Partial purification of (Ca2+ + Mg2+)-dependent ATPase from pig smooth muscle and reconstitution of an ATP-dependent Ca2+-transport system.

(CaMg)ATPase [(Ca2+ + Mg2+)-dependent ATPase] was partially purified from a microsomal fraction of the smooth muscle of the pig stomach (antrum). Membranes were solubilized with deoxycholate, followed by removal of the detergent by dialysis. The purified (CaMg)ATPase has a specific activity (at 37 degrees C) of 157 +/- 12.1 (7)nmol.min-1.mg-1 of protein, and it is stimulated by calmodulin to 255 +/- 20.9 (7)nmol.min.mg-1. This purification of the (CaMg)ATPase resulted in an increase of the specific activity by approx. 18-fold and in a recovery of the total enzyme activity of 55% compared with the microsomal fraction. The partially purified (CaMg)ATPase still contains some Mg2+-and (Na+ + K+)-dependent ATPase activities, but their specific activities are increased relatively less than that of the (CaMg)ATPase. The ratios of the (CaMg)ATPase to Mg2+- and (Na+ + K+)-dependent ATPase activities increase from respectively 0.14 and 0.81 in the crude microsomal fraction to 1.39 and 9.07 in the purified preparation. During removal of the deoxycholate by dialysis, vesicles were reconstituted which were capable of ATP-dependent Ca2+ transport.     

Characteristics of sarcoplasmic reticulum from normal and denervated rat skeletal muscle.

Denervation of rat skeletal muscle produces after 14 days a decrease in Ca2+ uptake of a heterogeneous population of sarcoplasmic-reticulum vesicles, when measured in the presence of oxalate. The Mg2+-dependent ATPase (Ca2+-independent) activity increased after the same period and the Ca2+ + Mg2+-dependent ATPase activity decreased. Concomitant with these changes, there was an increase in vesicle size and calcium content. The observations are discussed in terms of changes in altered membrane structure, manifested in the shift of the equilibrium of the ATPase from an enzyme involved in calcium transport to a phosphoenzyme giving rise to an increase in the Mg2+-dependent ATPase activity.     

Electron-cytochemical study of the localization and properties of ATPases in the isolated nuclei of rabbit skeletal muscle under normal conditions and in experimental muscular dystrophy]

The properties and localization of ATPase system in nuclei of skeletal muscle of normal rabbit and of those with experimental muscle dystrophy were studied by electron cytochemistry. The product of cytochemical reaction of ATP hydrolysis, which is a marker of ATPase activity localization in nuclear ultrastructures, was detected on the nuclear membrane, in chromatin and in the nucleolus, ATPase activity in the nuclei was detected in the presence of both, Mg2+ and Ca2+. Addition to the incubation medium, originally containing Mg2+, Na+ and K+, resulted in an increased formation of the product reaction in all the nuclear ultrastructures in both in the norm and under experimental muscle dystrophy. However, specific inhibitor of Mg2+, Na+, K+-ATPase--ouabain--suggests the absence in the nuclei of skeletal muscles of rabbit of transport ATPase working in the "Na-pump" system. The results of experiments with a specific complex of Ca2+--EGTA allow to suppose that Mg2+, Ca2+-ATPase of skeletal muscle nuclei of normal rabbits is localized in the nucleoplasm, whereas Mg2+-ATPase is found on the nuclear membrane. Using EGTA we failed to detected the localization of Mg2+, Ca2+-ATPase in nuclear ultrastructures upon experimental muscular dystrophy.     

ATPase activities, Ca2+ transport and phosphoprotein formation in sarcoplasmic reticulum subfractions of fast and slow rabbit muscles.

