Calix[4]arene C-90 selectively inhibits Ca2+,Mg2+-ATPase of myometrium cell plasma membrane

The supramolecular compound calix[4]arene C-90 (5,11,17,23-tetra(trifluoro)methyl(phenylsulfonylimino)-methylamino-25,26,27,28-tetrapropoxycalix[4]arene) is shown to efficiently inhibit the ATP hydrolase activity of Ca²⁺,Mg²⁺-ATPase in the myometrium cell plasma membrane fraction and also in a preparation of the purified enzyme solubilized from this subcellular fraction. The inhibition coefficient I _0.5 values were 20.2 ± 0.5 and 58.5 ± 6.4 μM for the membrane fraction and the solubilized enzyme, respectively. The inhibitory effect of calix[4]arene C-90 was selective comparatively to other ATPases localized in the plasma membrane: calix[4]arene C-90 did not influence the activities of Na⁺,K⁺-ATPase and “basal” Mg²⁺-ATPase. The inhibitory effect of calix[4]arene C-90 on the Ca²⁺,Mg²⁺-ATPase activity was associated with the cooperative action of four trifluoromethylphenyl sulfonylimine (sulfonylamidine) groups oriented similarly on the upper rim of the calix[4]arene macrocycle (the calix[4]arene “bowl”). The experimental findings seem to be of importance for studies, using calix[4]arene C-90, of membrane mechanisms of regulation of calcium homeostasis in smooth muscle cells and also for investigation of the participation of the plasma membrane Ca²⁺-pump in control of electro- and pharmacomechanical coupling in myocytes.     

Properties of an ATP-dependent Ca2+ pump and (Ca2+ + Mg2+)-ATPase in brain synaptosome membrane vesicles from the bertha armyworm,Mamestra configurata Wlk

Biochemical and kinetic properties under identical substrate and reaction conditions were obtained for an ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase in synaptosome membrane vesicles prepared from the brain of the moth, Mamestra configurata. Both the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase had single, high-affinity binding sites for ATP (K_m = 14 and 116 μM, respectively), Ca²⁺_free (K_m = 0.13 nM and 0.072 nM, respectively), and Mg²⁺ (K_m = 1.1 mM and 0.07 mM, respectively). Both systems were relatively little affected by K⁺ and were insensitive to ouabain, an inhibitor of (Na⁺ + K⁺)-ATPase. The results indicate that the ATP-dependent Ca²⁺ pump and (Ca²⁺ + Mg²⁺)-ATPase are functionally coupled in synaptic membranes and constitute a mechanism for Ca²⁺ transport in the brain of M. configurata. Although moth brain (Ca²⁺ + Mg²⁺)-ATPase is maximally active at nanomolar concentrations of free calcium ion, the enzyme retains at least one-half of its maximal activity at micromolar calcium concentrations, indicating either that the enzyme has two binding sites for calcium (a high-affinity site at nanomolar Ca²⁺_free and a low-affinity site at micromolar Ca²⁺_free), or that there are two enzymes with high and low affinity for calcium, respectively. Calcium extrusion from brain neurones of M. configurata may operate in a two-stage, concentration-dependent process in which a first stage, low-affinity pump reduces intraneuronal calcium to a concentration at which a second stage, high-affinity pump becomes activated.     

