Ca2+-activated ATPase of rat liver plasma membrane.

Rat liver plasma membranes hydrolyze ATP in the presence of Ca2+. The rate of hydrolysis is different when Mg2+ions are present in the incubation system. Several parameters differentiate Ca2+-ATPase from Mg2+-ATPase: a) the Km of ATP hydrolysis for Ca2+ (2.25 x 10(-4) M) is lower than for Mg2+ (2.14 x 10(-3) M); b) the shape of the activation curve is hyperbolic in the presence of Ca2+ and sigmoid in the presence of Mg2+; c) Mg2+-ATPase shows two different values of activation energy while Ca2+-ATPase presents only a single value; d) Ca2+-ATPase is inhibited, while Mg2+-ATPase is unaffected by cyclic AMP. Ca2+-ATPase is localized on the plasma membrane and is not inhibited by cysteine. It does not hydrolyze substrates different from nucleotides triphosphate, such as glucose-1-phosphate or alpha-glycero-phosphate. The enzyme is probably related to a mechanism of calcium transport.     

Ca2+-activated ATPase in microsomes from human liver

Human liver microsomal fractions exhibit ATP-supported Ca2+ uptake which is half-maximal at 7 X 10(-7) M free Ca2+ in the presence of oxalate. Ca2+ uptake is coupled to a Ca2+-stimulated ATPase activity, which is half-maximal at 4 X 10(-7) M free Ca2+. Catalysis involves formation of an Mr = 116,000 phosphoprotein with stability characteristics of an acylphosphate compound suggested to represent a phosphoryl protein intermediate of the Ca2+-ATPase. Phosphorylation is half-maximal at about 10(-6) M free Ca2+. The Mr = 116,000 protein is highly susceptible to proteolysis with trypsin. The phosphorylated active site was localized in an Mr = 58,000 primary tryptic fragment and in an Mr = 34,000 subfragment. Analyses on the mechanism of the Ca2+-ATPase suggest the following reaction sequence: formation of an ADP-reactive phosphoenzyme (Mr = 116,000) with bound Ca2+, which can transphosphorylate its Pi to ADP, giving rise to synthesis of ATP; reversible transformation of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, which cannot further react with ADP; hydrolytical cleavage, probably catalyzed by Mg2+, of the ADP-unreactive phosphoenzyme with liberation of Pi. Comparison with the Ca2+-transport ATPase in sarcoplasmic reticulum of skeletal muscle led us to suggest that the Mr = 116,000 Ca2+-ATPase belongs to the class of E1P . E2P-ATPases and might be operative as a Ca2+-transport ATPase at the level of the endoplasmic reticulum in human liver.     

Mg2+- or Ca2+-activated ATPase activities of plasma membranes isolated from vascular smooth muscle

Studies of ATP hydrolysis by various subcellular fractions isolated from rat mesenteric arteries and veins indicate that an apparent ATPase activity, which can be activated by Mg2+ or Ca2+, is primarily associated with the plasma membranes. Although both Mg2+-activated and Ca2+-activated ATPase activities under the optimal condition are substantially lower in venous than in arterial plasma membrane fraction, their dependence on the concentration of Mg2+ and Ca2+ are quite similar in arterial as well as venous plasma membrane fractions. No synergistic effect on ATP hydrolysis was observed in the presence of both Mg2+ and Ca2+. In addition, Mg2+-activated and Ca2+-activated ATPase activities show similar pH dependence, inhibition by deoxycholate, stability toward heat inactivation and substrate specificity. Furthermore, Mg2+-activated and Ca2+-activated ATPase activities were similarly reduced in vascular smooth muscles of spontaneously hypertensive rats. These results suggest that the activation of ATP hydrolysis by Mg2+ or Ca2+ may represent a single enzyme moiety in the plasma membrane of vascular smooth muscle. The possible involvement of such ATPase in the Ca2+ transport function of vascular smooth muscle is discussed.     

(Ca + Mg)-stimulated ATPase activity of a rat brain microsomal preparation.