Subfractionation of sarcoplasmic reticulum from fast-twitch and slow-twitch rabbit skeletal muscles was performed on a sucrose density gradient. Vesicle fractions were characterized by: measurement of (Ca2+,Mg2+)-dependent (extra) ATPase, Mg2+-dependent (basal) ATPase, Ca2+ uptake characteristics, polypeptide patterns in sodium dodecylsulphate polyacrylamide gel electrophoreses, phosphoprotein formation and electronmicroscopy of negatively stained samples. In fast-twitch muscle, low and high density vesicles were separated. The latter showed high activity of (Ca2+,Mg2+)-dependent ATPase, negligible activity of Mg2+-dependent ATPase, high initial rate and high capacity of Ca2+ uptake, high amount of phosphorylated 115000-Mr polypeptide, and appeared morphologically as thin-walled vesicles covered with particles of 4 nm in diameter. Low density vesicles had little (Ca2+,Mg2+)-dependent ATPase but high Mg2+-dependent ATPase. Although the initial rate of Ca2+ uptake was markedly lower, the total capacity of uptake was comparable with that of high density vesicles. Phosphorylated 115000-Mr polypeptide was detectable at low concentrations. Instead, 57000 and 47000-Mr polypeptides were characterized as forming stable phosphoproteins in the presence of ATP and Mg2+. Negatively stained, these vesicles appeared to have smooth surfaces. It is suggested that low density vesicles represent a Ca2+ sequestering system different from that of high density vesicles and that Mg2+-dependent (basal) ATPase as well as the 57000 and 47000-Mr polypeptides are part of the Ca2+ transport system within the low density vesicles. According to the results from slow-twitch muscle, Ca2+ sequestration by the sarcoplasmic reticulum functions in this muscle type only through the low density vesicles.     

Phosphorylation of uterine smooth muscle myosin permits actin-activation.

Myosin was purified from ovine uterine smooth muscle. The 20,000 dalton myosin light chain was phosphorylated to varying degrees by an endogenous Ca2+ dependent kinase. The kinase and endogenous phosphatases were then removed via column chromatography. In the absence of actin neither the size of the initial phosphate burst nor the steady state Mg2+-dependent ATPase activity were affected by phosphorylation. However, phosphorylation was required for actin to increase the Mg2+-dependent ATPase activity and for the myosin to superprecipitate with actin. Ca2+ did not affect the Mg2+-dependent ATPase activity in the presence or absence of action or the rate or extent of superprecipitation with actin once phosphorylation was obtained. These data indicate that: 1) phosphorylation of the 20,000 dalton myosin light chain controls the uterine smooth muscle actomyosin interaction, 2) in the absence of actin, phosphorylation does not affect either the ATPase of myosin or the size of the initial burst of phosphate and, 3) Ca2+ is important in controlling the light chain kinase but not the actomyosin interaction.     

Presence of a high-affinity Ca2+- and Mg2+-dependent ATPase in rat peritoneal mast-cell membranes.

Purified perigranular and plasma membranes isolated from rat peritoneal mast cells were examined for Ca2+- and Mg2+-dependent ATPase activity. Isolated perigranular membranes contained only a low-affinity Ca2+- or Mg2+-dependent ATPase (Km greater than 0.5 mM). The plasma membranes contained both a low-affinity Ca2+- or Mg2+-dependent ATPase (Km = 0.4 mM, Vmax. = 20 nmol of Pi/min per mg), as well as a high-affinity Ca2+- and Mg2+-dependent ATPase (Km = 0.2 microM, Vmax. = 6 nmol of Pi/min per mg).     

Inhibitory antibodies to plasmalemmal Ca2+-transporting ATPases. Their use in subcellular localization of (Ca2+ + Mg2+)-dependent ATPase activity in smooth muscle.

