Properties of a bicarbonate-stimulated ATPase from rat uterus

Crude subcellular fractions from rat uterus contain a HCO₃⁻-stimulated Mg²⁺-ATPase with properties analogous to those previously reported for the enzyme in gastric mucosa, pancreas, salivary gland and liver lysosome. Estradiol-17β treatment of ovariectomized rats resulted in an increase in uterine mitochondrial (HCO₃⁻ + Mg²⁺)-ATPase and Mg²⁺-ATPase activity. In an early response (105 min) to estradiol-17β treatment of ovariectomized rats, the lysosomal enzyme, β-N-acetylglucosaminidase increased in the nuclear and mitochondrial fractions and decreased in the microsomal and supernatant fractions.     

Effect of Glycine and Strychnine on Cl–-Activated Mg2+-ATPase from Bream Brain Microsomes (Abramis bramaL.)

The effect of glycine and strychnine on Mg²⁺-ATPase from the microsomal fraction of the bream (Abramis bramaL.) brain was studied. The glycine in the concentration range 10^–7–10^–4M activates the enzyme. The effect of glycine on Mg²⁺-ATPase is obviated by 100 M strychnine. The strychnine in the concentration range 5–90 M activates the basal Mg²⁺-ATPase but decreases the effect of the enzyme activation by 10^–4M glycine. The effect of Cl^–on Mg²⁺-ATPase depends on the substrate concentration (Mg²⁺-ATP) and is not observed in the presence of 100 M strychnine. A receptor-dependent pathway of glycine and strychnine action on Cl^–-activated Mg²⁺-ATPase from bream brain microsomes is proposed.     

Melafen effect on ATP-dependent accumulation of calcium in plasma membrane vesicles from cells of potato tubers

Synthetic growth regulator melafen (10⁻⁵–10⁻¹⁰ M) was tested for aneffect on the Ca²⁺ accumulation in plasma membrane vesicles (PMVs) isolated from potato Solanum tuberosum L. tubers at forced rest and sprouting. Melafen proved to regulate the Ca²⁺ accumulation in PMVs by changing the activity of Ca²⁺, Mg²⁺-ATPase of the plasma membrane, while no effect was observed with respect to Ca²⁺ outflow from vesicles. The melafen effect on Ca²⁺, Mg²⁺-ATPase activity depended on the physiological condition of tubers and the melafen concentration.     

Effect of Activators and Blockers of Ligand-Regulated Ion Channels on the Activity of the Cl−-Stimulated Mg2+-ATPase of the Plasma Membrane Fraction from Bream (Abramis brama L.) Brain

Effects of GABA, glycine, acetylcholine, and glutamate (agonists of the GABA_a/benzodiazepine, glycine, choline, and glutamate receptors, respectively) at concentrations in the range 10^–8-10^–4 M on the activity of basal Mg²⁺-ATPase of the plasma membrane fraction from bream brain and on its activation by Cl^– were investigated. GABA and glycine activated basal Mg²⁺-ATPase activity and suppressed its activation by Cl^–. Acetylcholine and glutamate activated basal Mg²⁺-ATPase to a lesser extent and did not suppress the activation of the enzyme by Cl^–.The activation of basal Mg²⁺-ATPase by neuromediators was decreased by blockers of the corresponding receptors (picrotoxin, strychnine, benztropine mesylate, and D-2-amino-5-phosphonovaleric acid). In addition, picrotoxin and strychnine eliminated the inhibiting effect of GABA and glycine, respectively, on the Cl^–-stimulated Mg²⁺-ATPase activity. Agonists of the GABA_a/benzodiazepine receptor–phenazepam (10^–8-10^–4 M) and pentobarbital (10^–6-10^–3 M)–activated the basal Mg²⁺-ATPase activity and decreased the Cl^–-stimulated Mg²⁺-ATPase activity. The dependence of both enzyme activities on ligand concentration is bell-shaped. Moreover, phenazepam and pentobarbital increased the basal Mg²⁺-ATPase activity in the presence of 10^–7 M GABA and did not influence it in the presence of 10^–4 M GABA and 10^–6 M glycine. The data suggest that in the fish brain membranes the Cl^–-stimulated Mg²⁺-ATPase interacts with GABA_a/benzodiazepine and glycine receptors but not with m-choline and glutamate receptors.     

