The effect of calmodulin on the phosphoprotein intermediate of Mg2+-dependent Ca2+-stimulated adenosine triphosphatase in human erythrocyte membranes.

The effect of calmodulin on the formation and decomposition of the Ca2+-dependent phosphoprotein intermediate of the (Mg2+ + Ca2+)-dependent ATPase in erythrocyte membranes was investigated. In the presence of 60 microM-Ca2+ and 25 microM-MgCl2, calmodulin (0.5-1.5 microgram) did not alter the steady-state concentration of the phosphoprotein, but increased its rate of decomposition. Higher calmodulin concentrations significantly decreased the steady-state concentration of phosphoprotein. Calmodulin (0.5-1.7 microgram) increased Ca2+-transport ATPase activity by increasing the turnover rate of its phosphoprotein intermediate. Increasing the MgCl2 concentration from 25 microM to 250 microM increased the (Mg2+ + Ca2+)-dependent ATPase activity, but decreased the concentration of the phosphoprotein intermediate. Similarly to calmodulin, MgCl2 increased the turnover rate of the Ca2+-transport ATPase complex (about 3-fold). At the higher MgCl2 concentration calmodulin did not further affect the decomposition of the phosphoprotein intermediate. It was concluded that both calmodulin and MgCl2 increase the turnover of the Ca2+-pump by enhancing the decomposition of the Ca2+-dependent phosphoprotein intermediate.     

Modulation of the activity of the (Ca2+ + Mg2+)-dependent adenosine triphosphatase of the human erythrocyte

We previously reported that the activity of the (Ca2+ + Mg2+)-dependent adenosine triphosphatase (ATPase) of the human erythrocyte membrane is inhibited by micromolar or nanomolar concentrations of cyclic AMP. Our further studies have now indicated that the inhibition of (Ca2+ + Mg2+)-dependent phosphohydrolase activity requires the participation of a membrane-associated cyclic AMP-dependent protein kinase and a membrane-associated protein substrate that is distinct from the ATPase itself. We have furthermore, identified a 20 kDa membrane protein which undergoes phosphorylation that is promoted by micromolar, but not millimolar, concentrations of cyclic AMP and which, when phosphorylated, undergoes dephosphorylation that is promoted by Ca2+. We suggest that this membrane component can participate in the modulation of the activity of the (Ca2+ + Mg2+)-dependent ATPase of the human erythrocyte.     

Characteristics of Ca2+ ion effect on the activity of Mg2+-dependent ATPase system in ghosts and reconstituted erythrocytes]

A good conformity if demonstrated of the kinetics of calcium ions effect on ATPase activity of human and rat erythrocyte ghosts. The increase of calcium concentration in the rat errythrocytes hemolysis medium (above 50-100 micrometer) results in a considerable aggregation of reconstructed vesicles. An activation of ouabaine-sensitive component of Mg2+-dependent ATPase under the increase of intracellular Ca2+ in reconstructed human erythrocytes is observed.     

Oxalate, calcium uptake and ATPase activity of sarcoplasmic reticulum vesicles.

Ca++-uptake and Mg++-Ca++-dependent ATPase activity of skeletal muscle sarcoplasmic reticulum vesicles were reciprocally affected by increasing the oxalate concentration from 0 to 4 mM. At 0-0.1 mM oxalate approximately 17% of the calcium was removed by the vesicles from the medium while the ATPase activity was maximal (approximately 0.66 mumoles Pi mg-1 protein min-1). Between 0.1 to 0.2 mM oxalate the ATPase activity was reduced to one-fifth but the uptake rose sharply and 100% of the 45Ca++ was removed from the medium. The uptake was maintained at this level at oxalate concentrations greater than 0.4 mM but the ATPase activity remained inhibited. The kinetics of Ca++-uptake and ATPase activity were also differentially affected by oxalate. In the presence of oxalate, ruthenium red had only a very slight inhibitory effect on the calcium uptake. Addition of 0.1 mM EGTA removed 80% of the Ca++ from preloaded vesicles within 10 min. The formation of insoluble Ca-oxalate salt on the surface of the vesicle is suggested by these results. Calculations based on the Ksp of the calcium oxalate salt are presented to show its formation and the possible speciation of a Ca-oxalate complex which may affect the Ca++-uptake and ATPase activity.     

