Occlusion of Ca2+ in soluble monomeric sarcoplasmic reticulum Ca2+-ATPase

Sarcoplasmic reticulum Ca2+-ATPase solubilized in monomeric form by nonionic detergent was reacted with CrATP in the presence of 45Ca2+. A Ca2+-occluded complex formed, which was stable during high performance liquid chromatography in the presence of excess non-radioactive Ca2+. The elution position corresponded to monomeric Ca2+-ATPase. It is concluded that a single Ca2+-ATPase polypeptide chain provides the full structural basis for Ca2+ occlusion.     

Interaction of alkaline metal ions with Ca(2+)-binding sites of Ca(2+)-ATPase of sarcoplasmic reticulum: 23Na-NMR studies.

The analysis of the 23Na-NMR signal shape variations in the presence of vesicles of light sarcoplasmic reticulum (SR) shows the existence of sodium sites on the membranes with Kd values of about 10 mM. Other monovalent cations displace Na+ from SR fragments in a competitive manner according to the row K+ greater than Rb+ greater than Cs+ greater than Li+. Calcium ions also reduce Na+ binding, the Na+ desorption curve being of a two-stage nature, which, as suggested, indicates the existence of two types of Ca(2+)-sensitive Na+ binding sites (I and II). Sites of type I and II are modified by Ca2+ in submicromolar and millimolar concentrations, respectively. Analysis of sodium (calcium) desorption produced by calcium (sodium) allowed us to postulate the competition of these two cations for sites I and identity of these sites to high-affinity Ca(2+)-binding ones on the Ca(2+)-ATPase. Sites I weakly interact with Mg2+ (KappMg approximately 30 mM). Reciprocal effects of sodium and calcium on binding of each other to sites II cannot be described by a simple competition model, which indicates nonhomogeneity of these sites. A portion of sites I (approximately 70%) interacts with Mg2+ (KappMg = 3-4 mM). The pKa value of sites II is nearly 6.0. The number of sites II is three times greater than that of sites I. In addition, sites with intermediate affinity for Ca2+ were found with Kd values of 2-5 microM. These sites were revealed due to the reducing of the sites II affinity for Na+ upon Ca2+ binding to SR membranes. It can thus be concluded that in nonenergized SR there are binding sites for monovalent cations of at least three types: (1) sites I (which also bind Ca2+ at low concentrations), (2) magnesium-sensitive sites II and (3) magnesium-insensitive sites II.     

Interaction of alkali metal ions with Ca2+-binding center of Ca2+- ATPase of the sarcoplasmic reticulum in skeletal muscles

Sodium ion interaction with sarcoplasmic reticulum (SR) membranes leads to considerable alterations of the [23Na]NMR lineshape. Na+ binding to SR in the presence of Ca2+ and H+ is well described by a model which postulates a competitive ion binding to high and low affinity sites of Ca2+-ATPase. The dissociation constant, Kd, for high and low affinity sites is 5 and 10 mM, respectively, for Na+ and (3-5).10(-8) and 1.5.10(-3) M, respectively, for Ca2+. The pK value for high and low affinity sites is 7.3 and 6.1, respectively. Other alkaline metal ions compete with Na+ for the low affinity sites of Ca2+-ATPase; their affinities decrease in the following order: Na+ = K+ greater than Rb+ greater than Cs greater than Li+. Some of the Na+ binding sites (approximately 10%) do not interact with Ca2+.     

Demonstration of two different reactive sulfhydryl groups in the ATP-binding sites of Ca2+-ATPase of sarcoplasmic reticulum by disulfides of thioinosine triphosphates

