ATP-driven Ca2+ transport in sealed plasma membrane vesicles prepared by aqueous two-phase partitioning from leaves of Commelina communis

Sealed plasma membrane vesicles were obtained in high purity from leaves of Commelina communis L. by aqueous two-phase partitioning. Based on the analysis of a range of markers, the preparations (U₃+U₃′ phases) were shown to be devoid of tonoplast, Golgi and thylakoid membranes, and showed only trace mitochondrial contamination. One-third of the vesicles were oriented inside out and exhibited ATP-driven ⁴⁵Ca²⁺ transport [? 15 pkat (mg protein)⁻¹]. Ca²⁺ uptake into the vesicles had a pH optimum of 7.2 and apparent K_m values for Ca²⁺ of 4.4 μM and for Mg-ATP of 300 μM. Ca²⁺ uptake, K⁺, Mg²⁺-ATPase (EC 3.6.1.3) activity as well as glucan synthase II (EC 2.4.1.34) activity were all maximal at the same equilibrium density (1.17 g cm⁻³) on continuous sucrose density gradients. The protonophore carbonylcyanide m-chlorophenylhydrazone (CCCP) did not inhibit the ATP-dependent Ca²⁺ transport into the vesicles, excluding a Ca²⁺/H⁺ exchange driven by a proton gradient. ATP-dependent Ca²⁺ uptake was inhibited by erythrosin B (I₅₀= 0.1 μM), ruthenium red (I₅₀= 30 μM), La³⁺ (I₅₀= 10 μM) and vanadate (I₅₀= 500 μM), but not by azide, cyanide and oligomycin. The calmodulin antagonists, trifluoperazine (I₅₀= 70 μM) and W-7 (I₅₀= 100 μM) were also inhibitory, However, this inhibition was not overcome by calmodulin. Trifluoperazine and W-7, on the other hand, stimulated Ca²⁺ efflux from the vesicles rather than inhibit Ca²⁺ uptake. Our results demonstrate the presence of a Ca²⁺-ATPase in the plasma membrane of C. communis. In the intact cell, the enzyme would pump Ca²⁺ out of the cell. Its high affinity for Ca²⁺ makes it a likely component involved in adjusting low cytoplasmic Ca²⁺ levels. No indications for a secondary active Ca²⁺/H⁺ transport mechanism in the plasma membrane of C. communis were obtained. Both, the nucleotide specificity and the sensitivity towards vanadate. distinguish the Ca²⁺-ATPase from the H⁺-translocating K⁺. Mg²⁺-ATPase in C. communis plasma membranes.     

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.     

Characterization of Exchange Inhibitory Peptide Effects on Na+/Ca2+ Exchange in Rat and Human Brain Plasma Membrane Vesicles

Abstract: The inhibitory effects of Na⁺/Ca²⁺ exchange inhibitory peptide (XIP), which corresponds to residues 219–238 of the Na⁺/Ca²⁺ exchange protein from canine heart, were studied in both rat and human brain plasma membrane vesicles. XIP had very high potency with respect to the inhibition of the initial velocity of intravesicular Na⁺-dependent Ca²⁺ uptake in both rat brain [IC₅₀ = 3.05 ± 0.69 µM (mean ± SE)] and human brain (IC₅₀ = 3.58 ± 0.58 µM). The maximal inhibition seen in rat brain vesicles was ～80%, whereas human brain vesicles were inhibited 100%. XIP also inhibited extravesicular Na⁺-dependent Ca²⁺ release, and the inhibitory effect was enhanced by increasing the extravesicular Na⁺ concentration. In contrast, the inhibitory effect of bepridil was competitive with respect to extravesicular Na⁺. When XIP was added at steady state (5 min after the initiation of intravesicular Na⁺-dependent Ca²⁺ uptake), it was found that the intravesicular Ca²⁺ content declined with time. Analysis of unidirectional fluxes for Ca²⁺ at steady state showed that 50 µM XIP inhibited Ca²⁺ influx and efflux ～85 and 70%, respectively. This result suggested that XIP inhibited both Na⁺/Ca²⁺ exchange and Ca²⁺/Ca²⁺ exchange but had no effect on the passive release pathway for Ca²⁺. The results suggest structural homology among cardiac, rat, and human brain exchangers in the XIP binding domain and that the binding of Na⁺ or other monovalent cations, e.g., K⁺, is required for XIP to have its inhibitory effect on Ca²⁺ transport.     