ATP Synthase of Chloroplast Thylakoid Membranes: A More in Depth Characterization of Its ATPase Activity

In contrast to everted mitochondrial inner membrane vesicles and eubacterial plasma membrane vesicles, the ATPase activity of chloroplast ATP synthase in thylakoid membranes is extremely low. Several treatments of thylakoids that unmask ATPase activity are known. Illumination of thylakoids that contain reduced ATP synthase (reduced thylakoids) promotes the hydrolysis of ATP in the dark. Incubation of thylakoids with trypsin can also elicit higher rates of ATPase activity. In this paper the properties of the ATPase activity of the ATP synthase in thylakoids treated with trypsin are compared with those of the ATPase activity in reduced thylakoids. The trypsin-treated membranes have significant ATPase activity in the presence of Ca²⁺, whereas the Ca²⁺-ATPase activity of reduced thylakoids is very low. The Mg²⁺-ATPase activity of the trypsinized thylakoids was only partially inhibited by the uncouplers, at concentrations that fully inhibit the ATPase activity of reduced membranes. Incubation of reduced thylakoids with ADP in Tris buffer prior to assay abolishes Mg²⁺-ATPase activity. The Mg²⁺-ATPase activity of trypsin-treated thylakoids was unaffected by incubation with ADP. Trypsin-treated membranes can make ATP at rates that are 75–80% of those of untreated thylakoids. The Mg²⁺-ATPase activity of trypsin-treated thylakoids is coupled to inward proton translocation and 10 mM sulfite stimulates both proton uptake and ATP hydrolysis. It is concluded that cleavage of the γ subunit of the ATP synthase by trypsin prevents inhibition of ATPase activity by the ε subunit, but only partially overcomes inhibition by Mg²⁺ and ADP during assay.     

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.     

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.     

Regucalcin modulates hormonal effect on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes

The interaction of various hormones and regucalcin on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of epinephrine (10^–6–10^–4 M), and insulin (10^–8–10^– M) in the reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity, while the enzyme activity was decreased significantly by calcitonin, (3×10^–8–3×10^–6 M). These hormonal effects, except for calcitonin, were clearly inhibited by the presence of vanadate (10^–4 M) which can inhibit the Ca²⁺-dependent phosphorylation of enzyme. Meanwhile, regucalcin (0.25 and 0.50 M), isolated from rat liver cytosol, elevated significantly (Ca²⁺–Mg²⁺)-ATPase activity in the plasma membranes, although this elevation was not inhibited by vanadate (10^–4 M). the epinephrine (10^–5 M) or phenylephrine (10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was disappeared in the presence of regucalcin; in this case the effect of regucalcin was also weakened. However, the inhibitory effect of calcitonin (3×10^–6 M) was not weakened by the presence of regucalcin (0.5 M). Moreover, GTP (10^–5 and 10^–4 M)-induced increase in (Ca²⁺–Mg²⁺)-ATPase activity was not seen in the presence of regucalcin (0.25 M). The present finding suggests that the activating mechanism of regucalcin on (Ca²⁺–Mg²⁺)-ATPase is not involved on GTP-binding protein which modulates the receptor-mediated hormonal effect in rat liver plasma membranes.     

Increase in calcium content and Ca2+-ATPase activity in the brain of fasted rats: Comparison with different ages

The effect of fasting on calcium content and Ca²⁺-ATPase activity in the brain tissues of 5 weeks and 50 weeks old rats was investigated. Brain calcium content and Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of the brain homogenate from young and elderly rats were significantly increased by overnight–fasting. These increases were appreciably restored by a single oral administration of glucose solution (400 mg/100 g body weight) to fasted rats. In comparison with young and elderly rats, brain calcium content and microsomal Ca²⁺-ATPase activity were significantly elevated by increasing ages. The effect of ageing was not seen in the brain mitochondrial Ca²⁺-ATPase activity. When calcium (50 mg/100 g) was orally administered to young and elderly rats, brain calcium content was significantly elevated. The calcium administration–induced increase in brain calcium content was greater in elderly r crease in Ca²⁺-ATPase activity in the microsomal and mitochondrial fractions of brain homogenates from young rats. In aged rats, the microsomal Ca²⁺-ATPase activity was not further enhanced by calcium administration, although the mitochondrial enzyme activity was significantly raised. The present study demonstrates that the fasting–induced increase in brain calcium content is involved in Ca²⁺-ATPase activity raised in the brain microsomes and mitochondria of rats with different ages, supporting a energy–dependent mechanism in brain calcium accumulation.     