The role of Mg++-ATPase (actomyosin-like protein) in maintaining the biconcave shape of erythrocytes.

Chemically different substances known to change the Mg++-ATPase activity in the red cell membrane, likewise alter the red cell shape. Normal human red cells retain their biconcave shape only when the activity of this enzyme remains unchanged. The present work deals with the possibility that Mg++-ATPase may cause certain tension in the membrane responsible for the biconcave shape of the erythrocyte.     

Ultrastructural localizations of adenosine triphosphatase activity in resting mammary gland.

Adenosine triphosphatase (ATPase) activity was localized at an ultrastructural level in the resting mammary glands of female BALB/c mice. A Mg++ dependent ATPase was localized in the plasma membranes of both the epithelial and myoepithelial cells of the mammary tubules. A second type of ATPase activity that was not Mg++-dependent but that was Na+ and K+ dependent was localized primarily in the plasma membranes of the myoepithelial cells. Preincubation with either ouabain or N-ethylmaleimide decreased the quantity of reaction product, indicating that both types of ATPase activity were sensitive to these inhibitors. Control media, containing adenosine triphosphate and Pb(NO3)2 without cations, demonstrated that the amount of nonezymatic hydrolysis was negligible. These differences in the cationic requirements for plasma membrane ATPase activity can be used to distinguish histochemically the epithelial from myoepithelial cells in mammary tissue.     

The inhibition of ATP-dependent shape change of human erythrocyte ghosts correlates with an inhibition of Mg(2+)-ATPase activity by fluoride and aluminofluoride complexes.

The vanadate-sensitive Mg(2+)-dependent ATPase activity of the human erythrocyte ghost is believed to be involved in the shape change events that convert echinocytic ghosts to smoothed forms (biconcave discs and stomatocytes). At physiological salt concentration, pH 7.4, 2 mM ATP, 5 mM Mg2+ and 1 mM EGTA, the Mg(2+)-ATPase activity of ghosts was inhibited strongly by millimolar concentrations of sodium fluoride: I50 = 1.31 +/- 0.23 mM (mean +/- S.D.; n = 12). The addition of aluminium chloride to 15 microM reduced the concentration of NaF required for 50% inhibition to 0.76 +/- 0.21 mM (n = 10). Aluminium alone had only a small inhibitory effect on the ATPase activity (13 +/- 9%; n = 10). Desferrioxamine, a strong chelator of tervalent aluminium ion, failed to reverse the inhibition by fluoride and reversed the inhibition in the presence of aluminium and fluoride back to those values obtained with fluoride alone. Of several metal salts tested only beryllium sulfate was able to replace aluminium as an effective inhibitor in the presence of fluoride. Inhibition of the Mg(2+)-ATPase activity by fluoride and the aluminofluoride complexes correlated with an inhibition of the rate of MgATP-dependent change in red cell ghost shape from echinocytes to smoothed forms. All gross morphological changes of the smoothing process were affected, including the production of discocytes, stomatocytes and endocyctic vesicles.     

Effects in vitro of mercury on rat brain Mg(++)-ATPase.