Antibodies directed against the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase [(Ca2+ + Mg2+)-dependent ATPase] from pig erythrocytes and from smooth muscle of pig stomach (antral part) were raised in rabbits. Both the IgGs against the erythrocyte (Ca2+ + Mg2+)-ATPase and against the smooth-muscle (Ca2+ + Mg2+)-ATPase inhibited the activity of the purified calmodulin-binding (Ca2+ + Mg2+)-ATPase from smooth muscle. Up to 85% of the total (Ca2+ + Mg2+)-ATPase activity in a preparation of KCl-extracted smooth-muscle membranes was inhibited by these antibodies. The (Ca2+ + Mg2+)-ATPase activity and the Ca2+ uptake in a plasma-membrane-enriched fraction from this smooth muscle were inhibited to the same extent, whereas in an endoplasmic-reticulum-enriched membrane fraction the (Ca2+ + Mg2+)-ATPase activity was inhibited by only 25% and no effect was observed on the oxalate-stimulated Ca2+ uptake. This supports the hypothesis that, in pig stomach smooth muscle, two separate types of Ca2+-transport ATPase exist: a calmodulin-binding ATPase located in the plasma membrane and a calmodulin-independent one present in the endoplasmic reticulum. The antibodies did not affect the stimulation of the (Ca2+ + Mg2+)-ATPase activity by calmodulin.     

Studies of gastric Ca2+-stimulated adenosine triphosphatase. I. characterization and general properties

Gastric microsomes do not contain any significant Ca2+-stimulated ATPase activity. Trypsinization of pig gastric microsomes in presence of ATP results in significant (2-3 fold) increase in the basal (with Mg2+ as the only cation) ATPase activity, with virtual elimination of the K+-stimulated component. Such treatment causes unmasking of latent Mg2+-dependent Ca2+-stimulation ATPase. Other divalent cations such as Sr2+, Ba2+, Zn2+, and Mn2+ were found ineffective as a substitute for Ca2+. Moreover, those divalent cations acted as inhibitors of the Ca2+-stimulated ATPase activity. The pH optimum of the enzyme is around 6.8. The enzyme has a Km of 70 microM for ATP and the Ka values for Mg2+ and Ca2+ are about 4 x 10(-4) and 10(-7) M, respectively. Studies with inhibitors suggest the involvement of sulfhydryl and primary amino groups in the operation of the enzyme. Possible roles of the enzyme in gastric H+ transport have been discussed.     

Properties of epsilon-ATP hydrolysis by CF1-ATPase from pea chloroplasts

The ATPase activity of CF1 isolated from pea chloroplasts with epsilon-ATP, the fluorescent analog of ATP and ATP used as substrates, in the presence of Mg2+, Ca2+ and sodium sulfite (stimulator of the ATPase activity) was studied. The rate of epsilon-ATP hydrolysis in the presence of Mg2+ is nearly two times as low as that of ATP; an addition of sodium sulfite to the reaction mixture increases the reaction rate without changing the above ratio. The rate of Ca2+-dependent hydrolysis of epsilon-ATP is rather low as compared to that in the presence of Mg2+. epsilon-ADP is a competitive inhibitor of Mg2+-dependent ATPase reaction and inhibits this process in the presence of Ca2+, the inhibition being of a mixed type. Modification of CF1 by covalent binding of epsilon-ADP results in a 70-80% decrease of the Mg2+-dependent ATPase activity, the Ca2+-dependent ATPase activity is changed only insignificantly thereby. The differences in the activation of ATP and epsilon-ATP hydrolyses by Ca2+ and Mg2+ can be accounted for by the existence of two sites in the active center of CF1, which are specific for Mg2+ and Ca2+, respectively. It is concluded that the binding of epsilon-ADP occurs in the Mg2+-dependent ATPase site of the active center.     