On the presence of inside-out plasma membrane vesicles and vanadate-inhibited K+,Mg2+-ATPase in microsomal fractions from wheat and maize roots

We have estimated the amount of inside-out plasma membrane (PM) vesicles in microsomal fractions from wheat (Triticum aestivum L. cv. Drabant) and maize (Zea mays L.) roots; non-latent activities of the PM markers vanadate-inhibited K⁺, Mg²⁺-ATPase (ΔVO₄-ATPase) and glucan synthase II (GS II, EC 2.4.1.34) were used as markers for inside-out PM vesicles, latent activities as markers for right-side-out PM vesicles, and specific staining with silicotungstic acid (STA) as a general marker for the PM. Separation of presumptive inside-out PM vesicles from right-side-out ones was achieved by counter-current-distribution (CCD) in an aqueous polymer two-phase system. Most of the GS II activity was latent and was found in material partitioning into the upper phase; a distribution which correlated well with that of STA-stained vesicles. Thus, most of the PM vesicles had a right-side-out orientation. ΔVO₄-ATPase, on the other hand, had a dual distribution (particularly pronounced in wheat) and was recovered both in material partitioning into the lower phase and into the upper phase. This indicates that ΔVO₄-ATPase activity was present also in membranes other than the PM. Additional evidence for this interpretation came from sucrose gradient centrifugation of wheat root material. This produced two peaks of ΔVO₄-ATPase activity with the membranes partitioning into the lower phase, none of which coincided with the peak obtained with right-side-out PM vesicles. Taken together, these results indicate that only very few inside-out PM vesicles are present in the microsomal fraction, and that ΔVO₄-ATPase as a marker for the PM, in contrast to GS II, may give quite misleading results with some plant materials. This stresses the need to use well-defined preparations of scaled, inside-out PM vesicles in solute uptake studies. The distribution of Ca²⁺-inhibited ATPase, on the other hand, agreed well with those of GS II and STA-stained vesicles both after CCD and sucrose gradient centrifugation, which suggests that Ca²⁺ inhibition may be a more specific property of the PM H⁺-ATPase than vanadate inhibition.     

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.     

Alteration of Ca2+ Fluxes in Brain Microsomes by K+ and Na+: Modulation by Sulfated Polysaccharides and Trifluoperazine

Abstract: Rat brain microsomes accumulate Ca²⁺ at the expense of ATP hydrolysis. The rate of transport is not modulated by the monovalent cations K⁺, Na⁺, or Li⁺. Both the Ca²⁺ uptake and the Ca²⁺-dependent ATPase activity of microsomes are inhibited by the sulfated polysaccharides heparin, fucosylated chondroitin sulfate, and dextran sulfate. Half-maximal inhibition is observed with sulfated polysaccharide concentrations ranging from 0.5 to 8.0 µg/ml. The inhibition is antagonized by KCl and NaCl but not by LiCl. As a result, Ca²⁺ transport by the native vesicles, which in the absence of polysaccharides is not modulated by monovalent cations, becomes highly sensitive to these ions. Trifluoperazine has a dual effect on the Ca²⁺ pump of brain microsomes. At low concentrations (20–80 µM) it stimulates the rate of Ca²⁺ influx, and at concentrations >100 µM it inhibits both the Ca²⁺ uptake and the ATPase activity. The activation observed at low trifluoperazine concentrations is specific for the brain Ca²⁺-ATPase; for the Ca²⁺-ATPases found in blood platelets and in the sarcoplasmic reticulum of skeletal muscle, trifluoperazine causes only a concentration-dependent inhibition of Ca²⁺ uptake. Passive Ca²⁺ efflux from brain microsomes preloaded with Ca²⁺ is increased by trifluoperazine (50–150 µM), and this effect is potentiated by heparin (10 µg/ml), even in the presence of KCl. It is proposed that the Ca²⁺-ATPase isoform from brain microsomes is modulated differently by polysaccharides and trifluoperazine when compared with skeletal muscle and platelet isoforms.     