Solubilization and reconstitution of ca pump from corn leaf plasma membrane

The Ca²⁺ transport system of corn (Zea mays) leaf plasma membrane is composed of Ca²⁺ pump and Ca²⁺/H⁺ antiporter driven by H⁺ gradient imposed by a H⁺ pump (M Kasai, S Muto [1990] J Membr Biol 114: 133-142). It is necessary for characterization of these Ca²⁺ transporters to establish the procedure for their solubilization, isolation, and reconstitution into liposomes. We attempted to solubilize and reconstitute the Ca²⁺ pump in the present study. A nonionic detergent octaethyleneglycol monododecyl ether (C₁₂E₈) was the most effective detergent for a series of extraction and functional reconstitution of the Ca²⁺ pump among seven detergents examined. This was judged from activities of ATP-dependent ⁴⁵Ca²⁺ uptake into liposomes reconstituted with the respective detergent-extract of the plasma membrane by the detergent dilution method. C₁₂E₈-extract of the plasma membrane was subjected to high performance liquid chromatography using a DEAE anion exchange column. Ca²⁺-ATPase was separated from VO₄³⁻-sensitive Mg²⁺-ATPase. These ATPases were separately reconstituted into liposomes, and their ATP-dependent Ca²⁺ uptake was measured. The liposomes reconstituted with the Ca²⁺-ATPase, but not with the VO₄³⁻-sensitive Mg²⁺-ATPase, showed ATP-dependent Ca²⁺ uptake. Nigericin-induced pH gradient (acid inside) caused only a little Ca²⁺ uptake into liposomes reconstituted with the Ca²⁺-ATPase, suggesting that the Ca²⁺/H⁺ antiporter was not present in the preparation. These results indicate that the Ca²⁺-ATPase actually functions as Ca²⁺ pump in the corn leaf plasma membrane.     

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A (Ca²⁺, Mg²⁺)-ATPase activity and a (Ca²⁺, Mg²⁺)-dependent phosphorylation from ATP have been found in plasma membrane fragments from squid optical nerves under conditions where contamination by intracellular organelles is unlikely. The properties of this (Ca²⁺, Mg²⁺)-ATPase activity are almost identical to those of the ATP-dependent uncoupled Ca²⁺ efflux observed in dialyzed squid giant axons. This gives further support to the notion that the mechanism responsible for maintaining the low levels of ionized Ca concentration in nerves at rest is not a Na⁺-Ca²⁺ exchange system but an ATP-driven uncoupled Ca²⁺ pump.     

Reconstitution of the energy-linked transhydrogenase activity in membranes from a mutant strain of Escherichia coli K12 lacking magnesium ion- or calcium ion-stimulated adenosine triphosphatase

1. We have isolated a mutant of Escherichia coli K12 (strain AN295) that forms de-repressed amounts of Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase. 2. The Mg²⁺,Ca²⁺-stimulated triphosphatase activity was separated from membrane preparations from strain AN295 by extraction with 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, resulting in a loss of the ATP-dependent transhydrogenase activity. The non-energy-linked transhydrogenase activity remained in the membrane residue. 3. The solubilized Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity from strain AN295 was partially purified by repeated gel filtration. The addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase to the membrane residue from strain AN295 reactivated the ATP-dependent transhydrogenase activity. 4. Strain AN296, lacking Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase activity, was derived by transducing the mutant allele, uncA401, into strain AN295. The ATP-dependent transhydrogenase activity was lost but the non-energy linked transhydrogenase was retained. 5. The ATP-dependent transhydrogenase activity in membrane preparations from strain AN296 (uncA⁻) could not be re-activated by the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295. However, after extraction by 5mm-Tris–HCl buffer containing EDTA and dithiothreitol, the ATP-dependent transhydrogenase activity could be re-activated by the addition of the purified Mg²⁺,Ca²⁺-stimulated adenosine triphosphatase from strain AN295 to the membrane residue from strain AN296 (uncA⁻).     

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.     

Characterization of Mg2+- and (Ca2+ + Mg2+)-ATPase activity in adipocyte endoplasmic reticulum