Mercuric chloride (Hg) in micromolar concentrations inhibited Mg(++)-dependent ATPase activity in rat brain microsomes. Inhibition was higher in oligomycin-sensitive (O.S.) than oligomycin-insensitive (O.I.) Mg(++)-ATPase. Hydrolysis of ATP with 15 and 50 micrograms of microsomal protein for 45 min without and with (2.10(-7M) Hg showed linear rates for 15-20 min. Altered pH vs activity demonstrated comparable inhibitions by Hg in buffered (neutral greater than acidic greater than basic) pH ranges. Inhibition of enzyme activity by Hg was found to be greater at 37 degrees C than at lower temperatures suggesting positive correlation trend. An uncompetitive inhibition with respect to the activation of Mg(++)-ATPase, O.S. Mg(++)-ATPase and O.I. Mg++ ATPase by ATP was indicated by a decrease in apparent Vmax and Km. Mg(++)-activation kinetic studies indicated that Hg causes uncompetitive inhibition of Mg(++)-ATPase and O.I. Mg(++)-ATPase and mixed inhibition of O.S. Mg(++)-ATPase. Inhibition was partially restored by repeated washings. These results indicate that the inhibition of microsomal Mg(++)-ATPase by Hg was pH, temperature, enzyme and Mg++ concentration dependent. Additionally, the data also suggest that O.S. compared to O.I. Mg(++)-ATPase is more sensitive to Hg toxicity.     

Na+, K+-dependent ATPase activity and effect of K+ on in vitro protein synthesis and NAD pyrophosphorylase in Toxoplasma gondii

Although Na+, K+-dependent ATPase activity was barely detectable in extracellular tachyzoites of Toxoplasma gondii, Mg++-dependent ATPase was readily demonstrable. Based on this finding, the effect of K+ on in vitro protein synthesis and NAD pyrophosphorylase of T. gondii was evaluated. Addition of KCl enhanced in vitro protein synthesis. NAD pyrophosphorylase also was activated by KCl. The optimal concentration of KCl for both enzymatic reactions was 150 mM. The supernatant fluid (15,000 g, 20 min) isolated from the homogenate of purified tachyzoites of T. gondii contained more Na+ than K+. Evaluation of leakage of Na+ and K+ from purified tachyzoites suggested that Na+ leaked more than K+. These observations suggest an important role of K+ in the regulation of the metabolism of T. gondii.     

A liquid diffraction analysis of sarcoplasmic reticulum. I. Compositional variation.

Intensities of x-ray scattering from a series of fragmented rabbit muscle sarcoplasmic reticulum (SR) samples have been measured over the range x = 0.05 to s = 0.25. By varying the relative concentrations of lipid and protein (chiefly the Mg++-dependent, Ca++- stimulated ATPase) in the membranes of this series, and by employing methods of analysis appropriate to the scattering from binary liquid mixtures, we have identified the separable contributions of protein and lipid, and the protein-lipid interaction contributions to the total scattering profiles. The shape of the protein term is consistent with scattering from a cylindrical ATPase particle 142 A in length and 35 A in diameter. These data imply that the dominant ATPase species is monomeric. The protein-lipid interaction term has been analyzed by a novel treatment based on a determination of the pair correlation function between the electrons of the protein molecule with the electrons of the lipid bilayer in terms of the asymmetry of the transbilayer disposition of the protein. Applied to our results, the analysis indicates a fully asymmetric disposition of ATPase, in which one end of the molecule is contiguous with either the lumenal or cytoplasmic surface of the bilayer.     

Evidence for the influence of the protein-phospholipid interface on sarcoplasmic reticulum Ca++ Mg++ ATPase activity.