1. The disulfide of thioinosine triphosphate, (SnoPPP)2, is a substrate of the Ca2+-pump and the Ca2+-ATPase of sarcoplasmic reticulum (Km = 400 microM). 2. Inactivation of Ca2+-ATPase by the beta,gamma-methylene diphosphonate analogue of the disulfide of thioinosine triphosphate, (SnoPP[CH2]P)2, in the presence of (Ca2+ + Mg2+ + K+) is preceeded by a dissociable enzyme inhibitor complex with a dissociation constant of 130 microM for a low-affinity binding site. ATP protected Ca2+-ATPase against the inactivation under these conditions with a dissociation constant of 140 microM. 3. Kinetic analysis of the inactivations of Ca2+-ATPase by (SnoPP[CH2]P)2 in the absence of Ca2+ and Mg2+ but the presence of K+ and EGTA led to the appearance of two nucleotide binding sites with two different inactivation velocities. Inactivation rate constants k2 were found for the rapid inactivating part (k2' = 1.44 X 10(-2) s-1) and the slow inactivating part (k2" = 1.15 X 10(-3) s-1). From the protective effect of ATP under these conditions a high-affinity (Kd = 48.78 microM) and a low-affinity ATP binding site (Kd = 114 microM) were apparent. 4. The affinity of the analogues to the enzyme is decreased in the sequence: (SnoPPP)2 > (SnoPP[NH]P)2 > (SnoPP[CH2]P)2 > (SnoP)2. 5. (SnoPPP)2-inactivated Ca2+-ATPase was reactivated by incubation with dithiothreitol. 6. Inactivation of Ca2+-ATPase by [gamma-32P](SnoPPP)2 in the presence of (Mg2+ + K+ + Ca2+) or (EGTA + K+) was accompanied by the incorporation of hydroxylamine-insensitive radioactivity into the acid-precipitable protein. The enzyme-bound [gamma-32P]SnoPPP was cleaved by dithiothreitol. 7. It is concluded that (SnoPPP)2 and its non-hydrolyzable analogues (SnoPP[NH]P)2 and (SnoPP[CH2]P)2 act as ATP affinity labels and form mixed disulfides with a sulfhydryl group within the active site.     

Inhibition of plasma membrane Ca(2+)-ATPase by CrATP. LaATP but not CrATP stabilizes the Ca(2+)-occluded state

The bidentate complex of ATP with Cr(3+), CrATP, is a nucleotide analog that is known to inhibit the sarcoplasmic reticulum Ca(2+)-ATPase and the Na(+),K(+)-ATPase, so that these enzymes accumulate in a conformation with the transported ion (Ca(2+) and Na(+), respectively) occluded from the medium. Here, it is shown that CrATP is also an effective and irreversible inhibitor of the plasma membrane Ca(2+)-ATPase. The complex inhibited with similar efficiency the Ca(2+)-dependent ATPase and the phosphatase activities as well as the enzyme phosphorylation by ATP. The inhibition proceeded slowly (T(1/2)=30 min at 37 degrees C) with a K(i)=28+/-9 microM. The inclusion of ATP, ADP or AMPPNP in the inhibition medium effectively protected the enzyme against the inhibition, whereas ITP, which is not a PMCA substrate, did not. The rate of inhibition was strongly dependent on the presence of Mg(2+) but unaltered when Ca(2+) was replaced by EGTA. In spite of the similarities with the inhibition of other P-ATPases, no apparent Ca(2+) occlusion was detected concurrent with the inhibition by CrATP. In contrast, inhibition by the complex of La(3+) with ATP, LaATP, induced the accumulation of phosphoenzyme with a simultaneous occlusion of Ca(2+) at a ratio close to 1.5 mol/mol of phosphoenzyme. The results suggest that the transport of Ca(2+) promoted by the plasma membrane Ca(2+)-ATPase goes through an enzymatic phospho-intermediate that maintains Ca(2+) ions occluded from the media. This intermediate is stabilized by LaATP but not by CrATP.     