The presence of an energy-dependent calcium uptake system in adipocyte endoplasmic reticulum (D. E. Bruns, J. M. McDonald, and L. Jarett, 1976, J. Biol. Chem.251, 7191–7197) suggested that this organelle might possess a calcium-stimulated transport ATPase. This report describes two types of ATPase activity in isolated microsomal vesicles: a nonspecific, divalent cation-stimulated ATPase (Mg²⁺-ATPase) of high specific activity, and a specific, calcium-dependent ATPase (Ca²⁺ + Mg²⁺-ATPase) of relatively low activity. Mg²⁺-ATPase activity was present in preparations of mitochondria and plasma membranes as well as microsomes, whereas the (Ca²⁺ + Mg²⁺)-ATPase activity appeared to be localized in the endoplasmic reticulum component of the microsomal fraction. Characterization of microsomal Mg²⁺-ATPase activity revealed apparent K_m values of 115 μm for ATP, 333 μm for magnesium, and 200 μm for calcium. Maximum Mg²⁺-ATPase activity was obtained with no added calcium and 1 mm magnesium. Potassium was found to inhibit Mg²⁺-ATPase activity at concentrations greater than 100 mm. The energy of activation was calculated from Arrhenius plots to be 8.6 kcal/mol. Maximum activity of microsomal (Ca²⁺ + Mg²⁺)-ATPase was 13.7 nmol ³²P/mg/min, which represented only 7% of the total ATPase activity. The enzyme was partially purified by treatment of the microsomes with 0.09% deoxycholic acid in 0.15 m KCl which increased the specific activity to 37.7 nmol ³²P/mg/min. Characterization of (Ca²⁺ + Mg²⁺)-ATPase activity in this preparation revealed a biphasic dependence on ATP with a Hill coefficient of 0.80. The apparent K_m^s for magnesium and calcium were 125 and 0.6–1.2 μm, respectively. (Ca²⁺ + Mg²⁺)-ATPase activity was stimulated by potassium with an apparent K_m of 10 mm and maximum activity reached at 100 mm potassium. The energy of activation was 21.5 kcal/mol. The kinetics and ionic requirements of (Ca²⁺ + Mg²⁺)-ATPase are similar to those of the (Ca²⁺ + Mg²⁺)-ATPase in sarcoplasmic reticulum. These results suggest that the (Ca²⁺ + Mg²⁺)-ATPase of adipocyte endoplasmic reticulum functions as a calcium transport enzyme.     

Interaction of phytohormones and synthetic growth regulator melafen in the control of Ca2+ translocation across the plasma membrane of potato (Solanum tuberosum L.) tuber cells

Interference of phytohormones (jasmonic, gibberellic, and abscisic acids) and synthetic growth regulator melafen on Ca²⁺ translocation across the membrane of plasma membrane vesicles prepared from dormant potato (Solanum tuberosum L.) tubers was studied. The activity of plasma membrane Ca²⁺, Mg²⁺-ATPase was stimulated by melafen and jasmonic and gibberellic acids and suppressed by abscisic acid. These substrances did not change the passive membrane permeability for Ca²⁺. The pattern of the effect of melafen on the activity of Ca²⁺,Mg²⁺-ATPase depended on the presence of phytohormones in incubation medium. When melafen and each phytohormone were simultaneously added to incubation medium, their effects were not additive, which indicates that the effects of the tested compounds on the Ca²⁺ uptake into the plasma membrane vesicles are interdependent. Apparently, the interaction between the phytohormones and plasma membrane components modulates the response to melafen.     

A Ca2+-ATPase and a Mg2+/H+-antiporter are present on tonoplast membranes from roots of Zea mays L.