Sarcoplasmic reticulum from the white hind leg muscle of the rabbit was examined with 31P nuclear magnetic resonance as a nonperturbing probe of phospholipid-protein interactions in the intact membrane. The phospholipids of the sarcoplasmic reticulum appear to inhabit two distinct environments: one very similar in behavior to pure phospholipid lamellar dispersions and the other immobilized by the protein in the membrane. Measurement of the population of the latter environment suggests that it is dependent on salt concentration and probably not due to the Ca++ Mg++ ATPase of the sarcoplasmic reticulum. This immobilization can be removed completely by papain proteolysis of the membrane protein, but only partially by trypsin treatment. The phospholipid composition of recombinants with the Ca++ Mg++ ATPase was varied in order to look for effects of the phospholipid-protein interface on enzymatic activity of the Ca++ Mg++ ATPase. Both transphosphatidylated phosphatidylethanolamine (from egg phosphatidylcholine) and bovine brain phosphatidylserine readily partitioned into the putative boundary layer, whereas under the same conditions soybean phosphatidylethanolamine was excluded. Only phosphatidylserine affected the activity of the enzyme, causing an inhibition that was proportional to the phosphatidylserine content, relative to phosphatidylcholine.     

Diamide inhibited (Ca++ + Mg++) and (Mg++) dependent ATPase in erythrocyte membranes: activity at different temperatures.

After inhibition of the monovalent cation dependent ATPase, a (Ca++ + Mg++) and a (Mg++) dependent ATPase activity can be detected. The inhibition due to diamide on the (Mg++) ATPase, assayed in the 12.5 degrees C - 30 degrees C temperature range, is almost complete. On the contrary the diamide induced inhibition of (Ca++ + Mg++) ATPase, in the same temperature range, is not complete and the residual activity increases with temperature. The reported data indicate that the ATPase activity induced by calcium is much less diamide-sensitive and -SH-dependent than that elicited by Mg++ alone.     

Differentiation of normal human mammary epithelial cells in culture: an ultrastructural study.

An ultrastructural and cytochemical study of normal human mammary epithelial cells cultured from post-weaning breast fluids is described. Cells were examined at the time of plating and at intervals up to 28 days in culture. Three different stages in the morphological differentiation of these cells in vitro were observed: (1) the first stage was the formation of a monolayer of single cells, which occurred between days 1 and 10 in culture. The cells in this stage were not interconnected by junctional complexes and lacked Mg++- dependent ATPase activity in the plasma membranes, but did contain a large quantity of lipid and exhibited some secretory characteristics. (2) The second stage, occurring at 10 to 16 days in culture, was characterized by the formation of junctional complexes, the appearance of Mg++-dependent ATPase in the plasma membrane and a decrease in the number of dense bodies with peroxidase activity. (3) The third stage, occurring at 16 to 28 days in culture, was characterized by the formation of stratified layers of epithelial cells, which were interconnected by a larger number of desmosomes with numerous pleomorphic microfilaments. The Mg++-dependent ATPase activity in the plasma membrane was retained and the dense bodies with peroxidase activity were rarely observed at this stage. During the last seven days were prominent in the cells of the stratified layer. After 28 days in the culture, the cells began to round up and slough off the culture plate.     

The effects of 3-methylindole on hemolysis, transport of Na+, and ATPase activities of bovine erythrocytes.

Biochemical effects of 3MI on cellular membranes were investigated. This study was conducted to examine the effects of 3MI on the hemolysis of erythrocytes, the transport of 22Na+ in resealed erythrocyte ghosts, and on the ATPase activities of erythrocyte membranes. The percent of hemolysis as a function of 3MI incubation time was sigmoidal. Seventy-five percent of the hemoglobin was released with the second 2 hr of incubation during which the concentration of 3MI in the cells reached a plateau of 2500 mug/ml of packed RBC. The effect of 3MI at a subhemolytic concentration on passive and active 22Na+ transport were not significant. The total and Mg2+-dependent ATPase activities in the membranes were significantly increased after 1 hr of incubation with 3MI at concentrations of 100, 200, 300, 400 and 500 mug/ml (P less than or equal to 0/ml (P smaller than or equal to 0.02).     

