Helminthosporium maydis T Toxin Decreased Calcium Transport into Mitochondria of Susceptible Corn

The effects of purified Helminthosporium maydis T (HmT) toxin on active Ca²⁺ transport into isolated mitochondria and microsomal vesicles were compared for a susceptible (T) and a resistant (N) strain of corn (Zea mays). ATP, malate, NADH, or succinate could drive ⁴⁵Ca²⁺ transport into mitochondria of corn roots. Ca²⁺ uptake was dependent on the proton electrochemical gradient generated by the redox substrates or the reversible ATP synthetase, as oligomycin inhibited ATP-driven Ca²⁺ uptake while KCN inhibited transport driven by the redox substrates. Purified native HmT toxin completely inhibited Ca²⁺ transport into T mitochondria at 5 to 10 nanograms per milliliter while transport into N mitochondria was decreased slightly by 100 nanograms per milliliter toxin. Malate-driven Ca²⁺ transport in T mitochondria was frequently more inhibited by 5 nanograms per milliliter toxin than succinate or ATP-driven Ca²⁺ uptake. However, ATP-dependent Ca²⁺ uptake into microsomal vesicles from either N or T corn was not inhibited by 100 nanograms per milliliter toxin. Similarly, toxin had no effect on proton gradient formation ([¹⁴C]methylamine accumulation) in microsomal vesicles. These results show that mitochondrial and not microsomal membrane is a primary site of HmT toxin action. HmT toxin may inhibit formation of or dissipate the electrochemical proton gradient generated by substrate-driven electron transport or the mitochondrial ATPase, after interacting with a component(s) of the mitochondrial membrane in susceptible corn.     

Identification and Characterization of the Ca-ATPase which Drives Active Transport of Ca at the Plasma Membrane of Radish Seedlings

In microsomes from 24-hour-old radish (Raphanus sativus L.) seedlings ATP-dependent Ca²⁺ uptake occurs only in inside-out plasma membrane vesicles (F Rasi-Caldogno, MC Pugliarello, MI De Michelis [1987] Plant Physiol 83: 994-1000). A Ca²⁺-dependent ATPase activity can be shown in the same microsomes, when assays are performed at pH 7.5. The Ca²⁺-dependent ATPase is stimulated by the Ca²⁺ ionophore A₂₃₁₈₇ and is localized at the plasma membrane. Ca²⁺-dependent ATPase activity and ATP-dependent Ca²⁺ uptake present very similar saturation kinetics with erythrosin B (50% inhibition at about 0.1 micromolar), free Ca²⁺ (half-maximal rate at about 70 nanomolar), and MgATP (K_m 15-20 micromolar). Ca²⁺ uptake can be sustained by GTP or ITP at about 60% the rate measured in the presence of ATP; only very low Ca²⁺ uptake is sustained by CTP or UTP and none by ADP. These results indicate that the Ca²⁺-ATPase described in this paper is the enzyme which drives active transport of Ca²⁺ at the plasma membrane of higher plants.     

Characterization of Ca Transport in Purified Endoplasmic Reticulum Membrane Vesicles from Lepidium sativum L. Roots

The characteristics of Ca²⁺ transport into endoplasmic reticulum vesicles isolated from roots of Lepidium sativum L. cv Krause have been investigated. The concentration of free Ca²⁺ and ATP needed for half-maximal activity were 2.5 and 73 micromolar, respectively, and the enzyme obeyed Michaelis-Menten-like kinetics. The pH maximum occurred at 7.5 and the activity was greatly reduced at either pH 7.0 or 8.0.

