Phenobarbital modulates the (Na+, K+)-stimulated ATPase and Ca2+-stimulated ATPase activities by increasing the bilayer fluidity of dog brain synaptosomal plasma membranes

The binding of [14C]phenobarbital into synaptosomal plasma membranes of dog brain follows a sigmoid path. The "best fit" curve of this binding is the one described by the Hill equation (r2 less than 0.93 and Hill coefficient, n = 1.32). (Na+, K+)-stimulated ATPase and Ca2+-stimulated ATPase activities are modulated by phenobarbital. Arrhenius plots of (Na+, K+, Mg2+)-dependent ATPase revealed that phenobarbital (2 mM) lowered the transition temperature and altered the Arrhenius activation energies of this enzyme. The allosteric inhibition by F- of the (Na+, K+)-stimulated ATPase was studied in control and phenobarbital-treated membranes. The lowering of the transition temperature and changes in Arrhenius activation energy about the transition temperature in combination with changes observed in the allosteric properties of the (Na+, K+)-stimulated ATPase by F-, produced by phenobarbital, would be expected if it is assumed that phenobarbital "fluidizes" synaptosomal plasma membranes.     

Cortisol effect on (Na++K+)-stimulated ATPase activity and on bilayer fluidity of dog brain synaptosomal plasma membranes

The binding of [¹⁴C]cortisol into dog brain synaptosomal plasma membranes (SPM) follows an exponential path described by the general formula y=a.e^bx. The specific activity of the SPM-bound (Na⁺+K⁺)-stimulated ATPase was linearly increased at different concentrations of cortisol. Changes in the allosteric properties of (Na⁺+K⁺)-stimulated ATPase by fluoride (F^–) (i. e. changes of Hill coefficients) indicate that cortisol increases the membrane fluidity. The fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene-labeled SPM decreased in cortisol treated SPM compared to untreated (control) SPM, which is consistent with a general increase in membrane fluidity. This increase of fluidity by cortisol may play a role in the physiological effects of this hormone in the brain.     

A Ca2+-stimulated ATPase activity in rabbit neutrophil membranes

An ATPase activity specifically stimulated by micromolar Ca²⁺ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca²⁺-ATPase activity with regard to neutrophil function.     

A Ca2+-stimulated ATPase activity in rabbit neutrophil membranes.

An ATPase activity specifically stimulated by micromolar Ca2+ concentrations has been identified in association with rabbit neurophil membranes. These studies provide the basis of further characterization of the Ca2+-ATPase activity with regard to neutrophil function.     

Ca2+-stimulated, Mg2+-dependent ATPase in bovine thyroid plasma membranes

An isolated plasma membrane fraction from bovine thyroid glands contained a Ca2+-stimulated, Mg2+-dependent adenosine triphosphatase ((Ca2+ + Mg2+)-ATPase) activity which was purified in parallel to (Na+ + K+)-ATPase and adenylate cyclase. The (Ca2+ + Mg2+)-ATPase activity was maximally stimulated by approx. 200 microM added calcium in the presence of approx. 200 microM EGTA (69.7 +/- 5.2 nmol/mg protein per min). In EGTA-washed membranes, the enzyme was stimulated by calmodulin and inhibited by trifluoperazine.     

High-affinity Ca2+-stimulated and Mg2+-dependent ATPase from rat osteosarcoma plasma membranes

Transplantable rat osteosarcoma plasma membrane preparations contain high-affinity and low-affinity calcium-stimulated ATPases. The high-affinity enzyme displayed a K0.5 for calcium of 0.03 microM, a Vmax of 99.2 nmol/min/mg, and a requirement for magnesium ions. It was not inhibited by 20 microM trifluoperazine nor stimulated by the addition of 2 ng of calmodulin. Lack of stimulation with exogenous calmodulin may be related to the high endogenous calmodulin content of the membrane preparations. The low-affinity Ca2+- or Mg2+-ATPase displayed a K0.5 for calcium of approximately 2.40 mM (Vmax of 185 nmol/min/mg) and a K0.5 for magnesium of approximately 2.75 mM (Vmax of 250 nmol/min/mg).     