The primary or secondary energized transport of Ca²⁺, Mg²⁺ and H⁺ into tonoplast membrane vesicles from roots of Zea mays L. seedlings was studied photometrically by using the fluorescent Ca²⁺ indicator Indo 1 and the pH indicator neutral red. The localization of an ATP-dependent, vanadate-sensitive Ca²⁺ pump on tonoplast-type vesicles was demonstrated by the co-migration of the Ca²⁺-pumping and tonoplast H⁺-pyrophosphatase (PP_iase) activity on continuous sucrose density gradients. In ER-membrane fractions, only a low Ca²⁺-pumping activity could be detected. The ATP-dependent Ca²⁺ uptake into tonoplast vesicles (using Ca²⁺ concentrations from 0.8–1 μM) was completely inhibited by the Ca²⁺ ionophore ionomycin (1 μM) whereas the protonophore nigericin (1 μM) which eliminates ATP-dependent intravesicular H⁺ accumulation had no effect. Vanadate (IC₅₀ = 43 μM) and diethylstilbesterol (IC₅₀ = 5.2 μM) were potent inhibitors of this type of Ca²⁺ transport. The nucleotides GTP, UTP, ITP, and ADP gave 27%–50% of the ATP-dependent activity (K _m = 0.41 mM). From these results, it was suggested that this ATP-dependent high-affinity Ca²⁺ transport mechanism is the only functioning Ca²⁺ transporter of the tonoplast under in-vivo conditions i.e. under the low cytosolic Ca²⁺ concentration. In contrast, the secondary energized Ca²⁺-transport mechanism of the tonoplast, the low-affinity Ca²⁺/H⁺-antiporter, which was reported to allow the uptake of Ca²⁺ in exchange for H⁺, functions chiefly as an Mg²⁺ transporter under physiological conditions because cytosolic Mg²⁺ is several orders of magnitude higher than the Ca²⁺ concentration. This conclusion was deduced from experiments showing that Mg²⁺ ions in a concentration range of 0.01 to 1 mM triggered a fast efflux of H⁺ from acid-loaded vesicles. Furthermore, the proton-pumping activity of the tonoplast H⁺-ATPase and H⁺-PP_iase was found to be influenced by Ca²⁺ differently from and independently of the Mg²⁺ concentration. Calcium was a strong inhibitor for the H⁺-PP_iase (IC₅₀ = 18 μM, Hill coefficient nH = 1.7) but a weak one for the H⁺-ATPase (IC₅₀ = 330 μM, nH = 1). From these results it is suggested that at the tonoplast membrane a functional interaction exists between (i) the Ca²⁺-and Mg²⁺-regulated H⁺-PP_iase, (ii) the newly described high-affinity Ca²⁺-AT-Pase, (iii) the low-affinity Mg²⁺(Ca²⁺)/H⁺-antiporter and (iv) the H²⁺-ATPase.     

Characterization of Mg2+-ATPase from sheep kidney medulla: purification.

Magnesium-dependent adenosine triphosphatase has been purified from sheep kidney medulla plasma membranes. The purification, which is based on treatment of a kidney plasma membrane fraction with 0.5% digitonin in 3 mm MgCl₂, effectively separates the Mg²⁺-ATPase from (Na⁺ + K⁺)-ATPase present in the same tissue and yields the Mg²⁺-ATPase in soluble form. The purified enzyme is activated by a variety of divalent cations and trivalent cations, including Mg²⁺, Mn²⁺, Ca²⁺, Co²⁺, Fe²⁺, Zn²⁺, Eu³⁺, Gd³⁺, and VO²⁺. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme shows two bands with R_f values corresponding to molecular weights of 150,000 and 77,000. The larger peptide is phosphorylated by [γ-³²P]ATP, suggesting that this peptide may contain the active site of the Mg²⁺-ATPase. The Mg²⁺-ATPase activity is unaffected by the specific (Na⁺ + K⁺)-ATPase inhibitor ouabain.     

(Ca2+ + Mg2+)-ATPase of density-separated human red cells. Effects of calcium and a soluble cytoplasmic activator (Calmodulin)

The effect of calcium and a soluble cytoplasmic activator on (Ca²⁺ + Mg²⁺)-ATPase of density-separated human red cells was investigated. At all calcium concentrations tested, dense (old) lysed cells and their isolated membranes displayed lower activities as compared to the light (young) cells and their membranes. Isolated membranes from all density red cell fractions showed two distinct (Ca²⁺ + Mg²⁺)-ATPase activities; one at low calcium and another at moderate calcium concentrations. At high calcium concentration, (Ca²⁺ + Mg²⁺)-ATPase activity of isolated membranes was low in all cell fractions. In contrast to the isolated membranes, lysed cells from all density fractions had a maximum (Ca²⁺ + Mg²⁺)-ATPase activity only at a low concentration of calcium, while moderate and high calcium concentrations produced low activity. Upon isolation of membranes, a substantial loss of (Ca²⁺ + Mg²⁺)-ATPase activity took place from all density cell fractions. Upon membrane isolation, the relative loss of (Ca²⁺ + Mg²⁺)-ATPase activity at low Ca²⁺ concentration was greater in older cells. The extent of stimulation of (Ca²⁺ + Mg²⁺)-ATPase by the activator at low calcium concentration was 3–4-fold greater in older cell membranes than in the young ones.These data suggest that the lower (Ca²⁺ + Mg²⁺)-ATPase activity in old cells could be accounted for by a selective loss of (Ca²⁺ + Mg²⁺)-ATPase activity at low Ca²⁺ concentration presumably due to reduced affinity of old cell membranes to activator protein.     