Effects of fatty acids on calcium-stimulated adenosine triphosphatase in rat submandibular gland microsomes.

Effects of fatty acids on Ca²⁺-ATPase and Mg²⁺-ATPase in the microsomal fraction of rat submandibular gland have been investigated. Saturated fatty acids had almost no effect, but unsaturated fatty acids inhibited both ATPases. Modes of inhibition by linoleic acid were as follows: competitive for calcium and ATP with Ca²⁺-ATPase; non-competitive for magnesium and ATP with Mg²⁺-ATPase     

Effects of cortisol and fluoride on ion-transporting ATPase activities in cultured osteoblastlike cells

Summary Na⁺,K⁺-ATPase, HCO ₃ ⁻ -ATPase, Ca²⁺,Mg²⁺,-ATPase, Ca²⁺-ATPase, and alkaline phosphatase activities were measured in cultures of osteoblastlike cells treated with fluoride and cortisol separately and in combinations. Low concentrations of cortisol increased HCO ₃ ⁻ -ATPase (10⁻¹¹ to 10⁻¹⁸ M cortisol) and alkaline phosphatase (10⁻¹¹ to 10⁻⁹ M cortisol) activities, but higher cortisol concentrations reduced these activities. Na⁺,K⁺-ATPase, Ca²⁺,Mg²⁺-ATPase, and Ca²⁺-ATPase activities tended only to be reduced by cortisol. Fluoride (10⁻⁶ and 5×10⁻⁶ M) increased HCO ₃ ⁻ -ATPase and alkaline phosphatase activities, but these activities were similar to controls in the presence of 10⁻⁵ M fluoride. Ca²⁺,Mg²⁺-ATPase activity was decreased and Na⁺,K⁺-ATPase activity was increased as the concentration of fluoride increased (10⁻⁶ to 10⁻⁵ M). Preliminary experiments with fluoride indicated that lower concentrations (10⁻⁷ M) were without effect. Cortisol concentrations of 10⁻⁹ and 10⁻⁸ M were chosen for studies with combinations of cortisol and fluoride because the effects of these concentrations on alkaline phosphatase activity were opposite, i.e. 10⁻⁹ M increased whereas 10⁻⁸ M decreased activity. Fluoride concentrations of 10⁻⁶, 5×10⁻⁶, and 10⁻⁵ M were chosen because a peak of alkaline phosphatase activity occurred at 5×10⁻⁶ M fluoride. Higher (10⁻⁴ M) and lower (10⁻⁷ M) fluoride concentrations were without effect. The effects of combinations of cortisol and fluoride depend on the enzyme activity measured. Fluoride (10⁻⁶ M) combined with cortisol (10⁻⁹ M) produced a peak of Na⁺,K⁺-ATPase activity. The increased activity obtained with all concentrations of fluoride alone was preserved when fluoride was combined with 10⁻⁸ M cortisol, although the activity tended to be reduced at 5×10⁻⁶ and 10⁻⁵ M fluoride. HCO ₃ ⁻ -ATPase activity was increased by fluoride combined with 10⁻⁸ M cortisol and decreased by fluoride combined with 10⁻⁹ M cortisol compared to the activities obtained with fluoride alone. The decrease in Ca²⁺,Mg²⁺-ATPase activity caused by fluoride alone was prevented by 10⁻⁹ and enhanced by 10⁻⁸ M cortisol, although all treatments produced the same activity at 10⁻⁵ M fluoride. Ca²⁺-ATPase activity tended to be increased by combinations of fluoride and cortisol, but significantly so only at 10⁻⁵ M fluoride in combinations with 10⁻⁸ and 10⁻⁹ M cortisol. Alkaline phosphatase activity was increased by fluoride combined with 10⁻⁹ M cortisol and decreased by fluoride combined with 10⁻⁸ M cortisol compared to the activities obtained with fluoride alone. These results suggest that the abilities of bone cells to regulate ion transport (as reflected in their ion-transporting ATPase activities) are modulated by glucocorticoids and fluoride. Inasmuch as these cells may regulate the ionic composition and concentrations of the bone extracellular fluid (ECF) in vivo, the modulation of their activities by cortisol and fluoride may result in altered bone ECF composition. This work was supported by Grant NAG-2-108 from the National Aeronautics and Space Administration, D.C., and Grant PO1 NS15767 from the National Institute of Neurological and Communicative Disorders and Stroke, Bethesda, MD.     