Effect of SH-reagents on ATPase systems of rabbit skeletal muscle nuclei]

The influence of sulfhydryl reagents on ATPase systems of rabbit sceletal muscles nuclei was studied. It is found that p-ChMB at low concentration similarly inhibits both Mg2+- and Mg2+, Ca2+-ATPases. p-ChMB at higher concentrations inhibits completely Mg2+, Ca2+-ATPase, while Mg2+- ATPase--only by 60%. N-EM is lesser specific inhibitor of SH-groups, than p-ChMB. The degree of nuclear ATPases inhibition by N-EM is practically identical. Using inhibitory analysis, two hypes of skeletal muscles nuclei SH-groups are found: easily reacting with N-EM, and those reacting with N-EM at more high concentrations, which are essential for ATPase ATP-hydrolysing activity. ATP defends Mg2+, Ca2+-ATPase, but not the Mg2+-ATPase from N-EM inhibitory action. Cysteine completely eliminates the inhibitory effect of p-ChMB on Mg2+-ATPase but only 40% on MG2+, Ca2+-ATPase. Mg2+, Ca2+-ATPase of nuclei is more sensitive to the sulfhydryl venoms action than Mg2+-ATPase.     

Ectonucleotide diphosphohydrolase activities in hemocytes of larval Manduca sexta

In this work, we describe the ability of living hemocytes from an insect (Manduca sexta, Lepidoptera) to hydrolyze extracellular ATP. In these intact cells, there was a low level of ATP hydrolysis in the absence of any divalent metal (8.24 +/- 0.94 nmol of Pi/h x 10(6) cells). The ATP hydrolysis was stimulated by MgCl2 and the Mg2+-dependent ecto-ATPase activity was 15.93 +/- 1.74 nmol of Pi/h x 10(6) cells. Both activities were linear with cell density and with time for at least 90 min. The addition of MgCl2 to extracellular medium increased the ecto-ATPase activity in a dose-dependent manner. At 5 mM ATP, half-maximal stimulation of ATP hydrolysis was obtained with 0.33 mM MgCl2. This stimulatory activity was not observed when Ca2+ replaced Mg2+. The apparent Km values for ATP-4 and Mg-ATP2- were 0.059 and 0.097 mM, respectively. The Mg2+-independent ATPase activity was unaffected by pH in the range between 6.6 and 7.4, in which the cells were viable. However, the Mg2+-dependent ATPase activity was enhanced by an increase of pH. These ecto-ATPase activities were insensitive to inhibitors of other ATPase and phosphatase activities, such as oligomycin, sodium azide, bafilomycin A1, ouabain, furosemide, vanadate, sodium fluoride, tartrate, and levamizole. To confirm the observed hydrolytic activities as those of an ecto-ATPase, we used an impermeant inhibitor, DIDS (4,4'-diisothiocyanostilbene-2,2'-disulfonic acid), as well as suramin, an antagonist of P2-purinoreceptors and inhibitor of some ecto-ATPases. These two reagents inhibited the Mg2+-independent and the Mg2+-dependent ATPase activities to different extents. Interestingly, lipopolysaccharide, a component of cell walls of gram-negative bacteria that increase hemocyte aggregation and phagocytosis, increased the Mg2+-dependent ecto-ATPase activity in a dose-dependent manner but did not modify the Mg2+-independent ecto-ATPase activity.     

A monoclonal antibody to the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle inhibits plasmalemmal (Ca2+ + Mg2+)-dependent ATPase activity.

A monoclonal antibody (2B3) directed against the calmodulin-binding (Ca2+ + Mg2+)-dependent ATPase from pig stomach smooth muscle was prepared. This antibody reacts with a 130,000-Mr protein that co-migrates on SDS/polyacrylamide-gel electrophoresis with the calmodulin-binding (Ca2+ + Mg2+)-ATPase purified from smooth muscle by calmodulin affinity chromatography. The antibody causes partial inhibition of the (Ca2+ + Mg2+)-ATPase activity in plasma membranes from pig stomach smooth muscle, in pig erythrocytes and human erythrocytes. It appears to be directed against a specific functionally important site of the plasmalemmal Ca2+-transport ATPase and acts as a competitive inhibitor of ATP binding. Binding of the antibody does not change the Km of the ATPase for Ca2+ and its inhibitory effect is not altered by the presence of calmodulin. No inhibition of (Ca2+ + Mg2+)-ATPase activity or of the oxalate-stimulated Ca2+ uptake was observed in a pig smooth-muscle vesicle preparation enriched in endoplasmic reticulum. These results confirm the existence in smooth muscle of two different types of Ca2+-transport ATPase: a calmodulin-binding (Ca2+ + Mg2+)-ATPase located in the plasma membrane and a second one confined to the endoplasmic reticulum.     