A [Ca2+ + Mg2+]-ATPase and active Ca2+ transport in the plasma membranes isolated from ram sperm flagella

Plasma membranes isolated from the flagella of ram ejaculated sperm were found to contain a [Ca²⁺ + Mg²⁺]-ATPase. Freeze-fracture electron microscopy showed the membranes occur as vesicles. The membrane vesicles actively accumulate Ca²⁺, uptake was reversed by the ionophore A23187 and inhibited by either ruthenium red or La³⁺. The plasma membranes contain two major proteins, designated proteins A and B, with molecular weights of 109,000 and 18,300 daltons, respectively. Protein B is not detected in plasma membranes isolated from ram epididymal sperm. The plasma membrane Ca²⁺ pump may be modulated by protein factors present in seminal plasma.     

Separation of the Mg-ATPases from the Ca-Phosphatase Activity of Microsomal Membranes Prepared from Barley Roots

Two methods for preparing membrane fractions from barley (Hordeum vulgare cv California Mariout 72) roots were compared in order to resolve reported differences between the characteristics of the plasma membrane ATPase of barley and that of other species. When microsomal membranes were prepared by a published procedure and applied to a continuous sucrose gradient, the membranes sedimented as a single broad band with a peak density of 1.16 grams per cubic centimeter (g/cm³). Activities of NADH cytochrome (Cyt) c reductase, Ca²⁺-ATPase, and Mg²⁺-ATPase were coincident and there was little ATP-dependent proton transport anywhere on the gradient. When the homogenization procedure was modified by increasing the pH of the buffer and the ratio of buffer to roots, the microsomal membranes separated as several components on a continuous sucrose gradient. A Ca²⁺-phosphatase was at the top of the gradient, NADH Cyt c reductase at 1.08 g/cm³, a peak of ATP-dependent proton transport at 1.09 to 1.12 g/cm³, a peak of nitrate-inhibited ATPase at 1.09 to 1.12 g/cm³, and of vanadate-inhibited ATPase at 1.16 g/cm³. The Ca²⁺-phosphatase had no preference for ATP over other nucleoside di- and tri-phosphates and was separated from the vanadate-inhibited ATPase on a sucrose gradient; approximately 70% of the Ca²⁺-phosphatase was removed from the microsomes by washing with 150 millimolar KCl. The vanadate-sensitive ATPase required Mg²⁺, was highly specific for ATP, and was not affected by the KCl wash. These results show that barley roots have a plasma membrane ATPase similar to that of other plant species.     

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.     

Effect of concanavalin A on the activity of membrane-bound and detergent-solubilized Mg2+-ATPase

Plasma membranes were isolated after binding liver and hepatoma cells to polylysine-coated polyacrylamide beads, and the effect of concanavalin A on the membrane-bound Mg²⁺-ATPase and the Mg²⁺-ATPase solubilized by octaethylene glycol monododecyl ether (C₁₂E₈) was studied. In the experiment of membranebound Mg²⁺-ATPase, plasma membranes were pretreated with Concanavalin A and the activity was assayed. Concanavalin A stimulated the activity of both liver and hepatoma enzymes assayed above 20°C. Concanavalin A abolished the negative temperature dependency characteristic of liver plasma membrane Mg²⁺-ATPase. On the other hand, Concanavalin A prevented the rapid inactivation due to storage at ?20°C, which was characteristic of hepatoma plasma membrane Mg²⁺-ATPase. With solubilized Mg²⁺-ATPase from liver plasma membranes, the negative temperature dependency was not observed. Concanavalin A, which was added to the assay medium, stimulated the activity of the enzyme solubilized in C₁₂E₈ at a high ionic strength. However, Concanavalin A failed to show any effect on the enzyme solubilized in C₁₂E₈ at a low ionic strength. With solubilized Mg²⁺-ATPase from hepatoma plasma membranes, Concanavalin A could not prevent the inactivation of the enzyme during incubation at ?20°C.     