The Ca²⁺-dependent modulation protein, calmodulin, was tested for its effect on Ca²⁺ transport into endoplasmic reticulum vesicles. Although the phenothiazine inhibitors chlorpromazine, fluphenazine, and trifluoperazine all inhibited Ca²⁺ transport activity with a half-maximal effect at approximately 35 micromolar, authentic bovine brain calmodulin did not alter the activity at concentrations of 0.5 to 8 micrograms per milliliter. Calmodulin also showed no influence on the time-dependent accumulation of Ca²⁺ into vesicles. The membranes did not contain endogenously bound calmodulin since washing with (ethylenebis[oxyethylenenitrile])tetraacetic acid or fluphenazine, treatments which disrupt calmodulin binding, did not alter Ca²⁺ transport activity. The inhibition of Ca²⁺ transport by phenothiazine drugs was likely related to their nonspecific interaction with the membrane. Thus, there was no indication that calmodulin regulated Ca²⁺ uptake into root endoplasmic reticulum.

     

Functional Reconstitution of an ATP-Driven Ca-Transport System from the Plasma Membrane of Commelina communis L

The protein(s) that constitute(s) the ATP-driven Ca²⁺-translocator of plasma membrane enriched vesicles obtained by aqueous two-phase partitioning from leaves of Commelina communis L. has/have been solubilized and reincorporated into tightly sealed liposomes. The reconstituted Ca²⁺-transport system was studied using ATP-driven ⁴⁵Ca²⁺ import into the proteoliposomes as a measure of activity. The detergent, 3-[(3-cholamidopropyl) dimethylammonio]-1-propane-sulfonate proved to be the most suitable and was used at 10 millimolar concentration, i.e. just above its critical micellar concentration. The presence of additional phospholipid (2 milligrams phosphatidylcholine per milliliter) and ATP (5 millimolar) improved the solubilization and/or reconstitution. The characteristics of the reconstituted system were similar to those of the plasma membrane-bound activity, including the apparent K_m for Ca²⁺ (5.2 micromolar), inhibition by relatively high levels of vanadate (IC₅₀ = 500 micromolar) and lacking response to added calmodulin. The reconstituted transport system was very strongly inhibited by erythrosine B (IC₅₀ = 0.01 micromolar) and had a low apparent K_m for ATP (11.4 micromolar). As in the plasma membrane vesicles, the protonophore carbonylcyanide m-chlorophenyl hydrazone did not affect Ca²⁺-transport detectably in the reconstituted system. However, low levels of the Ca²⁺-ionophore A 23187 instantaneously discharged 90% of the Ca²⁺ associated with the vesicles, proving that it had been accumulated in the intravesicular volume in soluble, freely exchangeable form. Ca²⁺-transport in the reconstituted system was thus primary active, through a Ca²⁺-translocating ATPase. The system reported here may serve as a valuable tool for purifying the Ca²⁺-ATPase and for studying structural and functional aspects of the purified enzyme.     

Transport Properties of the Tomato Fruit Tonoplast : III. Temperature Dependence of Calcium Transport

Calcium transport into tomato (Lycopersicon esculentum Mill, cv Castlemart) fruit tonoplast vesicles was studied. Calcium uptake was stimulated approximately 10-fold by MgATP. Two ATP-dependent Ca²⁺ transport activities could be resolved on the basis of sensitivity to nitrate and affinity for Ca²⁺. A low affinity Ca²⁺ uptake system (K_m > 200 micromolar) was inhibited by nitrate and ionophores and is thought to represent a tonoplast localized H⁺/Ca²⁺ antiport. A high affinity Ca²⁺ uptake system (K_m = 6 micromolar) was not inhibited by nitrate, had reduced sensitivity to ionophores, and appeared to be associated with a population of low density endoplasmic reticulum vesicles that contaminated the tonoplast-enriched membrane fraction. Arrhenius plots of the temperature dependence of Ca²⁺ transport in tomato membrane vesicles showed a sharp increase in activation energy at temperatures below 10 to 12°C that was not observed in red beet membrane vesicles. This low temperature effect on tonoplast Ca²⁺/H⁺ antiport activity could only by partially ascribed to an effect of low temperature on H⁺-ATPase activity, ATP-dependent H⁺ transport, passive H⁺ fluxes, or passive Ca²⁺ fluxes. These results suggest that low temperature directly affects Ca²⁺/H⁺ exchange across the tomato fruit tonoplast, resulting in an apparent change in activation energy for the transport reaction. This could result from a direct effect of temperature on the Ca²⁺/H⁺ exchange protein or by an indirect effect of temperature on lipid interactions with the Ca²⁺/H⁺ exchange protein.     