Opiates inhibit calmodulin activation of a high-affinity Ca2+-stimulated Mg2+-dependent ATPase in synaptic membranes

A high affinity Ca²⁺/Mg²⁺ ATPase has been identified and localized in synaptic membrane subfractions. This enzyme is stimulated by low concentrations of Ca²⁺ (1 M) believed to approximate the range of Ca²⁺ in the synaptosomal cytosol (0.1 to 5.0 M). The opiate agonist levorphanol, in a concentration-dependent fashion, inhibited Ca²⁺-stimulated ATP hydrolysis in lysed synaptic membranes. This inhibition was reversed by naloxone, while dextrorphan, the inactive opiate isomer, was without effect. Inhibition by levorphanol was most pronounced in a subfraction of synaptic membranes (SPM-1). The inhibition of Ca²⁺-stimulated ATP hydrolysis was characterized by a reduction inV _max for Ca²⁺. Levorphanol pretreatment reduced the Hill coefficient (H_N) of 1.5 to 0.7, suggesting cooperative interaction between the opiate receptor and the enzyme protein. Levorphanol, but not dextrorphan, also inhibited (28%) ATP-dependent Ca²⁺ uptake by synaptic membranes. Opiate ligand stereoisomers were tested for their effects on calmodulin stimulating of high affinity Ca²⁺/Mg²⁺ ATPase in synaptic membranes. Levorphanol (10 M), but not the inactive stereoisomer (+)dextrorphan, significantly inhibited (35%) the calmodulin-activated Ca²⁺-dependent ATP hydrolysis activity in a preparation of lysed synaptic membranes. Both Ca²⁺-dependent and calmodulin-dependent stimulation of the enzyme in the presence of optimal concentrations of the other co-substrate were inhibited by levorphanol (35–40%) but not dextrorphan. Inhibition of ATP hydrolysis was characterized by a reduction inV _max for both Ca²⁺ and calmodulin stimulation of the enzyme. Calmodulin stimulation of enzyme activity was most pronounced in SPM-1, the membrane fraction which also exhibits the maximal opiate inhibition (40%) of the Ca²⁺-ATPase. The results demonstrate that opiate receptor activation inhibits a high affinity Ca²⁺/Mg²⁺ ATPase in synaptic plasma membranes in a stereospecific fashion. The inhibition of the enzyme may occur by a mechanism involving both Ca²⁺ and calmodulin. Inhibition of calmodulin activation may contribute to the mechanism by which opiate ligands disrupt synaptosomal Ca²⁺ buffering mechanisms. Changes in the cytosolic distribution of synaptosomal Ca²⁺ following inhibition of Ca²⁺/Mg²⁺ ATPase may underlie some of the pharmacological effects of opiate drugs.     

Effects of chelating agents on the Ca2+-stimulated ATPase of rat liver plasma membranes

Using strictly controlled ionic conditions we have demonstrated, in agreement with previous findings (Lotersztajn et al. (1981) J. Biol. Chem. 256, 11209-11215; Lotersztajn, S. and Pecker, F. (1982) J. Biol. Chem. 257, 6638-6641) a Ca2+-stimulated ATPase in rat liver plasma membranes which is detectable at low free Mg2+ concentrations (normally fulfilled by endogenous levels) but not at free Mg2+ concentrations greater than about 10(-5) M. The findings reported here also suggest that this (Ca2+ + Mg2+)-ATPase is activated by EGTA or one of its liganded species. Furthermore, this is probably an intrinsic property of the enzyme as it was found to be independent of the isolation technique. The stimulation by EGTA appears to be a function both of free Ca2+ concentration and of one or more liganded species of EGTA and it is also inhibited at high free Mg2+ concentrations (approx. 10(-5) M). The specificity of the EGTA effect on ATPase activity is studied with respect to other, widely used, chelating agents namely HEEDTA, EDTA and CDTA. Of these, only CDTA shares the effect, although the concentration dependence of the activation is different from EGTA, suggesting that there is some degree of structural specificity involved rather than a generalised effect of complexed Ca2+.     

A protein activator of Mg2+-dependent, Ca2+-stimulated ATPase in human erythrocyte membranes distinct from calmodulin

The intracellular localization of aryl acylamidase (aryl-acylamide amidohydrolase, EC 3.5.1.13) in chicken kidney was investigated. By separation on density gradients of the silica sol Ludox AM, the enzyme was localized in the mitochondrial fraction. This mitochondrial fraction was shown to be substantially free of lysosomal contamination. Subfractionation of the purified mitochondria indicates that the enzyme is located on the outer membrane, can be solubilized, and may be a suitable marker enzyme for kidney mitochondria.     