Characterization of CrATP-induced calcium occlusion in membrane-bound and soluble monomeric sarcoplasmic reticulum Ca2+-ATPase

Occlusion of Ca2+ induced by beta, gamma-bidentate CrATP in membrane bound and in soluble monomeric sarcoplasmic reticulum Ca2+-ATPase was studied by previously developed filtration and HPLC techniques (Vilsen and Andersen (1986) Biochim. Biophys. Acta 855, 429-431). Activation of Ca2+ occlusion occurred at micromolar free Ca2+ and depended on the concentration of Ca2+, H+ and Mg2+ in a similar way as activation of Ca2+ transport and equilibrium Ca2+ binding to high-affinity Ca2+ transport sites. The slopes of the Ca2+ titration curves indicated that Ca2+ binding is a cooperative process both in membraneous and in soluble monomeric enzyme. At alkaline pH and absence of Mg2+, occlusion of Ca2+ was inhibited by 1 mM Ca2+ in membrane-bound, but not in soluble monomeric Ca2+-ATPase. Parallel studies of phosphorylation from [gamma-32P]CrATP indicated a stoichiometry of 2 mol Ca2+ occluded per mol Ca2+-dependent EP formed, at saturating as well as at desaturating Ca2+ concentrations. Tryptic digestion of the CrATP induced Ca2+ occluded complex indicated that it belongs to the E1 conformational class (E1P). In the absence of Ca2+ and Mg2+, but presence of CrATP the conformational state was E2. When Mg2+ was added together with CrATP at alkaline pH the conformation was shifted in direction of E1.     

Inactivation of (Na+ + K+)-ATPase by chromium(III) complexes of nucleotide triphosphates

(Na+ + K+)-ATPase from beef brain and pig kidney are slowly inactivated by chromium(III) complexes of nucleotide triphosphates in the absence of added univalent and divalent cations. The inactivation of (Na+ + K+)-ATPase activity was accompanied by a parallel decrease of the associated K+-activated p-nitrophenylphosphatase and a parallel loss of the capacity to form, Na+-dependently, a phosphointermediate from [gamma-32P]ATP. The kinetics of inactivation and of phosphorylation with [gamma-32P]CrATP and [alpha-32P]CrATP are consistent with the assumption of the formation of a dissociable complex of CrATP with the enzyme (E) followed by phosphorylation of the enzyme: formula: (see text). The dissociation constant of the CrATP complex of the pig kidney enzyme at 37 degrees C was 43 microM. The inactivation rate constant (k + 2 = 0.033 min-1) was in the range of the dissociation rate constant kd of ADP from the enzyme of 0.011 min-1. The phosphoenzyme was unreactive towards ADP as well as to K+. No hydrolysis of the native isolated phosphoenzyme was observed within 6 h under a variety of conditions, but high concentrations of Na+ reactivated it slowly. The capacity of the Cr-phosphoenzyme of 121 +/- 18 pmol/unit enzyme is identical with the capacity of the unmodified enzyme to form, Na+-dependently, a phosphointermediate. The Cr-phosphoenzyme behaved after acid denaturation like an acylphosphate towards hydroxylamine, but the native phosphoenzyme was not affected by it. ATP protected the enzyme against the inactivation by CrATP (dissociation constant of the enzyme ATP complex = 2.5 microM) as well as low concentrations of K+. CrATP was a competitive inhibitor of (Na+ + K+)-ATPase. It is concluded that CrATP is slowly hydrolyzed at the ATP-binding site of (Na+ + K+)-ATPase and inactivates the enzyme by forming an almost non-reactive phosphoprotein at the site otherwise needed for the Na+-dependent proteinkinase reaction as the phosphate acceptor site.     

Chromium(III)ATP inactivating (Na+ + K+)-ATPase supports Na+-Na+ and Rb+-Rb+ exchanges in everted red blood cells but not Na+,K+ transport