The Ca-Transport ATPase of Plant Plasma Membrane Catalyzes a nH/Ca Exchange

Microsomal vesicles from 24-hour-old radish (Raphanus sativus L.) seedlings accumulate Ca²⁺ upon addition of MgATP. MgATP-dependent Ca²⁺ uptake co-migrates with the plasma membrane H⁺-ATPase on a sucrose gradient. Ca²⁺ uptake is insensitive to oligomycin, inhibited by vanadate (IC₅₀ 40 micromolar) and erythrosin B (IC₅₀ 0.2 micromolar) and displays a pH optimum between pH 6.6 and 6.9. MgATP-dependent Ca²⁺ uptake is insensitive to protonophores. These results indicate that Ca²⁺ transport in these microsomal vesicles is catalyzed by a Mg²⁺-dependent ATPase localized on the plasma membrane. Ca²⁺ strongly reduces ΔpH generation by the plasma membrane H⁺-ATPase and increases MgATP-dependent membrane potential difference (Δψ) generation. These effects of Ca²⁺ on ΔpH and Δψ generation are drastically reduced by micromolar erythrosin B, indicating that they are primarily a consequence of Ca²⁺ uptake into plasma membrane vesicles. The Ca²⁺-induced increase of Δψ is collapsed by permeant anions, which do not affect Ca²⁺-induced decrease of ΔpH generation by the plasma membrane H⁺-ATPase. The rate of decay of MgATP-dependent ΔpH, upon inhibition of the plasma membrane H⁺-ATPase, is accelerated by MgATP-dependent Ca²⁺ uptake, indicating that the decrease of ΔpH generation induced by Ca²⁺ reflects the efflux of H⁺ coupled to Ca²⁺ uptake into plasma membrane vesicles. It is therefore proposed that Ca²⁺ transport at the plasma membrane is mediated by a Mg²⁺-dependent ATPase which catalyzes a nH⁺/Ca²⁺ exchange.     

Calcium pump activity of the renal plasma membrane and renal microsomes

ATP-dependent Ca²⁺ uptake distinct from that of the mitochondria is found in both plasma membrane and microsomal membranes of rat kidney. Activity attributed to these fractions is enhanced by ammonium oxalate and is apparently insensitive to NaN₃. In contrast, rat kidney mitochondrial Ca²⁺ uptake is blocked by NaN₃. The pH of optimal activity is significantly higher for the mitochondrial fraction. Microsomal membrane Ca²⁺ uptake differs from that of the plasma membrane. Microsomal membranes are four times as active as the plasma membrane at high (5 mM) ATP levels. Apparent K_m values for Mg²⁺-ATP differ in the two preparations with a higher affinity for Mg²⁺-ATP found in the plasma membrane Ca²⁺ uptake activity of the plasma membrane preparation is readily inhibited by Na⁺. Sucrose gradient density fractionation indicates that the observed microsomal membrane Ca²⁺ pump activity is associated with membrane vesicles derived from the endoplasmic reticulum. Ca²⁺ pump activity of both plasma membrane and microsomal fraction is depressed din the adrenalectomized rat. This activity is not restored by a single natriuretic dose of aldosterone.     

Calmodulin stimulation of calcium uptake and (Ca2+-Mg2+)-ATPase activities in microsomes from canine tracheal smooth muscle

The role of calmodulin in the regulation of microsomal ⁴⁵Ca²⁺ transport in canine tracheal smooth muscle was studied. Calmodulin stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities in microsomes treated with 0.5 mM EDTA and 0.5 mM EGTA. Oxalate also stimulated ATP-dependent ⁴⁵Ca²⁺ uptake and (Ca²⁺Mg²⁺)-ATPase activities and the stimulation was additive to the effects of calmodulin. The (Ca²⁺Mg²⁺)-ATPase and ATP-dependent ⁴⁵Ca²⁺ uptake activities are probably related as they exhibited similar [Ca²⁺]_free- and [calmodulin]-dependencies. These results indicate that calmodulin may play a role in the control of the cytosolic [Ca²⁺]_free in canine tracheal smooth muscle.     