Effects of Monovalent and Divalent Cations on Ca2+ Fluxes Across Chromaffin Secretory Membrane Vesicles

Abstract: Bovine chromaffin secretory vesicle ghosts loaded with Na⁺ were found to take up Ca²⁺ when incubated in K⁺ media or in sucrose media containing micromolar concentrations of free Ca²⁺. Li⁺- or choline⁺loaded ghosts did not take up Ca²⁺. The Ca²⁺ accumulated by Na⁺-loaded ghosts could be released by the Ca²⁺ ionophore A23187, but not by EGTA. Ca²⁺ uptake was inhibited by external Sr²⁺, Na ⁺, Li ⁺, or choline ⁺. All the ⁴⁵Ca²⁺ accumulated by Na⁺-dependent Ca²⁺ uptake could be released by external Na ⁺, indicating that both Ca²⁺ influx and efflux occur in a Na⁺-dependent manner. Na ⁺ -dependent Ca²⁺ uptake and release were only slightly inhibited by Mg²⁺. In the presence of the Na⁺ ionophore Monensin the Ca²⁺ uptake by Na ⁺-loaded ghosts was reduced. Ca²⁺ sequestered by the Na⁺-dependent mechanism could also be released by external Ca²⁺ or Sr²⁺ but not by Mg²⁺, indicating the presence of a Ca²⁺/Ca²⁺ exchange activity in secretory membrane vesicles. This Ca²⁺/Ca²⁺ exchange system is inhibited by Mg²⁺, but not by Sr²⁺. The Na ⁺ -dependent Ca²⁺ uptake system in the presence of Mg²⁺ is a saturable process with an apparent K_m of 0.28 μM and a V_max= 14.5 nmol min^?1 mg protein^?1. Ruthenium red inhibited neither the Na⁺/Ca²⁺ nor the Ca²⁺/Ca²⁺ exchange, even at high concentrations.     

Pharmacological Characterization of the Voltage-Dependent Ca2+ Channels Present in Synaptosomes from Rat and Chicken Central Nervous System

Abstract: The voltage-dependent calcium channels present in mammalian and chicken brain synaptosomes were characterized pharmacologically using specific blockers of L-type channels (1,4-dihydropyridines), N-type channels (ω-conotoxin GVIA), and P-type channels [funnel web toxin (FTX) and ω-agatoxin IVA]. K⁺-induced Ca²⁺ uptake by chicken synaptosomes was blocked by ω-conotoxin GVIA (IC₅₀ = 250 nM). This toxin at 5 µM did not block Ca²⁺ entry into rat frontal cortex synaptosomes. FTX and ω-agatoxin IVA blocked Ca²⁺ uptake by rat synaptosomes (IC₅₀ = 0.17 µl/ml and 40 nM, respectively). Likewise, in chicken synaptosomes, FTX and ω-agatoxin IVA affected Ca²⁺ uptake. FTX (3 µl/ml) exerted a maximal inhibition of 40% with an IC₅₀ similar to the one obtained in rat preparations, whereas with ω-agatoxin IVA saturation was not reached even at 5 µM. In chicken preparations, the combined effect of saturating concentrations of FTX (1 µl/ml) and different concentrations of ω-conotoxin GVIA showed no additive effects. However, the effect of saturating concentrations of FTX and ω-conotoxin GVIA was never greater than the one observed with ω-conotoxin GVIA. We also found that 60% of the Ca²⁺ uptake by rat and chicken synaptosomes was inhibited by ω-conotoxin MVIID (1 µM), a toxin that has a high index of discrimination against N-type channels. Conversely, nitrendipine (10 µM) had no significant effect on Ca²⁺ uptake in either the rat or the chicken. In conclusion, Ca²⁺ uptake by rat synaptosomes is potently inhibited by different P-type Ca²⁺ channel blockers, thus indicating that P-type channels are predominant in this preparation. In contrast, Ca²⁺ uptake by chicken synaptosomes is sensitive to ω-conotoxin GVIA, FTX, ω-agatoxin IVA, and ω-conotoxin MVIID. This suggests that a channel subtype with a mixed pharmacology is present in chicken synaptosomes.     