Stimulatory effect of hormonal signaling factors on (Ca2+−Mg2+)-ATPase activity in rat liver plasma membranes: Cross talk with regucalcin

The effect of hormonal signaling factors on (Ca²⁺–Mg²⁺)-ATPase activity in rat liver plasma membranes was investigated. The presence of inositol-glycan (10^–7–10^–5M), dibutyryl cAMP (10^–4 and 10^–3M) or inositol 1,4,5-trisphosphate (IP₃; 10^–6 and 10^–5 M) in the enzyme reaction mixture produced a significant increase in (Ca²⁺–Mg²⁺)-ATPase activity. These effects were completely inhibited by the presence of vanadate (10^–4 M), an inhibitor of the enzyme phosphorylation, and N-ethylmaleimide (5×10^–3 M), a SH group modifying reagent. Meanwhile, regucalcin, a Ca²⁺-binding protein isolated from rat liver cytosol, increased the enzyme activity by binding to the SH groups of (Ca²⁺–Mg²⁺)-ATPase in liver plasma membranes. The presence of regucalcin (0.25 M) with an effective concentration completely inhibited the effect of inositol-glycan (10^–5 M) to increase (Ca²⁺–Mg²⁺)-ATPase activity, while the effect of dibutyryl cAMP (10^–3M) or IP₃ (10^–5M) was not altered. The inositol-glycan effect was not modulated by the presence of dibutyryl cAMP or IP₃. Now, the preincubation of the plasma membranes with regucalcin did not modify the effect of inositol-glycan on the enzyme activity, suggesting that regucalcin competes with inositol-glycan for the binding to the plasma membranes. The present results suggest that there may be a cross talk with regucalcin and hormonal signaling factors in the regulation of (Ca²⁺–Mg²⁺)-ATPase activity in liver plasma membranes.     

The in vivo inhibition of transport enzyme activities by chloroquine in different organs of rat is reversible

The antimalarial drug chloroquine is found to inhibit Na⁺, K⁺-ATPase, Ca²⁺, Mg²⁺-ATPase, Ca²⁺-ATPase, pNPPase and acetylcholinesterase activities in different organs of rat in vivo when injected for a certain periods of time. The inhibition seems to be due to the changes in the level of phospholipid, cholesterol and the fatty acid of the lipid and the alteration of the fluidity of the microsomal membranes. However, the enzyme activities return to the normal level in about 2–3 weeks after the discontinuation of the drug suggesting that the drug effect is reversible.     

Properties of Cl−-Stimulated Mg2+-ATPase Sensitive to Ligands of Inhibitory Receptors in Brain Plasma Membranes of the Bream Abramis brama