Calcium Fluxes across the Plasma Membrane of Commelina communis L. Assayed in a Cell-Free System

The inside-out fraction of plasma membrane-rich vesicles prepared from leaves of Commelina communis L. by aqueous twophase partitioning was loaded with ⁴⁵Ca²⁺ through the action of the plasma membrane Ca²⁺-ATPase. While the Ca²⁺-loaded vesicles were tightly sealed, trifluoperazine (TFP) (effective concentration giving 50% of maximum effect [EC₅₀] = 70 micromolar) and W-7 (EC₅₀ = 100 micromolar), but to a much lesser extent, W-5 (EC₅₀ = 500 micromolar) led to a rapid efflux of ⁴⁵Ca²⁺ from the vesicles. This efflux could be blocked efficiently with low (<1 millimolar) concentrations of La³⁺, but it remained unaffected by the addition of calmodulin (CM). Further experiments with vesicles incubated in ⁴⁵Ca²⁺ in the absence of ATP, as well as experiments performed with control liposomes and nonloaded as well as Ca²⁺-loaded plasma membrane vesicles using the indicator dye arsenazo III showed, that TFP and W-7 and, again to a lesser extent, W-5 mobilized a pool of membrane-bound Ca²⁺ from the vesicles. No indications for a detergent effect of TFP and W-7 were obtained. The EC₅₀-values of these compounds for mobilizing membrane-associated Ca²⁺ (TFP = 100 micromolar, W-7 = 100 micromolar, W-5 = 500 micromolar) or for the triggering of Ca²⁺ release from Ca²⁺-loaded vesicles (see above) were very similar, suggesting a common basis of antagonist action on both processes. Our results suggest the presence of a Ca²⁺ channel in the plasma membrane of C. communis. The channel is obtained in a Ca²⁺-inactivated state after preparation and Ca²⁺-loading of the vesicles. The inactivation is removed by TFP or W-7, presumably due to the Ca²⁺-mobilizing effect of these compounds. The activated Ca²⁺ channel is La³⁺ sensitive and, in the cell, would allow for passage of Ca²⁺ into the cell. The possibility that TFP or W-7 act independent of CM, or through CM tightly associated with the plasma membrane, is discussed. The system described allows a cell free analysis of Ca²⁺ influx, displaying channel properties, in a higher plant.     

Effect of calmodulin antagonists on Ca2+ uptake by boar spermatozoa

Calcium uptake by washed boar sperm suspensions is markedly stimulated by the calmodulin antagonists trifluoperazine and calmidazolium. Both ⁴⁵Ca²⁺ uptake and net Ca²⁺ uptake are increased by these drugs. Drug stimulated Ca²⁺ uptake is blocked by verapamil (1 mM), by ruthenium red (25 μM) and by carbonyl cyanide p-trifluoromethoxyphenyl hydrazone. Calmodulin antagonists do not slow ATP-dependent Ca²⁺ extrusion from plasma membrane vesicles, and they do not inhibit plasma membrane Ca²⁺-ATPase. It is proposed that calmodulin is involved in the control of Ca²⁺ entry in boar spermatozoa. Most entering Ca²⁺ in uncapacitated spermatozoa is sequestered by mitochondria or rapidly extruded by plasma membrane pumps. In contrast to the uptake mechanism, ATP-dependent Ca²⁺ extrusion does not appear to be regulated by calmodulin.     