Toxins of Pseudomonas syringae pv. syringae affect H+-transport across the plasma membrane of maize

The activity of some phytotoxic metabolites of Pseudomonas syringae pv. syringae Van Hall strains B359 and B301 on in vivo and in vitro systems of H⁺-transport across the plasma membrane of maize (Zea mays L., hybrid Paolo) was investigated. In particular syringomycin, the first lipodepsinonapeptide isolated from Pss and already studied in plants and yeasts for its effects on several physiological systems, was compared with the recently described lipodepsipeptides with 22 or 25 amino acid residues, so called syringopeptins. The in vivo activity of the phytotoxins was tested on fusicoccin-stimulated H^?-extrusion from cuttings of maize roots, which was inhibited by both types of toxins, with syringomycin more efficient than the syringopeptins. In vitro the H⁺-ATPase activity of predominantly right-side-out plasma membrane vesicles purified by two-phase partitioning was stimulated by 10 μM syringomycin and inhibited by higher levels, in agreement with the results of others with preparations of dicotyledons. Also the inhibition of the phosphohydrolytic activity of inside-out vesicles of mung bean plasma membrane was confirmed for maize. In both types of vesicles the syringopeptirts were better inhibitors than syringomycin. The pH gradient formed on addition of ATP to predominantly (25% latency) inside-out vesicles was immediately and completely collapsed by syringomycin and syringopeptins; H⁺-pumping was prevented if the toxins were added before ATP. The inhibition was concentration dependent, but at very low concentrations the effect was inverted. The results of the present investigation, carried out with maize preparations, confirm and extend the evidence so far obtained with dicotyledons in favour of the plasma membrane as an important site of interaction of syringomycin with the plant cell. They also indicate that, except for some details, the effects of syringopeptins at the level of the plasma membrane are the same as those of syringomycin.     

Calcium Buffering and Free Ca2+ in Rat Brain Synaptosomes

Abstract: The effects of K⁺ depolarization and of stimulation by veratridine on apparent cytosolic free Ca²⁺ ([Ca²⁺]_cyt) and net Ca²⁺ accumulation were measured in isolated rat brain presynaptic nerve terminals (synaptosomes). [Ca²⁺]_cyt was determined with fura-2, and Ca²⁺ accumulation was measured with tracer ⁴⁵Ca. [Ca²⁺]_cyt was ～ 325 nM in synaptosomes incubated in the normal physiological salt solution under resting conditions. When [K⁺]₀, was increased from the normal 5 mM to 30 or 50 mM, ⁴⁵Ca uptake and [Ca²⁺]_cyt both increased within 1 s. Both increases were directly related to [Ca²⁺]₀ for [Ca²⁺]₀= 0.02–1.2 mM; however, the increase in ⁴⁵Ca uptake greatly exceeded the increase in [Ca²⁺]_cyt. With small Ca²⁺ loads ≤100 μmol/L of cell water, equivalent to the Ca²⁺ entry during a train of ≤60 impulses), the ⁴⁵Ca uptake exceeded the increase in [Ca²⁺]_cyt by a factor of nearly 1,000. This indicates that ～99.9% of the entering Ca²⁺ was buffered and/or sequestered within ～ 1 s. With larger Ca²⁺ loads, a larger fraction of the entering Ca²⁺ was buffered; ～99.97% of the load was buffered with loads of 250–425 μmol/L of cell water. The ratio between the total Ca²⁺ entry and the increase in [Ca²⁺]_cyt, the “calcium buffer ratio”β, was therefore ～ 3,500:1. This ratio was somewhat lower than the ratio of total intraterminal calcium: [Ca²⁺]_cyt, which ranged between ～7,300:1 and 12,800:1. When the synaptosomes were activated with 10 μM veratridine ([Ca²⁺]₀= 0.2–0.6 mM), ⁴⁵Ca influx and [Ca²⁺]_cyt increased progressively for ～10 s (β= 2,700:13,050:1) and then leveled off. Application of 10 μM tetrodotoxin before the introduction of veratridine prevented the increases in ⁴⁵Ca influx and [Ca²⁺]_cyt. Application of 10 μM tetrodotoxin after 5–10 s of exposure to veratridine caused both the [Ca²⁺]_cyt and the veratridine-stimulated ⁴⁵Ca within the terminals to decline, thereby demonstrating that the Ca²⁺ loading is reversible in the presence of extracellular Ca²⁺. These data show that synaptosomes are capable of buffering and metabolizing Ca²⁺ in a manner expected for intact neurons.     