The chromium(III) complex of ATP, an MgATP complex analogue, inactivates (Na+ + K+)-ATPase by forming a stable chromo-phosphointermediate. The rate constant k2 of inactivation at 37 degrees C of the beta, gamma-bidentate of CrATP is enhanced by Na+ (K0.5 = 1.08 mM), imidazole (K0.5 = 15 mM) and Mg2+ (K0.5 = 0.7 mM). These cations did not affect the dissociation constant of the enzyme-chromium-ATP complex. The inactive chromophosphoenzyme is reactivated slowly by high concentrations of Na+ at 37 degrees C. The half-maximal effect on the reactivation was reached at 40 mM NaCl, when the maximally observable reactivation was studied. However, 126 mM NaCl was necessary to see the half-maximal effect on the apparent reactivation velocity constant. K+ ions hindered the reactivation with a Ki of 70 microM. Formation of the chromophosphoenzyme led to a reduction of the Rb+ binding sites and of the capacity to occlude Rb+. The beta, gamma-bidentate of chromium(III)ATP (Kd = 8 microM) had a higher than the alpha, beta, gamma-tridentate of chromium(III)ATP (Kd = 44 microM) or the cobalt tetramine complex of ATP (Kd = 500 microM). The beta, gamma-bidentate of the chromium(III) complex of adenosine 5'-[beta, gamma-methylene]triphosphate also inactivated (Na+ + K+)ATPase. Although CrATP could not support Na+, K+ exchange in everted vesicles prepared from human red blood cells, it supported the Na+-Na+ and Rb+-Rb+ exchange. It is concluded that CrATP opens up Na+ and K+ channels by forming a relatively stable modified enzyme-CrATP complex. This stable complex is also formed in the presence of the chromium complex of adenosine 5'-[beta, gamma-methylene]triphosphate. Because the beta, gamma-bidentate of chromium ATP is recognized better than the alpha, beta, gamma-tridentate, it is concluded that the triphosphate site recognizes MgATP with a straight polyphosphate chain and that the Mg2+ resides between the beta- and the gamma-phosphorus. The enhancement of inactivation by Mg2+ and Na+ may be caused by conformational changes at the triphosphate site.     

Low-temperature studies of the sarcoplasmic reticulum calcium pump. Mechanisms of calcium binding

The mechanism of the sarcoplasmic reticulum Ca2+-ATPase was investigated at low temperatures (0 to -12 degrees C). Transient states of the enzyme were studied by two complementary techniques: intrinsic protein fluorescence and rapid filtration on Millipore filters. Intrinsic fluorescence was used to distinguish conformational states of the protein and to evaluate the rate of conversion between these states. Filtrations were used to measure the evolution of the active sites during the transition; the time resolution was 2-5 s. At sub-zero temperatures this time is shorter than the lifetime of most of the enzymatic states which have been detected. In this paper the mechanism of Ca2+ binding to the protein is investigated in the absence of nucleotides. Two basic experiments are described; (1) Kinetics of calcium binding and dissociation over a wide range of calcium concentration. (2) Kinetics of calcium exchange (45Ca2+ in equilibrium 40Ca2+) at constant concentration. The results obtained in the first series of experiments are consistent with a sequential binding to two interacting Ca2+ binding sites. Calcium ions have very fast access to a site with low apparent affinity (Kd approximately 25 microM). Occupation of this site induces a slow conformational change which increased its apparent affinity and reveals a second site of high apparent affinity. At equilibrium the two sites are not equivalent in terms of rate of exchange. Two different rates were detected k fast greater than 0.2 s-1, k slow approximately 0.015 s-1 at -10 degrees C. Removal of Ca2+ from the fast exchanging site by addition of EGTA accelerates the rate of release of the slow exchanging one. A model is proposed with two interacting Ca2+-binding sites. A set of parameters has been obtained which produces correctly the Ca2+-binding curve and the fluorescence level at equilibrium as well as the rate constants of the calcium-induced fluorescence changes over a very wide range of Ca2+ concentrations (0.02 to 150 microM). The non-equivalence of the two classes of site and the meaning of the initial low-affinity binding are discussed.     

Inactivation and phosphorylation of sarcoplasmic reticulum Ca(2+)-ATPase by Mg.ATP analogues Rh(III)-ATP and Co(III)-ATP.