Calpain I activates Ca2+ transport by the reconstituted erythrocyte Ca2+ pump

Summary Calpain I purified from human erythrocyte cytosol activates both the ATP hydrolytic activity and the ATP-dependent Ca²⁺ transport function of the Ca²⁺-translocating ATPase solubilized and purified from the plasma membrane of human erythrocytes and reconstituted into phosphatidylcholine vesicles. Following partial proteolysis of the enzyme by calpain I, both the initial rates of calcium ion uptake and ATP hydrolysis were increased to near maximal levels similar to those obtained upon addition of calmodulin. The proteolytic activation resulted in the loss of further stimulation of the rates of Ca²⁺ translocation or ATP hydrolysis by calmodulin as well as an increase of the affinity of the enzyme for calcium ion. However, the mechanistic Ca²⁺/ATP stoichiometric ratio was not affected by the proteolytic treatment of the reconstituted Ca²⁺-translocating ATPase. The proteolytic activation of the ATP hydrolytic activity of the reconstituted enzyme could be largely prevented by calmodulin. Different patterns of proteolysis were obtained in the absence or in the presence of calmodulin during calpain treatment: the 136-kDa enzyme was transformed mainly into a 124-kDa active ATPase fragment in the absence of calmodulin, whereas a 127-kDa active ATPase fragment was formed in the presence of calmodulin. This study shows that calpain I irreversibly activates the Ca²⁺ translocation function of the Ca²⁺-ATPase in reconstituted proteoliposomes by producing a calmodulin-independent active enzyme fragment, while calmodulin antagonizes this activating effect by protecting the calmodulin-binding domain against proteolytic cleavage by calpain.     

Characterization of a proton-translocating ATPase in microsomal vesicles from corn roots

Sealed microsomal vesicles were prepared from corn (Zea mays, Crow Single Cross Hybrid WF9-Mo17) roots by centrifugation of a 10,000 to 80,000g microsomal fraction onto a 10% dextran T-70 cushion. The Mg²⁺-ATPase activity of the sealed vesicles was stimulated by Cl⁻ and NH₄⁺ and by ionophores and protonophores such as 2 micromolar gramicidin or 10 micromolar carbonyl cyanide p-trifluoromethoxyphenyl hydrazone (FCCP). The ionophore-stimulated ATPase activity had a broad pH optimum with a maximum at pH 6.5. The ATPase was inhibited by NO₃⁻, was insensitive to K⁺, and was not inhibited by 100 micromolar vanadate or by 1 millimolar azide.

Quenching of quinacrine fluorescence was used to measure ATP-dependent acidification of the intravesicular volume. Quenching required Mg²⁺, was stimulated by Cl⁻, inhibited by NO₃⁻, was insensitive to monovalent cations, was unaffected by 200 micromolar vanadate, and was abolished by 2 micromolar gramicidin or 10 micromolar FCCP. Activity was highly specific for ATP. The ionophore-stimulated ATPase and ATP-dependent fluorescence quench both required a divalent cation (Mg²⁺ ≥ Mn²⁺ > Co²⁺) and were inhibited by high concentrations of Ca²⁺. The similarity of the ionophore-stimulated ATPase and quinacrine quench and the responses of the two to ions suggest that both represent the activity of the same ATP-dependent proton pump. The characteristics of the proton-translocating ATPase differed from those of the mitochondrial F₁F₀-ATPase and from those of the K⁺-stimulated ATPase of corn root plasma membranes, and resembled those of the tonoplast ATPase.

     

The spinach plasma membrane Ca2+ pump is a 120‐kDa polypeptide regulated by calmodulin‐binding to a terminal region