Antagonistic Effect of Na+ and Mg2+ on P2z Purinoceptor-Associated Pores in Dibutyryl Cyclic AMP-Differentiated NG108-15 Cells

Abstract: The effect of replacement of extracellular Na⁺ with N-methyl-d -glucamine (NMG) on P₂ receptor signaling pathways was investigated in dibutyryl cyclic AMP-differentiated NG108-15 cells. Benzoylbenzoic ATP (BzATP) dose-dependently increased the cytosolic Ca²⁺ concentration ([Ca²⁺]_i) with an EC₅₀ value of 230 µM. Replacement of Na⁺ with NMG as well as removal of Mg²⁺ from the bathing buffer potentiated ethidium bromide uptake, [Ca²⁺]_i increase, and ⁴⁵Ca²⁺ uptake in response to ATP or BzATP. In contrast, in the presence of 5 mM Mg²⁺ to limit the amount of ATP^4?, replacement of Na⁺ with NMG had no effect on the ATP-induced [Ca²⁺]_i increase but caused a markedly larger [Ca²⁺]_i increase when the calculated concentration of ATP^4? was >10 µM. The calculated EC₅₀ value for ATP^4? stimulation of the [Ca²⁺]_i increase was 23 µM in NG108-15 cells. In vascular smooth muscle cells, intracellular Ca²⁺ release was the major pathway for the ATP-induced [Ca²⁺]_i increase; both removal of Mg²⁺ and replacement of Na⁺ with NMG did not affect the action of ATP. These data suggest that ATP^4?-promoted pores are antagonized by Na⁺ and Mg²⁺ in dibutyryl cyclic AMP-differentiated NG108-15 cells.     

Plasma membrane Ca2+ transport: stimulation by soluble proteins.

Inside-out membrane vesicles were prepared from human red blood cells. In the presence of ATP, these vesicles took up ⁴⁵Ca²⁺ against a chemical gradient. The active transport of Ca²⁺ was increased by addition of an activator protein of (Ca²⁺+Mg²⁺)-ATPase isolated from the membrane-free hemolysate of human red blood cells. A closely related protein, the protein modulator of cyclic AMP phosphodiesterase from bovine brain, also increased the rate of active transport of ⁴⁵Ca²⁺. Addition of the calcium ionophore A23187 caused a rapid efflux of ⁴⁵Ca²⁺ from loaded, inside-out vesicles. When La³⁺ was added to the system in the presence of activator protein, the uptake of ⁴⁵Ca²⁺ was inhibited. Results are compatible with the interpretation that activity of the plasma membrane Ca²⁺ pump may be modulated by certain cytoplasmic proteins.     

Active calcium ion uptake by inside-out and right side-out vesicles of red blood cell membranes

The relationship between active extrusion of Ca⁺⁺ from red cell ghosts and active uptake of Ca⁺⁺ by isolated red cell membrane fragments was investigated by studying the Ca⁺⁺ uptake activities of inside-out and right side-out vesicles. Preparations A and B which had mainly inside-out and right side-out vesicles, respectively, were isolated from red cell membranes and were compared with respect to Ca⁺⁺ adenosine triphosphatase (ATPase) and ATP-dependent Ca⁺⁺ uptake activities. Preparation A had nearly eight times more inside-out vesicles and took up eight times more ⁴⁵Ca in the presence of ATP compared to preparation B. Separation of the ⁴⁵Ca-labeled membrane vesicles by density gradient centrifugation showed that the ⁴⁵Ca label was localized to the inside-out vesicle fraction. In addition, the ⁴⁵Ca taken up in the presence of ATP was lost during a subsequent incubation in the absence of ATP. The rate of ⁴⁵Ca loss was not influenced by the presence of EGTA, but was slowed in the presence of La⁺⁸ (0.1 mM) in the efflux medium. The results presented here support the thesis that the active uptake of Ca⁺⁺ by red cell membrane fragments is due to the active transport of Ca⁺⁺ into inside-out vesicles.     