ATPase activity of freshly prepared brain microsomes was stimulated 20% when 0.1 mm CaCl₂ was added in the presence of a “saturating” concentration of MgCl₂ (4 mm). This (Ca + Mg)-stimulated activity declined rapidly on storage. Treatment of the microsomes with 0.12% deoxycholate in 0.15 m KCl, followed by centrifugation and resuspension in sucrose, produced a preparation both stable on storage at ?15 °C and with an increased stimulation in the presence of CaCl₂. SrCl₂ was more effective than CaCl₂, but BaCl₂ was a poor activator. KCl and NaCl stimulated the (Ca + Mg)-ATPase activity by reducing substrate (ATP) inhibition. The K_m for ATP was 0.1 mm, a third that of the Mg-ATPase. CTP, ITP, and GTP could not substitute for ATP, although they were fair substrates for the Mg-ATPase. The energy of activation of the (Ca + Mg)-ATPase was 21 kcal, nearly twice that of the Mg-ATPase. After sucrose density-gradient centrifugation of the microsomal preparation, the (Ca + Mg)-ATPase activity was distributed with the (Na + K)-ATPase and not with the mitochondrial marker succinic dehydrogenase. Studies with ouabain, oligomycin, and azide distinguished the (Ca + Mg)-stimulated ATPase from (Na + K)- and mitochondrial ATPases. Sensitivity to ruthenium red suggested a link to Ca transport, although the microsomal ⁴⁵Ca accumulating system was much more sensitive to the inhibitor than was this ATPase activity.     

A Ca2(+)-activated, Mg2(+)-dependent ATPase with high affinities for both Ca2+ and Mg2+ in vascular smooth muscle microsomes: comparison with plasma membrane Ca2(+)-pump ATPase

A Ca2(+)-ATPase with a high affinity for free Ca2+ (apparent Km of 0.13 microM) was found and characterized in membrane fractions from porcine aortic and coronary artery smooth muscles in comparison with the plasma membrane Ca2(+)-pump ATPase purified from porcine aorta by calmodulin affinity chromatography. The activity of the high-affinity Ca2(+)-ATPase became enriched in a plasma membrane-enriched fraction, suggesting its localization in the plasma membrane. The enzyme was fully active in the absence of exogenously added Mg2+, but required a minute amount of Mg2+ for its activity as evidenced by the findings that it was fully active in the presence of 0.1 microM free Mg2+ but lost the activity in a reaction mixture containing trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid as a divalent cation chelator which has, unlike EGTA, high affinities for both Ca2+ and Mg2+. It was able to utilize a variety of nucleoside di- and triphosphates as substrates, such as ADP, GDP, ATP, GTP, CTP, and UTP, showing a broad substrate specificity. The activity of the enzyme was not modified by calmodulin (5, 10 micrograms/ml). Trifluoperazine, a calmodulin antagonist, had a partial inhibitory effect on the activity at 30 to 240 microM, but this inhibition could not be reproduced by a more specific calmodulin antagonist, W-7, indicating that this inhibition by trifluoperazine was not specific. Furthermore, the high-affinity Ca2(+)-ATPase activity was not modified either by low concentrations (0.5-9 microM) of vanadate or by 1-100 microM p-chloromercuribenzoic acid. Cyclic GMP, nitroglycerin, and nicorandil did not have any effect on the enzyme activity.(ABSTRACT TRUNCATED AT 250 WORDS)     

A high affinity Ca2+-stimulated and Mg2+-dependent ATPase in rat corpus luteum plasma membrane fractions

Plasma membrane fractions from rat corpus luteum contain two kinds of Ca2+-stimulated ATPase, one having a high affinity for Ca2+, the other a low affinity for Ca2+. The high affinity ATPase had a specific Ca2+ requirement with a K 1/2 of 0.2 to 0.3 microM; it had a Vmax of 105 nmol min-1 mg-1 and distributed, upon subcellular fractionation, with recognized plasma membrane enzymes. The properties of this enzyme indicate that it is a CA2+ extrusion pump. The low affinity pump (K 1/2 for Ca2+, about 15 microM) was nonspecific, being stimulated equally well by Ca2+ of Mg2+; its function is unknown. Although the high affinity ATPase resembled the erythrocyte Ca2+-pumping ATPase in the properties mentioned above, it differed in that it failed to respond to Mg2+ or calmodulin. The lack of response to Mg2+ was due to the enzyme's retention of endogenous Mg2+; it did, after incubation with chelators, show a Mg2+ requirement. However, we were unable to show any effect of added calmodulin or trifluoperazine. This failure may be related to the high content of tightly bound calmodulin in these membranes. Much of this calmodulin could not be extracted even by washing with 1 mM EGTA and/or 0.1% (w/v) Triton X-100. This enzyme, the erythrocyte enzyme, and the adipocyte plasma membrane Ca2+ ATPase all belong to the class of Ca2+ ATPases with plasma membrane distribution and high affinity for Ca2+, indicating that they are Ca2+ extrusion pumps. However, the data indicate that tissue-specific differences exist within this class, with the enzyme from adipocytes and rat corpus luteum belonging to a subclass in which the requirement for Mg2+ and any response to calmodulin are difficult to demonstrate.     