The spinach (Spinacia oleracea L.) leaf plasma membrane Ca²⁺-ATPase is regulated by calmodulin (3-fold stimulation) and limited proteolysis (trypsin; 4-fold stimulation). The plasma membrane Ca²⁺-ATPase was identified as a 120-kDa polypeptide on western immunoblots using two different antibodies. During trypsin treatment the 120-kDa band diminished and a new band appeared at 109 kDa. The appearance of the 109-kDa band correlated with the increase in enzyme activity following trypsin treatment. The stimulations by calmodulin and trypsin were not additive, suggesting that the 109-kDa polypeptide represents a Ca²⁺-ATPase lackin a terminal fragment involved in calmodulin regulation. This was confirmed by ¹²⁵I-calmodulin overlay studies where calmodulin labeled the 120-kDa band in the presence of Ca²⁺, while the 109-kDa band did not bind calmodulin. The effects of calmodulin and limited proteolysis on ATP-dependent accumulation of ⁴⁵Ca²⁺ in isolated inside-out plasma membrane vesicles were studied, and kinetical analyses performed with respect to Ca²⁺ and ATP. Calmodulin increased the V_max. for Ca²⁺ pumping 3-fold, and reduced K_m for Ca²⁺ from 1.6 to 0.9 µM. The K_m for ATP (11 µM) was not affected by calmodulin. The effects of limited proteolysis on the affinities for Ca²⁺ and ATP were similar to those obtained with calmodulin. Notably, however, limited proteolysis increased the V_max. for Ca²⁺ pumping to a higher extent than calmodulin, indicating incomplete calmodulin activation, or removal of an additional inhibitory site by trypsin.     

ATP-dependent CA2+ transport in endoplasmic reticulum isolated from roots ofLepidium sativum L.

Endoplasmic reticulum membranes were isolated from roots of garden cress (Lepidium sativum L. cv Krause) using differential and discontinuous sucrose gradient centrifugation. The endoplasmic reticulum fraction was 80% rough endoplasmic reticulum oriented with the cytoplasmic surface directed outward and contaminated with 12% unidentified smooth membranes and 8% mitochondria. Marker enzyme analysis showed that the activity for endoplasmic reticulum was enriched 2.4-fold over total membrane activity while no other organelle activity showed an enrichment. All evidence indicated that the fraction was composed of highly enriched endoplasmic reticulum membranes. Ca²⁺ uptake activity was measured using the filter technique described by Gross and Marmé (1978). The results of these experiments showed an ATP-dependent, oxalate-stimulated Ca²⁺ uptake into vesicles of the endoplasmic reticulum fraction. The majority of the transport activity was microsomal since specific inhibitors of mitochondrial Ca²⁺ transport (ruthenium red, LaCl₃ and oligomycin) inhibited the activity by only 25%. Sodium azide showed no inhibition. The transport was likely directly coupled to ATP hydrolysis since there was no inhibition with carbonylcyanidem-chlorophenylhydrazone. The transport activity was specific for ATP showing only 36% and 29% of the activity with inosine diphosphate and guanosine 5′-triphosphate, respectively. The results indicate a Ca²⁺ transport function located on the endoplasmic reciculum of garden cress roots.     

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.     

Detection of the ATP-Dependent Nonmitochondrial Calcium Store in a Cell Surface-Derived Vesicle Fraction from Isolated Rat Hepatocytes

In preceding studies, the IP₃-sensitive Ca²⁺store of the hamster insulinoma cell line, HIT, was detected in cell surface protrusions such as microvilli and related membrane structures [Lange, K., and Brandt, U. (1993)FEBS Lett.320, 183–188; and (1993)FEBS Lett.325, 205–209]. In this study, these experiments were extended on rat hepatocytes. We used the previously described shearing technique for isolating cell surface-derived vesicle fractions from freshly isolated and 48-h-cultured rat hepatocytes. As shown by Western blot analysis, these vesicles contained the hepatocyte-specific glucose transporter, GluT2, and actin, which are both typical microvillar components. Scanning electron microscopy revealed that a spherical vesicle population of uniform size (about 1 μm in diameter) originates from the hepatocyte microvilli. This vesicle fraction exhibited ATP-dependent and thapsigargin-sensitive Ca²⁺storage activity with properties identical to those of the known microsomal systems and of HIT cell surface-derived vesicles, except that the ATP-dependent Ca²⁺pool was insensitive to IP₃. Like HIT surface vesicles, hepatocyte surface vesicles rapidly took up ATP via a 4,4′-diisocyanostilbene-2,2′-disulfonic acid (DIDS)-sensitive anion pathway. Inhibition of ATP influx into the vesicles by DIDS also completely inhibited ATP-dependent Ca²⁺storage. Moreover, determination of efflux kinetics of Ca²⁺from passively (in the absence of ATP) loaded vesicles revealed a La³⁺-sensitive but IP₃-independent Ca²⁺pathway which rapidly equilibrated intravesicular free Ca²⁺with the external medium. Permeabilization of the vesicles with saponin (0.005%) opened an additional efflux pathway for Ca²⁺which is not La³⁺-sensitive. However, saponin treatment of vesicles preloaded with Ca²⁺in the presence of ATP did not affect the thapsigargin-sensitive vesicular Ca²⁺store but only released a small portion (about 20%) of the vesicular Ca²⁺that is not part of the thapsigargin-sensitive Ca²⁺pool. Also, the size of the saponin-releasable Ca²⁺pool was not affected by depletion of the thapsigargin-sensitive Ca²⁺store. These findings indicate that hepatocyte surface vesicles are readily permeable for Ca²⁺and ATP via cation and anion pathways. Consequently, Ca²⁺storage into these vesicles does not occur by concentrative Ca²⁺pumping but rather appears to be due to an internal, ATP-dependent mechanism of Ca²⁺sequestration. The presented data are in accord with the previously reported colocalization of the ATP-dependent Ca²⁺store and its functionally coupled, store-regulated Ca²⁺influx pathway in special cell surface organelles, the microvilli.     