Ca2+-dependent ATPase associated with plasma membrane from a calcareous alga, Serraticardia maxima (Corallinaceae, Rhodophyta)

The plasma membrane was isolated from a calcareous red alga, Serraticardia maxima (Yendo) Silva (Corallinaceae), by aqueous two-phase partitioning. Its purity was examined with marker enzymes, Mg²⁺-dependent ATPase, inosine diphosphatase, cytochrome c oxidase and NADH-cytochrome c reductase, as well as the sensitivity of Mg²⁺-dependent ATPase to vanadate, azide and nitrate. The results showed that the isolated plasma membrane was purified enough to study its functions. Electron microscopic observations on thin tissue sections revealed that most vesicles of the isolated plasma membrane were stained by the plasma membrane specific stain, phosphotungstic acid-chromic acid. Mg²⁺- or Ca²⁺-dependent ATPases were associated with the plasma membrane. Ca²⁺-dependent ATPase was activated at physiological cytoplasmic concentrations of Ca²⁺ (0.1–10 μmol/L). However, calmodulin (0.5 μmol/L) did not affect its activity. The pH optimum was 8.0, in contrast to 7.0 for Mg²⁺-dependent ATPase. The isolated plasma membrane vesicles were mostly right side-out. To test for H⁺-translocation, right side-out vesicles were inverted; 27% of vesicles were inside-out after treatment with Triton X-100. The inside-out plasma membrane vesicles showed reduction of quinacrine fluorescence in the presence of 1 mmol/L ATP and 100 μmol/L Ca²⁺. The reduced fluorescence was recovered with the addition of 10 mmol/L NH₄Cl, or 5 μmol/L nigericin plus 50 mmol/L KCl. UTP and CTP substituted for ATP, but ADP did not. Ca²⁺-dependent ATPase might pump H⁺ out in the physiological state. The acidification by this pump might be coupled with alkalinization at the calcifying sites, which induces calcification.     

Relationship Between Ca2+ Uptake and Catecholamine Secretion in Primary Dissociated Cultures of Adrenal Medulla

Abstract: Carbachol or elevated K⁺ stimulated ⁴⁵Ca²⁺ uptake into chromaffin cells two- to fourfold. The uptake was stimulated by cholinergic drugs with nicotinic activity, but not by those with only muscarinic activity. Ca²⁺ uptake and catecholamine secretion induced by the mixed nicotinic-muscarinic agonist carbachol were inhibited by the nicotinic antagonist mecamylamine, but not by the muscarinic antagonist atropine. Significant Ca²⁺ uptake occurred within 15 s of stimulation by carbachol or elevated K⁺ at a time before catecholamine secretion was readily detected. At later times the time course of secretion induced by carbachol or elevated K⁺ was similar to that of Ca²⁺ uptake. There was a close correlation between Ca²⁺ uptake and catecholamine secretion at various concentrations of Ca²⁺. The concentration dependencies for inhibition of both processes by Mg²⁺ or Cd²⁺ were similar. Ca²⁺ uptake saturated with increasing Ca²⁺ concentrations, with an apparent K_m for both carbachol-induced and elevated K⁺-induced Ca²⁺ uptake of approximately 2 mM. The Ca²⁺ dependency, however, was different for the two stimuli. The studies provide strong support for the notion that Ca²⁺ entry and a presumed increase in cytosolic Ca²⁺ concentration respectively initiates and maintains secretion. They also provide evidence for the existence of saturable, intracellular, Ca²⁺- dependent processes associated with catecholamine secretion. Ca²⁺ entry may, in addition, enhance nicotinic receptor desensitization and may cause inactivation of voltage-sensitive Ca²⁺ channels.     