Cell nuclear DNases: multiplicity and heterogeneity

In order to determine the ratio of activities of major endonucleases of rat liver chromatin, a stepwise fractionation of cell nuclear extracts by chromatography on phosphocellulose and gel filtration through Toyopearl HW60 was carried out. This procedure resulted in partially purified preparations of Ca2+,Mg2+-dependent endonuclease (55 +/- 10 kD), Ca2+,Mg2+-dependent endonuclease (30 +/- 10 kD), Mn2+-dependent endonuclease (30 +/- 5 kD) and acid cation-independent endonuclease. The Ca2+,Mg2+-dependent endonuclease with Mr of 55 +/- 10 kD made up to 57% of the nuclear extract activity in the presence of Ca2+ + Mg2+ and revealed a high calcium-magnesium synergism. Under the same experimental conditions, the 30 +/- 10 kD enzyme made up to 33% of the nuclear extract activity and revealed a low synergism. The activity of Mn2+-dependent endonuclease made up to 26% of the total nuclear extract activity in the presence of Mn2+, that of acid endonuclease--11% of the extract activity in 1 mM EDTA at pH 5.0. It was assumed that the low molecular weight Ca2+,Mg2+-dependent endonuclease represents a product of limited proteolysis of high molecular weight Ca2+,Mg2+-dependent endonuclease.     

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.     

Comparative study of calixarene effect on Mg2+ -dependent ATP-hydrolase enzymatic systems from smooth muscle cells of the uterus

Investigation the influence of calyx[4]arenes C-90, C-91, C-97 and C-99 (codes are indicated) on the enzymatic activity of four functionally different Mg2+ -dependent ATPases from smooth muscle of the uterus: actomyosin ATPase, transporting Ca2+, Mg2+ -ATPase, ouabain-sensible Na+, K+ -ATPase and basal Mg2+ -ATPase. It was shown that calixarenes C-90 and C-91 in concentration 100 microM act multidirectionally on the functionally different Mg2+ -dependent ATP-hydrolase enzymatic systems. These compounds activate effectively the actomyosin ATPase (Ka = 52 +/- 11 microM [Ukrainian character: see text] 8 +/- 2 microM, accordingly), at the same time calixarene C-90 inhibited effectively activity of transporting Ca2+, Mg2+ -ATPase of plasmatic membranes (I(0,5) = 34.6 +/- 6.4 microM), but influence on membrane-bound Na+, K+ -ATPase and basal Mg2+ -ATPase. Calixarene C-91 reduce effectively basal Mg2+ -ATPase activity, insignificantly activating Na+, K+ -ATPase but has no influence on transporting Ca2+, Mg2+ -ATPase activity of plasmatic membranes. Calixarenes C-97 and C-99 (100 microM), which have similar structure, have monodirectional influence on activity of three functionally different Mg2+-dependent ATPases of the myometrium: actomyosin ATPase and two ATPases, that related to the ATP-hydrolases of P-type--Ca2+, Mg2+ -ATPase and Na+, K+ -ATPase of plasmatic membranes. Basal Mg2+ -ATPase is resistant to the action of these two connections. Results of comparative experiments that were obtained by catalytic titration of calixarenes C-97 and C-99 by actomyosin ATPase (I(0,5) = 88 +/- 9 and 86 +/- 8 microM accordingly) and Na+, K+ -ATPase from plasmatic membranes (I(0,5) = 33 +/- 4 and 98 +/- 8 nM accordingly) indicate to the considerably more sensitiveness of Na+, K+ -ATP-ase to these calixarenes than ATPase of contractile proteins. Thus, it is shown that calixarenes have influence on activity of a number of important enzymes, involved in functioning of the smooth muscle of the uterus and related to energy-supplies of the process of the muscle contracting and support of intracellular ionic homeostasis. The obtained results can be useful in further researches, directed at the use of calixarenes as pharmaceutical substance, able to normalize the contractile function of the uterus at some pregnancy pathologies in women's.     