Altered coupling states between calcium transport and (Ca2+, Mg2+)-ATPase in the AS-30D Ascites hepatocarcinoma plasma membrane

Plasma membrane fractions from normal, regenerating liver and the AS-30D ascites hepatocarcinoma exhibited a high degree of enrichment when a set of plasma membrane enzyme markers were studied in comparison to the ones associated to the mitochondrial and cytosolic compartments. While the (Ca²⁺, Mg²⁺)-ATPase observed for the plasma membrane fraction isolated from normal liver showed an activity of 1.2 µmoles/mg/min, the regenerating liver and the AS-30D plasma membrane fractions presented a much lower ATPase activity (0.3 and 0.22 µmoles/mg/min respectively). Despite the differences in ATPase activity observed between models, the plasma membrane fraction from the AS-30D hepatocarcinoma presented a calcium transport activity similar to the value observed for the normal system (5.9 and 5.5 nmoles Ca²⁺/mg/10min, respectively). Interestingly, the ATP Pi exchange experiments carried out with the different plasma membrane fractions revealed that the (Ca²⁺, Mg²⁺)-ATPase contained in the plasma membrane from the AS-30D cells shows an exchange activity of 26 nmoles ATP Pi/mg/min, similar to the one observed for the enzyme from normal liver (30 nmoles ATP Pi/mg/min). Our results suggest that the plasma membrane from the transformed model presents a more efficient mechanism to regulate the movement of calcium through the calcium pump, with an optimum expenditure of energy.Dedicated to the memory of Catalina Mas Oliva and Valentín Mas Morera.     

The possibility that Ca2+-ATPase from the plasma membrane-rich fraction of bovine parotid gland is ecto-Ca2+-dependent nucleoside triphosphatase

• 1.1. Parotid plasma membrane nonpump low-affinity Ca²⁺-ATPase, which possesses high-affinity (Ca²⁺ + Mg²⁺ )-ATPase activity, was characterized.
• 2.2. Purified Ca²⁺-ATPase hydrolyzed the nucleoside triphosphates, GTP, ITP, CTP, UTP, TTP (67–93% of ATP) and nucleoside diphosphates, ADP. GDP, IDP, CDP, TDP (12–40% of ATP) but not AMP and p-NPP.
• 3.3. The maximum activities of Ca²⁺- and (Ca²⁺ +Mg²⁺ )-ATPases were obtained in the presence of 1 mM and 0.13 μ M Ca²⁺, respectively.
• 4.4. The K_m values for Ca²⁺ in Ca²⁺- and (Ca²⁺+ Mg²⁺ )-ATPases were 0.2 mM and 22 nM. respectively.
• 5.5. The activities of both Ca²⁺- and (Ca²⁺ + Mg²⁺ )-ATPases were found in the right-side-out-vesicles obtained from the plasma membrane-rich fraction.
• 6.6. These features suggest that Ca²⁺-ATPase is an ecto-Ca²⁺-dependent nucleoside triphosphatase.