Characterization of rat heart plasma membrane Ca2+/Mg2+ ATPase

The Ca2+/Mg2+ ATPase of rat heart plasma membrane was activated by millimolar concentrations of Ca2+ or Mg2+; other divalent cations also activated the enzyme but to a lesser extent. Sodium azide at high concentrations inhibited the enzyme by about 20%; oligomycin at high concentrations also inhibited the enzyme slightly. Trifluoperazine at high concentrations was found inhibitory whereas trypsin treatment had no significant influence on the enzyme. The rate of ATP hydrolysis by the Ca2+/Mg2+ ATPase decayed exponentially; the first-order rate constants were 0.14-0.18 min-1 for Ca2+ ATPase activity and 0.15-0.30 min-1 for Mg2+ ATPase at 37 degrees C. The inactivation of the enzyme depended upon the presence of ATP or other high energy nucleotides but was not due to the accumulation of products of ATP hydrolysis. Furthermore, the inactivation of the enzyme was independent of temperature below 37 degrees C. Con A when added into the incubation medium before ATP blocked the ATP-dependent inactivation; this effect was prevented by alpha-methylmannoside. In the presence of low concentrations of detergent, the rate of ATP hydrolysis was reduced while the ATP-dependent inactivation was accelerated markedly. Both Con A and glutaraldehyde decreased the susceptibility of Ca2+/Mg2+ ATPase to the detergent. These results suggest that the Ca2+/Mg2+ ATPase is an intrinsic membrane protein which may be regulated by ATP.     

Purification of the Ca2+-and Mg2+-requiring ATPase from rat brain synaptic plasma membrane.

A Ca2+-ATPase (Ca2+- and Mg2+-requiring ATPase) was purified from a synaptic plasma-membrane fraction of rat brain. This enzyme had properties similar to those of plasma-membrane Ca2+-ATPases from other organs: its splitting of ATP was dependent on both Ca2+ and Mg2+, it bound in a Ca2+-dependent fashion to calmodulin-Sepharose and it cross-reacted with specific antibodies raised against human erythrocyte-membrane Ca2+-ATPase. It had an apparent Mr of 138 000, similar to those of plasma-membrane ATPases from human erythrocyte and from dog heart sarcolemma. Previous high-Ca2+-affinity ATPases observed in brain had Mr 100 000; in at least one case, such an ATPase probably represented a different type of enzyme, derived from coated vesicles.     

A model for the phosphorylation of the Ca2+ + Mg2+-activated ATPase by phosphate.

We have shown that changes in fluorescence intensity for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate following the addition of Ca2+ can give the ratio of the two conformations (E1 and E2) of the ATPase. We show that the fluorescence response to Ca2+ is unaffected by Mg2+, phosphate or K+, implying that these ions bind equally well to the E1 and E2 conformations. A model is presented for phosphorylation of the ATPase by phosphate as a function of pH, Mg2+, K+ and Ca2+.     