Further Characterization of the Red Beet Plasma Membrane Ca-ATPase Using GTP as an Alternative Substrate

The GTP-driven component of Ca²⁺ uptake in red beet (Beta vulgaris L.) plasma membrane vesicles was further characterized to confirm its association with the plasma membrane Ca²⁺-translocating ATPase and assess its utility as a probe for this transport system. Uptake of ⁴⁵Ca²⁺ in the presence of GTP demonstrated similar properties to those previously observed for red beet plasma membrane vesicles utilizing ATP with respect to pH optimum, sensitivity to orthovanadate, dependence on Mg:substrate concentration and dependence on Ca²⁺ concentration. Calcium uptake in the presence of GTP was also strongly inhibited by erythrosin B, a potent inhibitor of the plant plasma membrane Ca²⁺-ATPase. Furthermore, after treatment with EGTA to remove endogenous calmodulin, the stimulation of ⁴⁵Ca²⁺-uptake by exogenous calmodulin was nearly equivalent in the presence of either ATP or GTP. Taken together these results support the proposal that GTP-driven ⁴⁵Ca²⁺ uptake represents the capacity of the plasma membrane Ca²⁺-translocating ATPase to utilize this nucleoside triphosphate as an alternative substrate. When plasma membrane vesicles were phosphorylated with [γ-³²P]-GTP, a rapidly turning over, 100 kilodalton phosphorylated peptide was observed which contained an acyl-phosphate linkage. While it is proposed that this peptide could represent the catalytic subunit of the plasma membrane Ca²⁺-ATPase, it is noted that this molecular weight is considerably lower than the 140 kilodalton size generally observed for plasma membrane Ca²⁺-ATPases present in animal cells.     

Calcium- and calmodulin-regulated breakdown of phospholipid by microsomal membranes from bean cotyledons

Evidence for the involvement of Ca²⁺ and calmodulin in the regulation of phospholipid breakdown by microsomal membranes from bean cotyledons has been obtained by following the formation of radiolabeled degradation products from [U-¹⁴C]phosphatidylcholine. Three membrane-associated enzymes were found to mediate the breakdown of [U-¹⁴C] phosphatidylcholine, viz. phospholipase D (EC 3.1.4.4), phosphatidic acid phosphatase (EC 3.1.3.4), and lipolytic acyl hydrolase. Phospholipase D and phosphatidic acid phosphatase were both stimulated by physiological levels of free Ca²⁺, whereas lipolytic acyl hydrolase proved to be insensitive to Ca²⁺. Phospholipase D was unaffected by calmodulin, but the activity of phosphatidic acid phosphatase was additionally stimulated by nanomolar levels of calmodulin in the presence of 15 micromolar free Ca²⁺. Calmidazolium, a calmodulin antagonist, inhibited phosphatidic acid phosphatase activity at IC₅₀ values ranging from 10 to 15 micromolar. Thus the Ca²⁺-induced stimulation of phosphatidic acid phosphatase appears to be mediated through calmodulin, whereas the effect of Ca²⁺ on phospholipase D is independent of calmodulin. The role of Ca²⁺ as a second messenger in the initiation of membrane lipid degradation is discussed.     