Detection of a Na+-activated, furosemide-inhibited ATPase activity in rat intestinal mucosa

An ouabain-insensitive, Mg++-dependent, Na+-stimulated ATPase activity which is inhibited by furosemide was found in mucosal homogenate of rat small intestine. The subcellular localization of this ATPase activity was studied by means of isolated purified brush borders and basolateral plasma membranes. The results suggest a nearly identical distribution of Na+-activated and (Na+K+)-activated ATPase within the epithelial cells. Under conditions of alloxan and streptozotocin diabetes an increase of both ATPase activities can be found only in the basolateral plasma membranes. These observations agree well with the convective model of intestinal absorption.     

Calcium ion-dependent phosphorylation of human erythrocyte membranes.

Calcium ions promote the rapid transfer of the terminal phosphate of ATP to a protein of human erythrocyte membranes. The concentration of Ca2+ for half-maximal effect is 7 muM. At nonlimiting ATP concentrations the level of 32P incorporated by the membranes is independent of the presence or absence of Mg2+. The number of phosphorylating sites in a single erythrocyte membrane is about 700. The influence of pH on the rate of hydrolysis of the bound phosphate and its rapid release on exposure to hydroxylamine are both consistent with an acylphosphate bond. The phosphate in the protein undergoes rapid turnover. Enzymatic splitting of the phosphate is stimulated by Mg2+ but not by Ca2+. It is proposed that Mg2+ accelerates the splitting of the phosphate by favoring the conversion of the phosphoprotein from a state of low reactivity to a state of high reactivity towards water. The reactions described probably are intermediate steps in the hydrolysis of ATP catalyzed by the Ca2+-dependent ATPase of human erythrocyte membranes.     

The influence of adenosine 3',5'-monophosphate upon the activity of the membrane-associated (Ca+ + Mg2+)-dependent adenosine triphosphatase of the human erythrocyte

The phosphohydrolase activity of the membrane-associated (Ca2+ + Mg2+)-dependent adenosine triphosphatase (ATPase) of the human erythrocyte can be inhibited by micromolar of nanomolar concentrations of cyclic AMP. Millimolar concentrations of cyclic AMP are less effective. The inhibitory effect of cyclic AMP is potentiated in the presence of the phosphodiesterase inhibitor, theophylline.     

Effects of divalent cations on dynein cross bridging and ciliary microtubule sliding

We recently demonstrated that addition of the divalent cation Mg++ to demembranated cilia causes the dynein arms to attach uniformly to the B subfibers. We have now studied the dose-dependent relationship between Mg++ or Ca++ and dynein bridging frequencies and microtubule sliding in cilia isolated from Tetrahymena. Both cations promote efficient dynein bridging. Mg++-induced bridges become saturated at 3 mM while Ca++-induced bridges become saturated at 2 mM. Double reciprocal plots of percent bridging vs. the cation concentration (0.05-10 mM) suggest that bridging occurs in simple equilibrium with the cation concentration. When microtubule sliding (spontaneous disintegration in 40 mM N-2-hydroxyethylpiperazine-N'-2-ethane sulfonic acid (HEPES), 0.1 mM ATP at pH 7.4) is assayed (A350 nm) relative to the Mg++ or Ca++ concentration, important differential effects are observed. 100% Disintegration occurs in 0.5-2 mM Mg++ and the addition of 10 mM Mg++ does not inhibit the response. The addition of 0.05-10 mM Ca++ to cilia reactivated with 0.1 mM ATP causes a substantial reduction in disintegration at low Ca++ concentrations and complete inhibition at concentrations greater than 3 mM. When Ca++ is added to cilia reactivated with 2 mM Mg++ and 0.1 mM ATP, the percent disintegration decreases progressively with the increasing Ca++ concentration. The addition of variable concentrations of Co++ to Mg++-activated cilia causes a similar but more effective inhibition of the disintegration response. These observations, when coupled with the relatively high concentrations of Ca++ or Co++ needed to inhibit disintegration, suggest that inhibition results from simple competition for the relevant cation-binding sites and thus may not be physiologically significant. The data do not yet reveal an interpretable relationship between percent disintegration, percent dynein bridging, and percent ATPase activity of both isolated dynein and whole cilia. However, they do illustrate that considerable (sliding) disintegration (60%) can occur under conditions that reveal only 10-15% attached dynein cross bridges.     