The interaction of sarcoplasmic reticulum Ca(2+)-ATPase with the Mg.ATP analogues Rh(H2O)4ATP and Co(NH3)4ATP have been examined. Co(NH3)4ATP slowly inactivates Ca(2+)-ATPase in a first order process, with a rate constant of 1.13 x 10(-3) s-1 and an apparent inactivation constant, KI, of 32 mM. Rh(H2O)4ATP likewise inactivates sarcoplasmic reticulum Ca(2+)-ATPase, but the plot of reciprocal apparent inactivation rate constants versus 1/[Rh(H2O)4ATP] is biphasic. The chi-intercepts of this plot yield apparent inactivation constants for the inhibition of Ca(2+)-ATPase by Rh(H2O)4ATP of KI1 = 30 microM and KI2 = 221 microM. The corresponding values of k2, the maximal first-order rate constant for inhibition in these two phases, are 1.16 and 2.19 x 10(-4)s-1. Tridentate Rh(H2O)3ATP also inhibits Ca(2+)-ATPase, but only after much longer incubation times. Ca(2+)-ATPase inactivation is accompanied by incorporation of radioactivity from gamma-32P into an acid-precipitable enzyme. Both processes were dependent on the presence of Ca2+ ions and were quenched by excess ATP. The first-order rate constant for inactivation of Ca(2+)-dependent ATPase activity in this experiment was 2.19 x 10(-4)s-1, and the first-order rate constant for Ca(2+)-dependent E-P formation was 2.07 x 10(-4)s-1, in excellent agreement with the value for inactivation. A linear relationship is observed between ATPase inactivation and E-P formation. Moreover, atomic absorption analysis demonstrates that the phosphorylation of Ca(2+)-ATPase by Rh(H2O)4ATP is accompanied by incorporation and tight binding of rhodium, with a stoichiometry of one rhodium incorporated per ATPase molecule phosphorylated. The characteristics of ATPase inactivation and phosphorylation (i.e., Ca2+ dependence, ATP competition, agreement of rate constants, and stoichiometric rhodium incorporation) suggest that Rh(H2O)4ATP is binding to the catalytic nucleotide site on Ca(2+)-ATPase and producing a highly stable, phosphorylated intermediate.     

Interaction of CrATP with the phosphorylation site of the sarcoplasmic reticulum ATPase.

The chromium moiety of gamma,beta-bidentate CrATP slowly accepts a ligand from the sarcoplasmic reticulum Ca-ATPase to form an exchange inert coordination complex (k + 1 = 0.083 min-1; k - 2 = 0.003 min-1, 37 degrees C, 100 microM CaCl2). The stability of the Cr3+ coordinate bonds allowed the complex to be isolated by filtration techniques at neutral pH without acid precipitation. We found 4-5 nmol of [gamma-32P]CrATP to bind to 1 mg of sarcoplasmic reticulum protein with the subsequent occlusion of 7-8 nmol of 45Ca2+. At 37 degrees C, the CrATP.ATPase complex could be formed in the absence of Ca2+, although the reaction was 2-3 times slower than in the presence of Ca2+. Inhibition by Pi, by orthovanadate, and by fluorescein 5'-isothiocyanate verified that the bound CrATP was at the catalytic site. The site of CrATP attachment was found to be on the A tryptic fragment, possibly on the A2 subfragment. It was determined that Ca2+ binding to high affinity sites on the enzyme controls the rate by which the Cr3+ moiety accepts the ligand from the enzyme. The rate of change in the EPR spectrum of iodoacetamide spin-labeled ATPase was shown to follow the rate of ligand acceptance, rather than the binding of Ca2+ and substrate per se. This particular change has been attributed to the formation of an activated complex that is immediately precursory to phosphorylation and indicates here that this complex cannot be properly formed until the metal has been chelated by the enzyme. It is concluded that control over metal chelation (Cr3+ here, Mg2+ in the normal mechanism) is one means by which Ca2+ activates the enzyme.     

Cyclic AMP-dependent protein kinase phosphorylation of cardiac (Na+ + K+)-ATPases. Effect on calcium binding.