Effects of in vivo ethanol administration on Ca2+/Mg2+ ATPase and ATP-dependent Ca2+ uptake activity in synaptosomal membranes

1. Acute administration of ethanol (4 g/kg, i.p.) to mice inhibits the sequestration of calcium into endoplasmic reticulum-like organelles in synaptosomal membranes.
2. Ethanol administration inhibits both Ca²⁺-stimulated adenosine triphosphate hydrolysis and ATP-dependent calcium uptake in the vesicles at time of loss of righting reflex.
3. At recovery of righting reflex, the Ca²⁺-ATPase activity returns to normal levels, while the ATP-dependent uptake remains inhibited.
4. The effect of ethanol is specific for the sequestration (active transport) of calcium since calcium binding to synaptic membranes is not altered.
5. Alteration in mechanisms responsible for synaptosomal buffering of cytosolic Ca²⁺ levels by in vivo ethanol may contribute to altered transmitter release rates following ethanol adminstration.

     

A direct colorimetric assay for Ca2+ -stimulated ATPase activity

A simple and rapid colorimetric assay for measuring the high affinity Ca2+-ATPase activity in subcellular fractions is presented. With this method a one-step addition of a malachite green/molybdate/polyvinyl alcohol reagent to the assay mixture at the end of the incubation period is all that is required for the spectrophotometric quantification of the phosphomolybdate-malachite green complex. The presence of polyvinyl alcohol allows the quantification of released phosphate without having to separate it from protein. We have validated this assay by characterizing the high affinity Ca2+-ATPase activity in isolated rat liver microsomes. Comparable Ca2+-ATPase activities in rat liver microsomes and adipocyte plasma membranes were found when measured with this colorimetric assay and an isotopic assay. This method is applicable to the measurement of other types of ATPase activities.     

Ouabain-insensitive Na+-stimulated ATPase activity of basolateral plasma membranes from guinea-pig kidney cortex cells: II. Effect of Ca2+

The ouabain-insensitive, Mg²⁺-dependent, Na⁺-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca²⁺ to the assay medium. The Ca²⁺ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily ‘deactivated’ by reducing the free Ca²⁺ concentration of the assay medium to values lower than 1 μM; and as a stable component, which can be ‘deactivated’ by preincubating the membranes for periods of 3–4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca²⁺. The $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca²⁺ on the Na⁺-stimulated ATPase is on the $\text{V}{}_{\mathrm{<mtext>max</mtext>}}$, and not on the $\text{K}{}_{\mathrm{<mtext>a</mtext>}}$ of the system for Na⁺. The activating effect of Ca²⁺ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca²⁺ concentration may modulate the ouabain-insensitive, Na⁺-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na⁺ accompanied by Cl^? and water that these cells show, and to which the Na⁺-ATPase has been associated as being responsible for the energy supply of this mode of Na⁺ extrusion.     

Ouabain-insensitive Na+-stimulated ATPase activity of basolateral plasma membranes from guinea-pig kidney cortex cells. II. Effect of Ca2+

The ouabain-insensitive, Mg2+-dependent, Na+-stimulated ATPase activity present in fresh basolateral plasma membranes from guinea-pig kidney cortex cells (prepared at pH 7.2) can be increased by the addition of micromolar concentrations of Ca2+ to the assay medium. The Ca2+ involved in this effect seems to be associated with the membranes in two different ways: as a labile component, which can be quickly and easily 'deactivated' by reducing the free Ca2+ concentration of the assay medium to values lower than 1 microM; and as a stable component, which can be 'deactivated' by preincubating the membranes for periods of 3-4 h with 2 mM EDTA or EGTA. Both components are easily activated by micromolar concentrations of Ca2+. The Ka of the system for Na+ is the same, 8 mM, whether only the stable component or both components, stable and labile, are working. In other words, the activating effect of Ca2+ on the Na+-stimulated ATPase is on the Vmax, and not on the Ka of the system for Na+. The activating effect of Ca2+ may be related to some conformational change produced by the interaction of this ion with the membranes, since it can also be obtained by resuspending the membranes at pH 7.8 or by ageing the preparations. Changes in the Ca2+ concentration may modulate the ouabain-insensitive, Na+-stimulated ATPase activity. This modulation could regulate the magnitude of the extrusion of Na+ accompanied by Cl- and water that these cells show, and to which the Na+-ATPase has been associated as being responsible for the energy supply of this mode of Na+ extrusion.     

