction curve in relation to ATP concentration. Ca²⁺-deficient enzyme with high basal activity showed a biphasic reaction curve and Mg²⁺-activation was seen only at high ATP concentrations. Activation of membrane-associated ATPase by various cations was decreased or lost during Ca²⁺ starvation. The basal ATPase activity of Ca²⁺-deficient enzyme increased for various substrates including pyrophosphate, p-nitrophenyl phosphate, glucose-6 phosphate, β-glycerophosphate, AMP, ADP and ATP. Mg²⁺-activation was found only for ADP and ATP in both the complete and Ca²⁺-deficient enzymes, but the activation for ATP was greatly reduced by Ca²⁺ starvation. The heat inactivation curves for basal and Mg²⁺-activated ATPase did not differ much between the complete and Ca²⁺-deficient enzyme. The delipidation of membrane-associated enzyme by acetone affected the protein content and the basal activity slightly, but inhibited the Mg²⁺-activated ATPase activity clearly with somewhat different behaviour between the complete and Ca²⁺-deficient enzyme.     

Partial purification of plant plasma membrane K+, Mg2+-ATPase by ion exchange chromatography

A purification procedure is presented which differs in three respects from other procedures for the purification of plant plasma membrane H⁺-pumping ATPase (EC 3.6.1.35) from various plants. Soybean ( Glycine max L. cv. Williams) hypocotyls were homogenized in the presence of physiological ionic strength and plasma membrane vesicles were purified by aqueous polymer two-phase partitioning. Plasma membrane vesicles were then solubilized in one step by using non-ionic detergent (either Triton X-100 or C₁₂E₈). The Mg-ATPase was separated by ion exchange chromatography from other solubilized membrane proteins. ATPase molecules bound to phosphocellulose fibers were eluted by a 0–1 M gradient of NaCl. The NaCl-eluted fractions contained a Mg-ATPase which showed the characteristics of Mg-ATPase present in the plasma membranes. The specific activity of the partially purified enzyme was 2–5 μmol mg⁻¹ min⁻¹ when it was reconstituted into proteoliposomes. This value is in good agreement with data obtained by other purification methods in the literature.