Diacylglycerol kinase in plasma membranes from wheat

Diacylglycerol kinase activity was demonstrated in highly purified plasma membranes isolated from shoots and roots of dark-grown wheat (Triticum aestivum L.) by aqueous polymer two-phase partitioning. The active site of the diacylglycerol kinase was localized to the inner cytoplasmic surface of the plasma membrane using isolated inside-out and right-side-out plasma membrane vesicles from roots. The enzyme activity in plasma membrane vesicles from shoots showed a broad pH optimum around pH 7. The reaction was Mg²⁺ and ATP dependent, and maximal activity was observed around 0.5 mM ATP and 3 mM MgCl₂. The Mg²⁺ requirement could be substituted only partially by Mn²⁺ and not at all by Ca²⁺. The phosphorylation of endogenous diacylglycerol was strongly inhibited by detergents indicating an extreme dependence of the lipid environment. Inositol phospholipids stimulated the activity of diacylglycerol kinase in plasma membranes from shoots and roots, whereas the activity was inhibited by R59022, a putative inhibitor of several diacylglycerol kinase isoenzymes involved in uncoupling diacylglycerol activation of mammalian protein kinase C.     

Sealed inside-out and right-side-out plasma membrane vesicles : optimal conditions for formation and separation

Plasma membrane preparations of high purity (about 95%) are easily obtained by partitioning in aqueous polymer two-phase systems. These preparations, however, mainly contain sealed right-side-out (apoplastic side out) vesicles. Part of these vesicles have been turned inside-out by freezing and thawing, and sealed inside-out and right-side-out vesicles subsequently separated by repeating the phase partition step. Increasing the KCI concentration in the freeze/thaw medium as well as increasing the number of freeze/thaw cycles significantly increased the yield of inside-out vesicles. At optimal conditions, 15 to 25% of total plasma membrane protein was recovered as inside-out vesicles, corresponding to 5 to 10 milligrams of protein from 500 grams of sugar beet (Beta vulgaris L.) leaves. Based on enzyme latency, trypsin inhibition of NADH-cytochrome c reductase, and H⁺ pumping capacity, a cross-contamination of about 20% between the two fractions of oppositely oriented vesicles was estimated. Thus, preparations containing about 80% inside-out and 80% right-side-out vesicles, respectively, were obtained. ATPase activity and H⁺ pumping were both completely inhibited by vanadate (K_i ≈ 10 micromolar), indicating that the fractions were completely free from nonplasma membrane ATPases. Furthermore, the polypeptide patterns of the two fractions were close to identical, which shows that the vesicles differed in sidedness only. Thus, preparations of both inside-out and right-side-out plasma membrane vesicles are now available. This permits studies on transport, signal transduction mechanisms, enzyme topology, etc., using plasma membrane vesicles of either orientation.     

Ca2+ responses to acetylcholine and adenosine triphosphate in the otocyst of chick embryo

The action of acetylcholine and adenosine triphosphate (ATP) on cytoplasmic Ca²⁺ concentration ([Ca²⁺]_i) was studied in the otocyst epithelium of embryonic day 3 chicks with Ca²⁺-sensitive fluorescence measurements. Increases in [Ca²⁺]_i were evoked by the bath application of acetylcholine (1 μM or higher). The rise in [Ca²⁺]_i was due to the release of Ca²⁺ from intracellular Ca²⁺ stores, since the Ca²⁺ response occurred even in a Ca²⁺-free medium. The Ca²⁺ response to acetylcholine was mediated by muscarinic receptors. Atropine of 1 μM abolisehd the response to 10 μM acetylcholine; muscarine and carbamylcholine (100 μM each) evoked Ca²⁺ rises. Increases in [Ca²⁺]_i were also evoked by the bath application of ATP (10 μM or higher). The Ca²⁺ rise by ATP was evoked even in a Ca²⁺-free medium. Adenosine (500 μM) did not cause any Ca²⁺ response. Suramin and reactive blue 2 (200 μM each) completely blocked the Ca²⁺ response to 500μM ATP. Uridine triphosphate (500 μM) caused comparable Ca²⁺ responses with those to 500 μM ATP. These results suggested the involvement of P_2U purinoceptors. The potentiation of Ca²⁺ rise was observed when acetylcholine and ATP were co-applied at submaximal concentrations (10 μM and 100 μM, respectively). We conclude that undifferentiated cells in the otocyst epithelium have CaCa²⁺ mobilizing systems activated by acetylcholine and ATP. © 1995 John Wiley & Sons, Inc.     