Ca2+-stimulated ATPase: inactivation by Ca2+ and mechanism

Summary The preincubation of the rat red blood cell membranes in the presence of low Ca²⁺ levels causes an irreversible inhibition of the Ca²⁺-stimulated ATPase activity. The inactivation is dependent on the Ca²⁺ concentration and the apparent Ki is identical to the Ca²⁺ concentration needed to reach the half-maximal activity of the enzyme. This fact and the energy of activation (E_a = 13.8 Kcal/mol) for the inhibition suggest that Ca²⁺ inactivates the Ca²⁺-stimulated ATPase by binding to the same site which it normally occupies to activate the enzyme. It is concluded that the Ca²⁺-stimulated ATPase is in a dynamic equilibrium between two states: a stable ATP-bound state and an unstable ATP-free state.     

High affinity Ca2+-stimulated Mg2+-dependent ATPase in rat brain synaptosomes, synaptic membranes, and microsomes

High affinity Ca2+-stimulated Mg2+-dependent ATPase activity of nerve ending particles (synaptosomes) from rat brain tissue appears to be associated primarily with isolated synaptic plasma membranes. The synaptic membrane (Ca2+ + Mg2+)-ATPase activity was found to exhibit strict dependence on Mg2+ for the presence of the activity, a high affinity for Ca2+ (K0.5 = 0.23 microM), and relatively high affinities for both Mg2+ and ATP (K0.5 = 6.0 microM for Mg2+ and KM = 18.9 microM for ATP). These kinetic constants were determined in incubation media that were buffered with the divalent cation chelator trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid. The enzyme activity was not inhibited by ouabain or oligomycin but was sensitive to low concentrations of vanadate. The microsomal membrane subfraction was the other brain subcellular fraction with a high affinity (Ca2+ + Mg2+)-ATPase activity which approximated that of the synaptic plasma membranes. The two membrane-related high affinity (Ca2+ + Mg2+)-ATPase activities could be distinguished on the basis of their differential sensitivity to vanadate at concentrations below 10 microM. Only the synaptic plasma membrane (Ca2+ + Mg2+)-ATPase was inhibited by 0.25-10 microM vanadate. The studies described here indicate the possible involvement of both the microsomal and the neuronal plasma membrane (Ca2+ + Mg2+)-ATPase in high affinity Ca2+ transport across membranes of brain neurons. In addition, they suggest a means by which the relative contributions of each transport system might be evaluated based on their differential sensitivity to inhibition by vanadate.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.     

Effect of tricyclohexylhydroxytin on synaptosomal Ca2+-dependent ATP hydrolysis and rat brain subcellular calmodulin

Effect of tricyclohexylhydroxytin (plictran) on Ca2+-ATPase activity was studied in rat brain synaptosomes under in vitro and in vivo conditions. Plictran inhibited basal Ca2+-ATPase activity with an IC50 value of 6 nM suggesting its interaction with calcium transport phenomenon. Plictran inhibited calmodulin (CaM) activated Ca2+-ATPase in a concentration-dependent manner. A complete reversal of calmodulin activation of Ca2+-ATPase was observed with 2-3 nM plictran. A 50 per cent decrease of CaM activated Ca2+-ATPase was observed with 0.5 nM plictran, a concentration at which no significant effect was observed on basal enzyme activity. Of all the brain fractions studied, calmodulin levels in P2 fractions alone were reduced significantly to about 75 per cent of control values in plictran treated rats. The synaptosomal Ca2+-ATPase was also decreased by 35 per cent, 42 per cent and 65 per cent in 10, 20 and 40 mg plictran kg-1 day-1 treated rats for 3 days respectively. The activity levels of Ca2+-ATPase in 10 and 20 mg plictran kg-1 day-1 treated rats were restored to normal level by exogenously added calmodulin. These results suggest that plictran may disrupt synaptic function by altering calcium and calmodulin regulated processes in the central nervous system.     

Arylisothiocyanate modification of sarcoplasmic Ca2+-stimulated ATPase

The related probes phenylisothiocyanate andp-sulfophenylisothiocyanate possess comparable reactivity with nucleophiles but are dissimilar in their solubility characteristics. The reagents are utilized to topologically characterize the sites of covalent interaction with the Ca²⁺-stimulated ATPase of sarcoplasmic reticulum membranes. The hydrophobic probe phenylisothiocyanate binds covalently to the membrane-integrated protein. The extent of covalent interaction of this probe is reduced to a limited level of label incorporation by either preincubation withp-sulfophenylisothiocyanate or by exposing the labeled protein to alkaline reductive conditions. With respect to the chemical nature a dual interaction of phenylisothiocyanate is postulated. Phenylisothiocyanate modifies the Ca²⁺-ATPase hydrophobically. In addition, aqueous-exposed nucleophiles (cysteine thiols) interact with both arylisothiocyanates. Inhibition of the Ca²⁺-stimulated ATPase activity is effected by either probe.