1. Calcium binding to (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) preparations from beef and pig heart preparations of varying degrees of purity was measured. 2. Binding was inhibited by Mg2+, Na+ and K+. Inhibition by Na+ and K+ appeared to be due to an ionic strength effect. 3. Four classes of binding sites were identified with Kd values for calcium of about 0.03, 1, 15 and 200 micrometer. 4. Cyclic AMP-dependent phosphorylation of the enzyme by protein kinase (ATP: protamine O-phosphotransferase, EC 2.7.1.70) had no effect on (Na+ + K+)-ATPase activity. 5. Phosphorylation also had no effect on either Kd or Bmax for calcium binding at any of the four sites whether measured in the presence of absence of NaCl or KCl. 6. It is concluded that previous reports of an effect of phosphorylation on calcium binding to a (Na+ + K+)-ATPase preparation may have been due to the presence of membrane material not directly associated with (Na+ + K+)-ATPase.     

Calcium-dependent calcium occlusion in the sarcoplasmic reticulum Ca2+-ATPase. Its enhancement by phosphorylation of the enzyme

45Ca2+-40Ca2+ exchangeability of 45Ca bound to the calcium transport sites of unphosphorylated sarcoplasmic reticulum Ca2+-ATPase at equilibrium has been found to be heterogeneous: Half of the bound calcium is [Ca2+]-dependent in a slowly exchangeable (k less than 0.3 s-1), "occluded" state in the Ca2+-ATPase, and the other calcium is [Ca2+]-independent in a rapidly exchangeable (k approximately 0.3 s-1), "unoccluded" state (Nakamura, J. (1986) Biochim. Biophys. Acta 870, 495-501). In this paper, the two different forms of exchangeable calcium were studied after phosphorylation of the enzyme by ATP without added Mg2+ at pH 7.0 and 0 degree C. By the phosphorylation, the degree of the occlusion became higher (k less than 0.03 s-1). The unoccluded calcium was, however, not significantly affected. The more highly occluded calcium exchanged at the same rate as the decay rate of the phosphoenzyme (EP) in the steady state at a ratio of about 1:1. The occluded calcium was relieved by dephosphorylation of EP by ADP. These results suggest that 1 mol of ADP-sensitive EP more highly occluded 1 mol of calcium, already occluded before phosphorylation. After transformation of ADP-sensitive EP to its ADP-insensitive form by the addition of 20 mM Mg2+ at pH 8.8, the unoccluded calcium was rapidly (k = 0.1-0.3 s-1) released from the transformed EP. However, the occluded calcium was maintained in an occluded state in which the calcium was slowly (k approximately 0.01 s-1) released from the EP without exchange. The results suggest that calcium occlusion in the ADP-sensitive EP is not relieved by the loss of ADP sensitivity of the EP itself.     

Interaction of nucleotides and cations with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum as determined by fluorescence changes of bound 1-anilino-8-naphthalenesulfonate

The changes in fluorescence of 1-anilino-8-naphthalenesulfonate (ANS-) have been used to determine binding of ligands to the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles, isolated from rabbit skeletal muscle. ANS- binds to sarcoplasmic reticulum membranes with an apparent Kd of 3.8 X 10(-5) M. The binding of ANS- had no effect on Ca2+ transport or Ca2+-dependent ATPase activity. EGTA, by binding endogenous Ca2+, increased the fluorescence intensity of bound ANS- by 10-12%. Subsequent addition of ATP, ADP, or Ca2+, in the presence or absence of Mg2+, reversed this change of fluorescence. The binding parameters, as determined by these decreases in fluorescence intensity, were as follows: for ATP, Kd = 1.0 X 10(-5) M, nH = 0.80; for ADP, Kd = 1.2 X 10(-5) M, nH = 0.89; and for Ca2+, Kd = 3.4 X 10(-7) M, nH = 1.8. The binding parameters for ITP and for the nonhydrolyzable analogue, adenyl-5'-yl-beta, gamma-methylene)diphosphate, were similar to those of ATP, but GDP, IDP, CDP, AMP, and cAMP had lower apparent affinities. Millimolar concentrations of pyrophosphate also decreased the fluorescence of bound ANS-, whereas orthophosphate caused a small (2-3%) increase in fluorescence in Ca2+-free media. Vanadate, in the presence of EGTA, decreased the fluorescence of bound ANS-with half-maximal effect at 4 X 10(-5) M. The changes of fluorescence intensity of bound ANS- appear to reflect conformational changes of the (Ca2+, Mg2+)-ATPase, consequent to ligand binding, with the low and high fluorescence intensity species corresponding to the E1 and E2 conformations, respectively. These appear to reflect similar conformational states of the (Ca2+, Mg2+)-ATPase to those reported by changes in intrinsic tryptophan fluorescence (DuPont, Y. (1976) Biochem, Biophys. Res. Commun. 71, 544-550).     