     

Calcium-pumping ATPases in vesicles from carrot cells : stimulation by calmodulin or phosphatidylserine, and formation of a 120 kilodalton phosphoenzyme

Ca²⁺-ATPases keep cytoplasmic [Ca²⁺] low by pumping Ca²⁺ into intracellular compartments or out of the cell. The transport properties of Ca²⁺-pumping ATPases from carrot (Daucus carota cv Danvers) tissue culture cells were studied. ATP-dependent Ca²⁺ transport in vesicles that comigrated with an endoplasmic reticulum marker, was stimulated three- to fourfold by calmodulin. Cyclopiazonic acid (a specific inhibitor of the sarcoplasmic/endoplasmic reticulum Ca²⁺-ATPase) partially inhibited oxalate-stimulated Ca²⁺ transport activity; however, it had no effect on calmodulin-stimulated Ca²⁺ uptake driven by ATP or GTP. The results would suggest the presence of two types of Ca²⁺-ATPases, an endoplasmic reticulum- and a plasma membrane-type. Interestingly, incubation of membranes with [gamma³²P]ATP resulted in the formation of a single acyl [³²P]phosphoprotein of 120 kilodaltons. Formation of this phosphoprotein was dependent on Ca²⁺, but independent of Mg²⁺. Its enhancement by La³⁺ is characteristic of a phosphorylated enzyme intermediate of a plasma membrane-type Ca-ATPase. Calmodulin stimulated Ca²⁺ transport was decreased by W-7 (a calmodulin antagonist), ML-7 (myosin light chain kinase inhibitor) or thyroxine. Acidic phospholipids, like phosphatidylserine, stimulated Ca²⁺ transport, similar to their effect on the erythrocyte plasma membrane Ca²⁺-ATPase. These results would indicate that the calmodulin-stimulated Ca²⁺ transport originated in large part from a plasma membrane-type Ca²⁺ pump of 120 kilodaltons. The possibility of calmodulin-stimulated Ca²⁺-ATPases on endomembranes, such as the endoplasmic reticulum and secretory vesicles, as well as the plasma membrane is suggested.     

Activatory effect of regucalcin on hepatic plasma membrane (Ca2+-Mg2+)-ATPase is impaired by liver injury with carbon tetrachloride administration in rats

The alteration of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver of rats administered orally carbon tetrachloride (CCl₄) solution was investigated. Rats received a single oral administration of CCl₄ (10, 25 and 50%, 1.0 ml/100 g body weight), and 3 or 24 h later they were sacrificed. CCl₄ administration caused a remarkable elevation of liver calcium content and a corresponding increase in liver plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity, indicating that the increased Ca²⁺ pump activity is partly involved in calcium accumulation in liver cells. Moreover, the participation in regucalcin, which is an intracellular activating factor on the enzyme, was examined by using anti-regucalcin IgG. The plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity increased by CCl₄ administration was not entirely inhibited by the presence of anti-regucalcin IgG (1.0 and 2.5 ug/ml) in the enzyme reaction mixture. However, the effect of regucalcin (0.25–1.0 uM) to activate (Ca²⁺-Mg²⁺)-ATPase in the liver plasma membranes of normal rats was not revealed in the liver plasma membranes obtained from CCl₄-administered rats. Also, the effect of regucalcin was not seen when the plasma membranes were washed with 1.0 mM EGTA, indicating that the disappearance of regucalcin effect is not dependent on calcium binding to the plasma membranes due to liver calcium accumulation. Now, the presence of dithiothreitol (5 mM) or heparin (20 ug/ml) caused a remarkable elevation of the plasma membrane (Ca²⁺-Mg²⁺)-ATPase activity in the liver obtained from CCl₄-administered rats. Thus, the regucalcin effect differed from that of dithiothreitol or heparin. The present study suggests that the impairment of regucalcin effect on Ca²⁺ pump activity in liver plasma membranes is partly contribute to hepatic calcium accumulation induced by liver injury with CCl₄ administration.     

Substrate specificity of the erythrocyte Ca2+-ATPase

In the absence of Mg²⁺, the observed activity of the erythrocyte plasma membrane Ca²⁺-ATPase is due to the hydrolysis of CaATP at a low rate. In the presence of Mg²⁺, the activity of the enzyme is much higher, but it is inhibited by high levels of free Mg²⁺. This inhibition appears to be due to competition of Mg²⁺ and Ca²⁺ for a site on the enzyme, rather than for ATP.