Ca2+-stimulated,Mg2+-independent ATP hydrolysis and the high affinity Ca2+-pumping ATPase. Two different activities in rat kidney basolateral membranes

The Mg²⁺-dependency of Ca²⁺-induced ATP hydrolysis is studied in basolateral plasma membrane vesicles from rat kidney cortex in the presence of CDTA and EGTA as Mg²⁺- and Ca²⁺-buffering ligands. ATP hydrolysis is strongly stimulated by Mg²⁺ with a K_m of 13 μ M in the absence or presence of 1 μ M free Ca²⁺. At free Mg²⁺ concentrations of 1 μ M and lower, ATP hydrolysis is Mg²⁺ -independent, but is strongly stimulated by submicromolar Ca²⁺ concentrations K_m  0.25 μM, V_max  24 μmol P_i/h per mg protein). The Ca²⁺-stimulated ATP hydrolysis strongly decreases at higher Mg²⁺ concentrations. The Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis is not affected by calmodulin or trifluoperazine and shows no specificity for ATP over ADP, ITP and GTP. In contrast, at high Mg²⁺ concentrations calmodulin and trifluoperazine affect the high affinity Ca²⁺-ATPase activity significantly and ATP is the preferred substrate. Control studies on ATP-dependent Ca²⁺-pumping in renal basolaterals and on Ca²⁺-ATPase in erythrocyte ghosts suggest that the Ca²⁺-pumping enzyme requires Mg²⁺. In contrast, a role of the Ca²⁺-stimulated Mg²⁺-independent ATP hydrolysis in active Ca²⁺ transport across basolateral membranes is rather unlikely.     

A Ca/H Antiport System Driven by the Proton Electrochemical Gradient of a Tonoplast H-ATPase from Oat Roots

Two types of ATP-dependent calcium (Ca²⁺) transport systems were detected in sealed microsomal vesicles from oat roots. Approximately 80% of the total Ca²⁺ uptake was associated with vesicles of 1.11 grams per cubic centimeter and was insensitive to vanadate or azide, but inhibited by NO₃⁻. The remaining 20% was vanadate-sensitive and mostly associated with the endoplasmic reticulum, as the transport activity comigrated with an endoplasmic reticulum marker (antimycin A-insensitive NADH cytochrome c reductase), which was shifted from 1.11 to 1.20 grams per cubic centimeter by Mg²⁺.

Like the tonoplast H⁺-ATPase activity, vanadate-insensitive Ca²⁺ accumulation was stimulated by 20 millimolar Cl⁻ and inhibited by 10 micromolar 4,4′-diisothiocyano-2,2′-stilbene disulfonic acid or 50 micromolar N,N′-dicyclohexylcarbodiimide. This Ca²⁺ transport system had an apparent K_m for Mg-ATP of 0.24 millimolar similar to the tonoplast ATPase. The vanadate-insensitive Ca²⁺ transport was abolished by compounds that eliminated a pH gradient and Ca²⁺ dissipated a pH gradient (acid inside) generated by the tonoplast-type H⁺-ATPase. These results provide compelling evidence that a pH gradient generated by the H⁺-ATPase drives Ca²⁺ accumulation into right-side-out tonoplast vesicles via a Ca²⁺/H⁺ antiport. This transport system was saturable with respect to Ca²⁺ (K_m apparent = 14 micromolar). The Ca²⁺/H⁺ antiport operated independently of the H⁺-ATPase since an artifically imposed pH gradient (acid inside) could also drive Ca²⁺ accumulation. Ca²⁺ transport by this system may be one major way in which vacuoles function in Ca²⁺ homeostasis in the cytoplasm of plant cells.