Properties of Cl^–-stimulated Mg²⁺-ATPase in the brain plasma membranes of the bream Abramis brama L. were studied; this enzyme is composed of basal Mg²⁺-ATPase activity that can be stimulated by 40–80% by Cl^– ions (Cl^–-ATPase). These anions stimulate the basal Mg²⁺-ATPase starting with 8 mM concentration, their maximal effect being observed at a concentration of 30–100 mM. The Cl^–-ATPase activity was found at a low molarity of HEPES-Tris buffer (< 30 mM) but was not revealed at a high molarity (> 30 mM). The basal Mg²⁺-ATPase activity was detected in the whole studied pH range (5.5–9.0), with maximum at pH 7.2–7.8 values, whereas optimum to reveal Cl^–-ATPase was at high and low H⁺ concentrations (pH 6.0 and 8.5, respectively). At physiological pH values (7.2–7.5) the Cl^–-ATPase activity was not revealed, but was detected after preincubation of the enzyme with 10 µM GABA. The basal Mg²⁺-ATPase, like Cl^–-ATPase, hydrolyzed ATP with a maximal rate, while CTP, ITP, and ADP only slightly, and did not hydrolyze GTP and AMP. The Cl^–-ATPase activity decreased in the presence of divalent cations in the following order: Mg²⁺ > Co²⁺ > Mn²⁺ = Cd²⁺ > Al³⁺ = Cu²⁺, and it was not found in the presence of Ca²⁺ and Zn²⁺. Anions of halogen series activated the basal Mg²⁺-ATPase in the descending order: Cl^– > Br^– > J^– > F^–. Among other monovalent anions, HCO₃ ^– activated the enzyme, NO₃ ^– practically had no effect, and SCN^– inhibited its activity. Blockers of Cl^– transport (ethacrinic acid, furosemide, and SITS) and GABA-receptor ligands (pentobarbital, diazepam, and picrotoxin) suppressed the enzyme activity. Out of SH-reagents, PCMB inhibited the enzyme, while NEM did not affect it. The H⁺-ATPase blocker oligomycin inhibited the enzyme, while the blocker of Na⁺,K⁺-ATPase ouabain and the blocker of Ca²⁺,Mg²⁺-ATPase ruthenium red had no effect. The properties of the Cl^–-stimulated Mg²⁺-ATPase of fish brain are discussed in comparison with those of the rat brain Cl^–-ATPase. The conclusion is made that the bream brain enzyme differs markedly from Cl^–-ATPase (the ATP-dependent Cl^–-pump) of mammalian brain.     

Effects of delta9-tetrahydrocannabinol on ATPases in mouse brain subcellular fractions.

The effect of (?)-Δ⁹-THC on the activities of Mg²⁺?, Na⁺?K⁺? and Mg²⁺Ca²⁺-ATPases were studied in mouse brain subcellular fractions. $\text{In vitro}$treatment with Δ⁹-THC produced a dose dependent stimulation of Mg²⁺ ATPase in the crude mitochondrial fraction and its subfractions and a dose-related inhibition of this activity in the microsomal fraction. Na⁺-K⁺- and Mg²⁺-Ca²⁺-ATPase activities were inhibited in a dose-related manner in all subcellular fractions studied.     

The effect of glucagon on the kinetics of hepatic mitochondrial calcium uptake

Summary Previous work by this and other laboratories has shown that glucagon administration stimulates calcium uptake by subsequently isolated hepatic mitochondria. This stimulation of hepatic mitochondrial Ca²⁺ uptake byin vivo administration of glucagon was further characterized in the present report. Maximal stimulation of mitochondrial Ca²⁺ accumulation was achieved between 6–10 min after the intravenous injection of glucagon into intact rats. Under control conditions, Ca²⁺ uptake was inhibited by the presence of Mg²⁺ in the incubation medium. Glucagon treatment, however, appeared to obliterate the observed inhibition by Mg²⁺ of mitochondrial Ca²⁺ uptake. Kinetic experiments revealed the usual sigmoidicity associated with initial velocity curves for mitochondrial calcium uptake. Glucagon treatment did not alter this sigmoidal relationship. Glucagon treatment significantly increased the V_max for Ca²⁺ uptake from 292±22 to 377±34 nmoles Ca²⁺ /min per mg protein (n=8) but did not affect the K_0.5, (6.5–8.6 μM). Since the major kinetic change in mitochondrial Ca²⁺ uptake evoked by glucagon is an increase in V_max, the enhancement mechanism is likely to be an increase either in the number of active transport sites available to Ca²⁺ or in the rate of Ca²⁺ carrier movement across the mitochondrial membranes.     