Inhibition of poly(ADP-ribose) polymerase as a possible reason for activation of Ca2+/Mg2+-dependent endonuclease in thymocytes of irradiated rats

The molecular mechanism of activation of Ca2+/Mg2+-dependent endonuclease in thymocytes of irradiated rats was studied. Thymocyte nuclei of control and irradiated rats were pre-incubated with NAD under conditions favourable for poly ADP-ribosylation. Pre-incubation results in a decrease in the rate of autolytic DNA digestion by Ca2+/Mg2+-dependent endonuclease of 6-7- and 2-3-fold for control and irradiated animals, respectively. The activity of Ca2+/Mg2+-nuclease extracted from the nuclei pre-incubated with NAD is also considerably decreased. The presence of nicotinamide and thymidine in the preincubation medium prevents the suppression of Ca2+/Mg2+-nuclease activity. In the experiments performed with isolated nuclei and permeabilized thymocytes the synthesis of poly(ADP-ribose) does not significantly change within 1 h after irradiation at a dose of 10 Gy, whereas 2 and 3 h after the exposure it decreases by 35-40 and 45-55 per cent, respectively. The activity of poly(ADP-ribose) glycohydrolase in this period is similar to that in the controls. The average size of the de novo synthesized chains of poly(ADP-ribose) increases from 11 to 17 ADP-ribose units by the second hour after irradiation. Inhibition of poly(ADP-ribose) polymerase in the postirradiation period preceded the internucleosomal fragmentation of chromatin. The results suggest that activation of Ca2+/Mg2+-nuclease in irradiated thymocytes is accounted for by the disturbance of its poly ADP-ribosylation.     

Laser Raman characterization of conformational changes in sarcoplasmic reticulum induced by temperature, Ca2+, and Mg2+

Laser Raman spectroscopy has been used to examine the conformations of the protein and phospholipid components of sarcoplasmic reticulum from rabbit white skeletal muscle. The phospholipid component is shown to have the conformation of fluid, liquid-crystalline lipids, even at 10 degrees C, and no breaks in the lipid conformation are observed in the range of 10-37 degrees C. Protein (predominantly the Ca2+-dependent ATPase) conformation is shown to contain very little beta-sheet structure under all conditions. Absolute content of alpha-helix and random coil or beta-turn could not be determined because of interference in the amide I and III regions. However, the Ca2+-ATPase in sarcoplasmic reticulum appears to undergo a conformational change at 15-18 degrees C which involves removal of a portion of the tryptophan residues from an aqueous environment and an increase in alpha-helical content. This conformation change coincides with a change in slope of Arrhenius plots of ATP hydrolysis activity. Increasing concentrations of Ca2+ and Mg2+ appear to slightly decrease the alpha-helical content of sarcoplasmic reticulum protein.     

Investigation of adenosinetriphosphatase activity of rat liver and thymus cell nuclei]

The activity of ATPase was studied in highly purified rat liver and thymus cell nuclei, HCO3-, CO3(2-) and SO3(2-) stimulated nuclear ATPase in 1.5--2 times. HSO3- did not affect the enzyme activity, and NO3-, J-, ClO4-,F- and SCN- inhibited it. Bicarbonate increased V and decreased Ka for ATP. SCN- inhibited HCO3--ATPase activity non-competitively with respect to HCO3-. Mg2+-ATPase activity did not depend on pH, and HCO3-component of the activity was decreased under alkaline pH. Mg2+, Mn2+ and Co2+ increased the initial ATPase activity and helped its stimulation with HCO3-. Ba2+, Ni2+ and Zn2+ inhibited the ATPase activity, and Ca2+ did not affect it, Nuclear ATPase is sensitive to 2,4-dinitrophenol and DNAase. It is suggested that cell nuclei have their own H+-ATPase differing for some characteristics from mitochondrial H+-ATPase.     