Agents inducing high Mg2+-ATPase activity of isolated coupling factor 1 from spinach chloroplasts

Conditions are reported under which purified coupling factor 1 (CF1) from spinach chloroplasts exhibits Mg2+-dependent ATPase activity of about 120 mumoles/min/mg protein. It is shown that CF1, partially activated by treatment with heat and dithiothreitol (DTT), can be further activated by octyl glucoside. The Mg2+-dependent ATPase activity increases linearly as a function of the concentration of octyl glucoside from about 20 mumoles/min/mg protein in the absence of detergent to 120 mumoles/min/mg protein in the presence 15 mM octyl glucoside. This concentration is below the critical micellar concentration (CMC) of the detergent, indicating that the monomeric form is responsible for the activation. Without treatment with heat and DTT, the Mg2+-dependent ATPase activity of CF1 is virtually zero, but can be stimulated by octyl glucoside. In this case, however, only concentrations around CMC give a substantial increase in activity (about 50 mumoles/min/mg at 28 mM octyl glucoside). Concentrations higher than CMC inhibit both latent and heat-activated CF1.     

Calmodulin confers calcium sensitivity on ciliary dynein ATPase

Extraction of demembranated cilia of Tetrahymena by Tris-EDTA (denoted by the suffix E) yields 14S-E and 30S-E dyneins with ATPase activities that are slightly increased by Ca++. This effect is moderately potentiated when bovine brain calmodulin is added to the assay mixture. Extraction with 0.5 M KCl (denoted by the suffix K) yeilds a 14S-K dynein with a low basal ATPase activity in the presence of Ca++. Subsequent addition of calmodulin causes marked activation (up to 10- fold) of ATPase activity. Although 14S-K and 14S-E dyneins have Ca++- dependent ATPase activities that differ markedly in the degree of activation, the concentration of calmodulin required for half-maximal saturation is similar for both, approximately 0.1 microM. Both 30S-K and 30S-E dyneins, however, require approximately 0.7 microM bovine brain calmodulin to reach half-maximal activation of their Ca++- dependent ATPase activities. Tetrahymena calmodulin is as effective as bovine brain calmodulin in activating 30S dynein , but may be slightly less effective than the brain calmodulin in activating 14S dynein. Rabbit skeletal muscle troponin C also activates the Ca++-dependent ATPase activity of 30S dynein and, to a lesser extent, that of 14S dynein, but in both cases is less effective than calmodulin. The interaction of calmodulin with dynein that results in ATPase activation is largely complete in less than 1 min, and is prevented by the presence of low concentrations of ATP. Adenylyl imidodiphosphate can partially prevent activation of dynein ATPase by calmodulin plus Ca++, but at much higher concentrations than required for prevention by ATP. beta, gamma-methyl-adenosine triphosphate appears not to prevent this activation. The presence of Ca++-dependent calmodulin-binding sites on 14S and 30S dyneins was demonstrated by the Ca++-dependent retention of the dyneins on a calmodulin-Sepharose-4B column. Gel electrophoresis of 14S dynein that had been purified by the affinity-chromatography procedure showed that presence of two major and one minor high molecular weight components. Similar analysis of 30S dynein purified by this procedure also revealed on major and one minor high molecular weight components that were different from the major components of 14S dynein. Ca++-dependent binding sites for calmodulin were shown to be present on axonemes that had been extracted twice with Tris-EDTA or with 0.5 M KCl by the use of 35S-labeled Tetrahymena calmodulin. It is concluded that the 14S and 30S dyneins of Tetrahymena contain Ca++- dependent binding sites for calmodulin and the calmodulin mediates the Ca++-regulation of the dynein ATPases of Tetrahymena cilia.