Effect of cadmium on ciliary and ATPase activity in the gills of freshwater mussel Anodonta cygnea

1. Cadmium (≤ 50 μM) decreases the heat resistance (39°C) of the activity of frontal cilia in the Anodonta cygnea gills incubated in dechlorinated tap water, while in the presence of added 2 mM Ca²⁺ the minimal acting concentration of cadmium rises up to 100 μM.2. The inhibitory effect of Cd²⁺ (1.5 mM) on the ATPase activity measured in the gill microsomal fraction is temperature dependent and increases as follows: ouabain insensitive Na²⁺- or K⁺-ATPase (no inhibition), Ca²⁺-ATPase (50% inhibition), Mg²⁺-ATPase (100% inhibition).3. Cadmium itself (≤ 50 μM) added to microsomal suspension stimulates the H⁺-sensitive ATP hydrolysis resembling on its pH-dependence the Mg²⁺- but not Ca²⁺-ATPase activity.4. Cd²⁺ can mimic the effect of Mg²⁺ as a cofactor required for activation of the ouabain-insensitive Na⁺- or K⁺-ATPase. Monovalent cations fail to activate the ATPase when Mg²⁺ is substituted by Ca²⁺.5. One of the mechanisms underlying the toxicity of Cd²⁺ to Anodonta gills could be based upon an interaction of Cd²⁺ with Mg²⁺-ATPase followed by suppression of the ciliary activity.     

Activating effect of regucalcin on (Ca2+-Mg2+)-ATPase in rat liver plasma membranes: relation to sulfhydryl group

The activating mechanism of regucalcin, a calcium-binding protein isolated from rat liver cytosol, on (Ca²⁺–Mg²⁺)-ATPase in the plasma membranes of rat liver was investigated. (Ca²⁺–Mg²⁺)-ATPase activity was markedly increased by a sulfhydryl (SH) group protecting reagent dithiothreitol (DTT; 2.5 and 5 mM as a final concentration), while the enzyme activity was significantly decreased by a SH group modifying reagent N-ethylmaleimide (NEM; 0.5–5 mM). The effect of DTT (5 mM) to increase the enzyme activity was clearly blocked by NEM (5 mM). Regucalcin (0.25–1.0 M) significantly increased (Ca²⁺-Mg²⁺)-ATPase activity. This increase was completely blocked by NEM (5 mM). Meanwhile, digitonin (0.04%), which can solubilize the membranous lipids, significantly decreased (Ca²⁺–Mg²⁺)-ATPase activity. Digitonin did not have an effect on the DTT (5 mM)-increased enzyme activity. However, the effect of regucalcin (0.25 M) increasing (Ca²⁺–Mg²⁺)-ATPase activity was entirely blocked by the presence of digitonin. The present results suggest that regucalcin activates (Ca²⁺–Mg²⁺)-ATPase by the binding to liver plasma membrane lipids, and that the activation is involved in the SH groups which are an active site of the enzyme.     

The effect of ionic strength on a Mg2+-ATPase and its relevance to the determination of (Na++K+)-ATPase

A ouabain-insensitive Mg²⁺-ATPase present in a microsomal fraction prepared from the dog submandibular gland was studied. This Mg²⁺-ATPase was inhibited by increasing concentrations of NaCl, KCl, RbCl and CsCl. The addition of an osmotically equal amount of sucrose was without effect. This inhibition was obtained over a pH range of from 6.3 to 8.8. The Mg²⁺-ATPase present in microsomes treated with NaI showed a similar inhibition. These results indicate that it is advisable to keep the ionic strength constant in solutions used to obtain (Na⁺+K⁺)-ATPase activities.     

Effect of Naloxone on the Activity of Cl–-Activated Mg2+-ATPase from Plasma Membrane Fraction of Bream Brain (Abramis brama L.) in the Presence of GABAa-ergic Substances

We studied the effect of naloxone—an antagonist of the opioid receptors—on sensitivity of Cl^–-activated Mg²⁺-ATPase from the plasma membrane fraction of bream brain (Abramis brama L.) to GABA_a-ergic substances. Preincubation of the plasma membranes with 1–100 M naloxone increased the basal Mg²⁺-ATPase activity and suppressed its activation by chloride ions. The same effects were observed in the presence of the agonists of GABA_a/benzodiazepine receptors: 0.1–100 M GABA, 1–500 M pentobarbital, and 0.1–100 M phenazepam. Naloxone (10 M) inhibited activation of the basal Mg²⁺-ATPase by the studied ligands and restored the enzyme sensitivity to Cl^–. However, the effect of naloxone was not observed in the presence of high concentrations of pentobarbital (500 M) and phenazepam (100 M). The obtained data show that naloxone modulates the activity of Cl^–-activated Mg²⁺-ATPase from the plasma membranes of bream brain and antagonizes the GABA_a receptor ligands.