A Ca(2+)-ATPase from rat parotid gland plasma membranes has the characteristics of an ecto-ATPase.

A Ca(2+)-ATPase with an apparent Km for free Ca2+ = 0.23 microM and Vmax = 44 nmol Pi/mg/min was detected in a rat parotid plasma membrane-enriched fraction. This Ca(2+)-ATPase could be stimulated without added Mg2+. However, the enzyme may require submicromolar concentrations of Mg2+ for its activation in the presence of Ca2+. On the other hand, Mg2+ could substitute for Ca2+. The lack of a requirement for added Mg2+ distinguished this Ca(2+)-ATPase from the Ca(2+)-transporter ATPase in the plasma membranes and the mitochondrial Ca(2+)-ATPase. The enzyme was not inhibited by several ATPase inhibitors and was not stimulated by calmodulin. An antibody which was raised against the rat liver plasma membrane ecto-ATPase, was able to deplete this Ca(2+)-ATPase activity from detergent solubilized rat parotid plasma membranes, in an antibody concentration-dependent manner. Immunoblotting analysis of the pellet with the ecto-ATPase antibody revealed the presence of a 100,000 molecular weight protein band, in agreement with the reported ecto-ATPase relative molecular mass. These data demonstrate the presence of a Ca(2+)-ATPase, with high affinity for Ca2+, in the rat parotid gland plasma membranes. It is distinct from the Ca(2+)-transporter, and immunologically indistinguishable from the plasma membrane ecto-ATPase.     

Interaction of Cd2+ with the calmodulin-activated (Ca2+ + Mg2+)-ATPase activity of human erythrocyte ghosts

Treatment of erythrocyte ghosts with micromolar concentrations of Cd2+ results in a noncompetitive inhibition of the calmodulin-dependent (Ca2+ + Mg2+)-ATPase activity. Higher concentrations of Cd2+ are required for inhibition of the (Ca2+ + Mg2+)-ATPase activity of calmodulin-depleted ghosts. The interaction of Cd2+ is time-dependent with an apparent rate constant around 0.12/min. The inhibition is relieved by addition of EGTA with a rate constant around 0.15/min. If Cd2+ is allowed to interact with calmodulin prior to the association of the protein with the ghosts, the inhibition is mainly competitive. The results suggest that the inhibitory effect caused by Cd2+ is due to an interaction with calmodulin. The slow interaction of Cd2+ suggests that calmodulin bound to the (Ca2+ + Mg2+)-ATPase is inaccessible to Cd2+.     

Characterisation of thapsigargin-releasable Ca(2+) from the Ca(2+)-ATPase of sarcoplasmic reticulum at limiting [Ca(2+)