Purification and composition of Ca2+/Mg2+ ATPase from rat heart plasma membrane

The Ca²⁺/Mg²⁺ ATPase, which is activated by millimolar concentrations of Ca²⁺ or Mg²⁺, was solubilized from rat heart plasma membrane by employing lysophosphatidylcholine, CHAPS, Nal, EDTA and Tris-HCI at pH 7.4. The enzyme was purified by sucrose density gradient, Affi-Gel Blue column and Sepharose 6B column chromatography. The purified enzyme was seen as a single peptide band in the sodium dodecyl sulfate polyacrylamide gel electrophoresis with a molecular weight of about 90,000. The apparent molecular weight of the holoenzyme as determined under non-dissociating conditions by gel filtration on Sepharose 6B column was about 180,000 indicating two subunits. The enzyme was insensitive to ouabain, verapamil, vanadate, oligomycin, N,N-dicyclohexylcarbodiimide and NaN₃, but was markedly inhibited by 20 µM gramacidin S and 50 µM trifluoperazine. Analysis of the purified Ca²⁺/Mg²⁺ ATPase revealed the presence of 17 amino acids where leucine, glutamic acid and aspartic acid were the major components and histidine, cysteine and methionine were the minor components. The purified enzyme was associated with 19.7 µmol phospholipid/mg protein which was 60 times higher than the phospholipid content in plasma membrane. The cholesterol content in the purified enzyme preparation was 0.75 µmol/mg protein and this represented an 8-fold enrichment over plasma membrane. The glycoprotein nature of the enzyme was evident from the positive periodic acid-Schiff staining of the purified Cau2+/Mg^ATPase in the sodium dodecyl sulfate polyacrylamide gel. The polysaccharide content of the enzyme was enriched 8-fold over plasma membrane; neurominidase treatment decreased the polysaccharide content. Concanavalin A prevented the ATP-dependent inactivation of the purified Ca²⁺/Mg²⁺ ATPase and was found to bind to the purified enzyme with a K_D of 576 nM and Bmax of 4.52 nmol/mg protein. The results indicate that Ca²⁺/Mg²⁺ ATPase is a glycoprotein and contains a large amount of lipids.     

A kinetic model for Ca2+ efflux mediated by the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

We present a model for Ca2+ efflux from vesicles of sarcoplasmic reticulum (SR). It is proposed that efflux is mediated by the Ca2+ + Mg2+-activated ATPase that is responsible for Ca2+ uptake in this system. In the normal ATPase cycle of the ATPase, phosphorylation of the ATPase is followed by a conformational change in which the Ca2+-binding sites change from being outward-facing and of high affinity to being inward-facing and of low affinity. To mediate Ca2+ efflux, it is proposed that the ATPase can adopt a conformation in which the Ca2+-binding sites are of low affinity but still outward-facing. It is shown that experimental data on the rates of Ca2+ efflux can be simulated in terms of this model, with Ca2+-binding-site affinities previously proposed to explain ATPase activity [Gould, East, Froud, McWhirter, Stefanova & Lee (1986) Biochem. J. 237, 217-227]. Effects of Mg2+ and adenine nucleotides on efflux rates are explained. It is suggested that Ca2+ efflux from SR mediated by the ATPase could be important in excitation-contraction coupling in skeletal muscle.     

Conformational transitions in the Ca2+ + Mg2+-activated ATPase and the binding of Ca2+ ions.

We have studied the fluorescence of the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum labelled with fluorescein isothiocyanate. The change in intensity of fluorescein fluorescence caused by addition of Ca2+ to the labelled ATPase can be interpreted in terms of a two-conformation model for the ATPase, one conformation (E1) having a high affinity for Ca2+, the other (E2) a low affinity. Effects of Ca2+ as a function of pH allow an estimate of the effect of pH on the E1/E2 ratio, consistent with kinetic studies. A model is presented for binding of Ca2+ to the ATPase as a function of pH that is consistent both with the data on the E1/E2 equilibrium and with literature data on Ca2+ binding.     

A kinetic model for the Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum.

The Ca2+ + Mg2+-activated ATPase of sarcoplasmic reticulum exhibits complex kinetics of activation with respect to ATP. ATPase activity is pH-dependent, with similar pH-activity profiles at high and low concentrations of ATP. Low concentrations of Ca2+ in the micromolar range activate the ATPase, whereas activity is inhibited by Ca2+ at millimolar concentrations. The pH-dependence of this Ca2+ inhibition and the effect of the detergent C12E8 (dodecyl octaethylene glycol monoether) on Ca2+ inhibition are similar to those observed on activation by low concentrations of Ca2+. On the basis of these and other studies we present a kinetic model for the ATPase. The ATPase is postulated to exist in one of two conformations: a conformation (E1) of high affinity for Ca2+ and MgATP and a conformation (E2) of low affinity for Ca2+ and MgATP. Ca2+ binding to E2 and to the phosphorylated form E2P are equal. Proton binding at the Ca2+-binding sites in the E1 and E2 conformations explains the pH-dependence of Ca2+ effects. Binding of MgATP to the phosphorylated intermediate E1'PCa2 and to E2 modulate the rates of the transport step E1'PCa-E2'PCa2 and the return of the empty Ca2+ sites to the outside surface of the sarcoplasmic reticulum, as well as the rate of dephosphorylation of E2P. Only a single binding site for MgATP is postulated.