A Mg2+-independent high-affinity Ca2+-stimulated adenosine triphosphatase in the plasma membrane of rat stomach smooth muscle. Subcellular distribution and inhibition by Mg2+

Plasma membrane enriched fraction isolated from the fundus smooth muscle of rat stomach displayed Ca2+-stimulated ATPase activity in the absence of Mg2+. The Ca2+ dependence of such an ATPase activity can be resolved into two hyperbolic components with a high affinity (Km = 0.4 microM) and a low affinity (Km = 0.6 mM) for Ca2+. Distribution of these high-affinity and low-affinity Ca2+-ATPase activities parallels those of several plasma membrane marker enzyme activities but not those of endoplasmic reticulum and mitochondrial membrane marker enzyme activities. Mg2+ also stimulates the ATPase in the absence of Ca2+. Unlike the Mg2+-ATPase and low-affinity Ca2+-ATPase, the plasmalemmal high-affinity Ca2+-ATPase is not sensitive to the inhibitory effect of sodium azide or Triton X-100 treatment. The high-affinity Ca2+-ATPase is noncompetitively inhibited by Mg2+ with respect to Ca2+ stimulation. Such an inhibitory effect of Mg2+ is potentiated by Triton X-100 treatment of the membrane fraction. Calmodulin has little effect on the high-affinity Ca2+-ATPase activity of the plasma membrane enriched fraction with or without EDTA pretreatment. Findings of this novel, Mg2+-independent, high-affinity Ca2+-ATPase activity in the rat stomach smooth muscle plasma membrane are discussed with those of Mg2+-dependent, high-affinity Ca2+-ATPase activities previously reported in other smooth muscle plasma membrane preparations in relation to the plasma membrane Ca2+-pump.     

Characterization of the Ca2+- and Mg2+-Dependent ATPases in Electrophorus Electroplax Microsomes

Electrophorus electroplax microsomes were examined for Ca2+- and Mg2+-dependent ATPase activity. In addition to the previously reported low-affinity ATPase, a high-affinity (Ca2+,Mg2+)-ATPase was found. At low ATP and Mg2+ concentrations (200 microM or less), the high-affinity (Ca2+,Mg2+)-ATPase exhibits an activity of 18 nmol Pi mg-1 min-1 with 0.58 microM Ca2+. At higher ATP concentrations (3 mM), the low-affinity Ca2+-ATPase predominates, with an activity of 28 nmol Pi mg-1 min-1 with 1 mM Ca2+. In addition, Mg2+ can also activate the low-affinity ATPase (18 nmol Pi mg-1 min-1). The high-affinity ATPase hydrolyzes ATP at a greater rate than it does GTP, ITP, or UTP and is insensitive to ouabain, oligomycin, or dicyclohexylcarbodiimide inhibition. The high-affinity enzyme is inhibited by vanadate, trifluoperazine, and N-ethylmaleimide. Added calmodulin does not significantly stimulate enzyme activity; rinsing the microsomes with EGTA does not confer calmodulin sensitivity. Thus the high-affinity ATPase from electroplax microsomes is similar to the (Ca2+,Mg2+)-ATPase reported to be associated with Ca2+ transport, based on its affinity for calcium and its response to inhibitors. The low-affinity enzyme hydrolyzes all tested nucleoside triphosphates, as well as diphosphates, but not AMP. Vanadate and N-ethylmaleimide do not inhibit the low-affinity enzymes. The low-affinity enzyme reflects a nonspecific nucleoside triphosphatase, probably an ectoenzyme.