ATP-dependent Ca2+ transport and Ca2+-ATPase activities in an enriched plasma membrane fraction from gypsy moth larval midgut tissue

A subcellular fraction enriched in plasma membranes was obtained from gypsy moth (Lymantria dispar) larval midgut tissue. Using [⁴⁵Ca]²⁺ as a tracer, Ca²⁺ transport activity by membrane vesicles in the enriched fraction was measured and shown to be ATP-dependent, with a very high affinity for Ca²⁺ (apparent K_m for [Ca²⁺ _free]

1 Abbreviations used: [Ca²⁺free] = concentration of free (unbound) calcium ion;CaM = calmodulin; F = fraction; IOV = inside-out membrane vesicles; W-5 = N-(6-aminohexyl)-1-naphthalenesulfonamide; W-7 = N-(6-aminohexyl)-5-chloro-1-naphthalenesulfonamide.

= 22 nM). Ca²⁺ transport was abolished upon addition of the calcium ionophore, A23187. Ca²⁺-stimulated, Mg²⁺-dependent ATPase activity peaked between 100 and 200 nM Ca²⁺_free. Ca²⁺-Mg²⁺-ATPase activity was inhibited by vanadate, 2 phenothiazine drugs (trifluoperazine and chlorpromazine), and the naphthalene sulfonamide, W-7; the related compound, W-5, and ouabain had a negligible effect. These results suggest the presence of a high affinity plasma membrane Ca²⁺ pump in gypsy moth larval midgut cells and are discussed in light of earlier work involving calcium transport in isolated midguts of larval Hyalophora cecropia. Ionic and other conditions that characterize the midgut physiology of larval Lepidoptera (e.g., luminal pH; electrochemical gradient for Ca²⁺; effect of certain ions and inhibitors on Ca²⁺ transport) contrast significantly with those found in adult Diptera. The implications that these differences may have for calcium regulation are discussed. © 1992 Wiley-Liss, Inc.     

Characterisation of a Ca2+-dependent ATP hydrolysis associated with the vacuolar membrane of wheat roots

Microsomal fractions from wheat tissues exhibit a higher level of ATP hydrolytic activity in the presence of Ca²⁺ than Mg²⁺. Here we characterise the Ca²⁺-dependent activity from roots of Triticum aestivum lev. Troy) and investigate its possible function. Ca²⁺-dependent ATP hydrolysis in the microsomal fraction occurs over a wide pH range with two slight optima at pH 5.5 and 7.5. At these pHs the activity co-migrates with the major peak of nitrate-inhibited Mg²⁺. Cl-ATPase on continuous sucrose gradients indicating that it is associated with the vacuolar membrane. Ca²⁺-dependent ATP hydrolysis can be distinguished from an inhibitory effect of Ca²⁺ on the plasma membrane K⁺, Mg²⁺-ATPase following microsomal membrane separation using aqueous polymer two phase partitioning. The Ca²⁺-dependent activity is stimulated by free Ca²⁺ with a K_m of 8.1 μM in the absence of Mg²⁺ ([CaATP] = 0.8 mM). Vacuoiar membrane vacuolar preparations contain a higher Ca²⁺-dependent than Mg²⁺-dependent ATP hydrolysis, although the two activities are not directly additive. The nucleotide specificity of the divalent ion-dependent activities in vacuolar membrane-enriched fractions was low. hydrolysis of CTP and UTP being greater than ATP hydrolysis with both Ca²⁺ and Mg²⁺ The Ca²⁺-dependent activity did discriminate against dinucleotides, and mononucleotides. and failed to hydrolyse phosphatase substrates. Despite low nucleotide specificity the Mg²⁺-dependent activity functioned as a bafilomycin sensitive H⁺-pump in vacuolar membrane vesicles. Ca²⁺-dependent ATP hydrolysis was not inhibited by the V-, P-, or F-type ATPase inhibitors bafilomycin. vanadate and azide, respectively. nor by the phosphatase inhibitor molybdate, but was inhibited 20% at pH 7.5 by K⁺. Possible functions of Ca²⁺-dependent hydrolysis as a H⁺-pump or a Ca²⁺-pump was investigated using vacuolar membrane vesicles. No H⁺ or Ca²⁺ translocating activity was observed under conditions when the Ca²⁺-dependent ATP hydrolysis was active.