The Ca(2+) binding sites of the Ca(2+)-ATPase of skeletal muscle sarcoplasmic reticulum (SR) have been identified as two high-affinity sites orientated towards the cytoplasm, two sites of low affinity facing the lumen, and a transient occluded species that is isolated from both membrane surfaces. Binding and release studies, using (45)Ca(2+), have invoked models with sequential binding and release from high- and low-affinity sites in a channel-like structure. We have characterised turnover conditions in isolated SR vesicles with oxalate in a Ca(2+)-limited state, [Ca(2)](lim), where both high- and low-affinity sites are vacant in the absence of chelators (Biochim. Biophys. Acta 1418 (1999) 48-60). Thapsigargin (TG), a high-affinity specific inhibitor of the Ca(2+)-ATPase, released a fraction of total Ca(2+) at [Ca(2+)](lim) that accumulated during active transport. Maximal Ca(2+) release was at 2:1 TG/ATPase. Ionophore, A23187, and Triton X-100 released the rest of Ca(2+) resistant to TG. The amount of Ca(2+) released depended on the incubation time at [Ca(2+)](lim), being 3.0 nmol/mg at 20 s and 0.42 nmol/mg at 1000 s. Rate constants for release declined from 0. 13 to 0.03 s(-1). The rapidly released early fraction declined with time and k=0.13 min(-1). Release was not due to reversal of the pump cycle since ADP had no effect; neither was release impaired with substrates acetyl phosphate or GTP. A phase of reuptake of Ca(2+) followed release, being greater with shorter delay (up to 200 s) following active transport. Reuptake was minimal with GTP, with delays more than 300 s, and was abolished by vanadate and at higher [TG], >5 microM. Ruthenium red had no effect on efflux, indicating that ryanodine-sensitive efflux channels in terminal cisternal membranes are not involved in the Ca(2+) release mechanism. It is concluded that the Ca(2+) released by TG is from the occluded Ca(2+) fraction. The Ca(2+) occlusion sites appear to be independent of both high-affinity cytoplasmic and low-affinity lumenal sites, supporting a multisite 'in line' sequential binding mechanism for Ca(2+) transport.     

Two-step internalization of Ca2+ from a single E approximately P.Ca2 species by the Ca2+-ATPase

Phosphorylation by ATP of E.*Ca2 (sarcoplasmic reticulum vesicles (SRV) with bound 45Ca2+) during 5-10 ms leads to the occlusion of 2 *Ca2+/EPtot [quench by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) alone] in both "empty" (10 microM free Ca2+in) or "loaded" SRV (20-40 mM free Ca2+in). The rate of Ca2+ "internalization" from the occluded E approximately P.*Ca2 was measured by using an ADP + EGTA quench; a *Ca2+ ion that is not removed by this quench is defined as internalized. In the presence of 20-40 mM unlabeled Ca2+ inside SRV, 1 *Ca2+/EPtot is internalized from 45Ca-labeled E approximately P.*Ca2 with a first-order rate constant of kl = 34 s-1. Empty SRV take up 2 *Ca2+/EPtot with the same initial rate, but the overall rate constant is kobsd = 17 s-1. The apparent rate constant (kb = 17 s-1) for internalization of the second *Ca2+ is inhibited by [Ca]in, with K0.5 approximately 1.3 mM and a Hill coefficient of n = 1.1. These data show that the two Ca2+ ions are internalized sequentially, presumably from separate sequential sites in the channel. [32P]EP.Ca2 obtained by rapid mixing of E.Ca2 with [gamma-32P]ATP and EGTA disappears in a biphasic time course with a lag corresponding to approximately 34 s-1, followed by EP* decay with a rate constant of approximately 17 s-1. This shows that both Ca2+ ions must be internalized before the enzyme changes its specificity for catalysis of phosphoryl transfer to water instead of to ADP. Increasing the concentration of ATP from 0.25 to 3 mM accelerates the rate of 45Ca2+ internalization from 34 to 69 s-1 for the first Ca2+ and from 17 to 34 s-1 for the second Ca2+. High [ATP] also accelerates both phases of [32P]EP.Ca2 disappearance by the same factor. The data are consistent with a single form of ADP-sensitive E approximately P.Ca2 that sequentially internalizes two ions. The intravesicular volume was estimated to be 2.0 microL/mg, so that one turnover of the enzyme gives 4 mM internal [Ca2+].     

Existence of high- and low-affinity vanadate-binding sites on Ca(2+)-ATPase of the sarcoplasmic reticulum.

The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca(2+)-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1 microM, and the low-affinity sites one of 36 microM. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca(2+)-ATPase. These results indicate that 1 mol of Ca(2+)-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxy-ethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13 microM. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca(2+)-ATPase. Vanadate binding to the solubilized Ca(2+)-ATPase was competitively inhibited by inorganic phosphate.(ABSTRACT TRUNCATED AT 250 WORDS)