Biphasic kinetics of sarcoplasmic reticulum Ca2+-ATPase and the detergent-solubilized monomer

The mechanism of ATP hydrolysis was studied at 0 degrees C and pH 7.5 using purified leaky vesicles of sarcoplasmic reticulum Ca2+-ATPase and enzyme solubilized in monomeric form with high concentrations of octaethylene glycol monododecyl ether (C12E8). The enzyme reaction of membranous Ca2+-ATPase was characterized by an initial burst in the hydrolysis of ATP and modulated by millimolar concentrations of ATP. For detergent-solubilized Ca2+-ATPase no burst and moderate low affinity modulation was observed, but the reaction was activated both at low (phosphorylating) and intermediate (K0.5 = 0.06 mM) ATP concentrations. A study of the partial reactions indicated that the effects of detergent and ATP were attributable to activation of the E1P----E2P transition which was rate-limiting. E32P dephosphorylation of membranous Ca2+-ATPase and the detergent-solubilized monomer comprised both a slow and a rapid component. The inhibitory effect of high Ca2+ was correlated with the development of a dominant contribution of slow phase dephosphorylation and with ATP-induced extra binding of Ca2+ binding which presumably takes place at the phosphorylation site (ECaP). Ca2+ was bound with lesser affinity to detergent-solubilized Ca2+-ATPase but with qualitatively the same characteristics as to membranous ECaP. Either Ca2+ or Mg2+ was required for dephosphorylation, also after detergent solubilization. It is concluded that ATP hydrolysis occurs by the same steps for membranous and monomeric Ca2+-ATPase and involves formation of either EMgP or ECaP as reaction intermediates, leading to biphasic kinetics, which, therefore, cannot be taken as evidence of an oligomeric function of the enzyme.     

Effects of adenyl-5'-imidodiphosphate and vanadate Ion on the intermolecular cross-linking of Ca2(+)-ATPase in the sarcoplasmic reticulum membrane with N,N'-(1,4-phenylene)bismaleimide

The functional significance of the molecular interaction of Ca2(+)-ATPase in the sarcoplasmic reticulum (SR) membrane was examined using intermolecular cross-linking of Ca2(+)-ATPase with N,N'-(1,4-phenylene)bismaleimide (PBM). When SR vesicles were allowed to react with 1 mM PBM at pH 7 and 23 degrees C for various intervals and subjected to SDS-PAGE, the amount of the major band of monomeric ATPase decreased with a half life of about 20 min. Higher orders of oligomers were concurrently formed without accumulation of any particular species of oligomer. When SR vesicles were allowed to react with 1 mM PBM in the presence of 1 mM adenyl-5'-imidodiphosphate (AMP-PNP), the rate of oligomerization was markedly reduced and the amount of dimeric Ca2(+)-ATPase increased with time. After 1 h, more than 40% of the Ca2(+)-ATPase had accumulated in the dimeric form. When 1 mol of fluorescein isothiocyanate (FITC) was bound per mol of ATPase, the effects of AMP-PNP on the cross-linking with PBM were completely abolished. When SR vesicles were treated with PBM in the presence of 0.1 mM vanadate in Ca2+ free medium, the oligomerization of the Ca2(+)-ATPase by PBM was strongly inhibited. The vanadate effect on the cross-link formation was completely removed by the presence of Ca2+ and AMP-PNP in the reaction medium. When SR vesicles were pretreated with PBM in the presence of AMP-PNP and digested with trypsin for a short time, the dimeric ATPase was degraded to a peptide with an apparent molecular mass of about 170 kDa.(ABSTRACT TRUNCATED AT 250 WORDS)     

Vanadate oligomer inhibition of passive and active Ca2+ translocation by the Ca2+ pump of sarcoplasmic reticulum

'Monovanadate' containing mainly monomeric, dimeric and tetrameric vanadate species or 'decavanadate', containing mainly decameric vanadate species inhibits the passive and the active efflux of Ca2+ through the sarcoplasmic reticulum calcium pump. When the efflux of Ca2+ by sarcoplasmic reticulum vesicles is not associated with ATP synthesis both vanadate solutions inhibit the passive efflux of Ca2+. However, only 'decavanadate' exerts noticeable effects when the efflux of Ca2+ is associated with ATP synthesis being the active efflux of Ca2+ almost completely inhibited by decameric species concentration as low as 40 microM.     

Equilibrium between monomers and oligomers of soluble Ca2+-ATPase during the functional cycle

Molecular sieve HPLC shows that soluble sarcoplasmic reticulum Ca2+-ATPase at low concentrations of the non-ionic detergent octaethylene glycol monododecyl ether exists as monomers in equilibrium with dimers and higher oligomers. Binding of vanadate or ATP as well as phosphoenzyme turnover shifts the equilibrium towards the monomer. This suggests that the Ca2+-pump cycle can occur without transient self-association of Ca2+-ATPase peptides.     

Protein kinase C-dependent phosphorylation of sarcolemmal Ca2(+)-ATPase isolated from bovine aortic smooth muscle

We examined the effect of protein kinase C (PKC)-dependent phosphorylation on Ca2+ uptake and ATP hydrolysis by microsomal as well as purified sarcolemmal Ca2(+)-ATPase preparations isolated from bovine aortic smooth muscle. The phosphorylation was performed by treating these preparations with PKC and saturating concentrations of ATP (or ATP-gamma S), Ca2+, and 12-O-tetradecanoyl phorbol-13-acetate (TPA) at 37 degrees C for 10 min. In microsomes, treatment with PKC enhanced a portion of the Ca2+ uptake activity inhibitable by 10 microM vanadate, by up to about 30%. On the other hand, Ca2(+)-dependent ATPase activity in the purified Ca2(+)-ATPase preparation was stimulated by up to twofold. Up to twofold stimulation by PKC was also observed for the Ca2+ uptake by proteoliposomes reconstituted from purified sarcolemmal Ca2(+)-ATPase and phospholipids. Since these effects were evident only at Ca2+ concentrations between 0.1 to 1.0 microM, we concluded that it was the affinity of the Ca2(+)-ATPase for Ca2+ that was increased by the PKC treatment. Under conditions in which PKC increased Ca2+ pump activity, the sarcolemmal Ca2(+)-ATPase was phosphorylated to a level of about 1 mol per mol of the enzyme. There was good parallelism between the ATPase phosphorylation and the extent of enzyme activation. These results strongly suggest that the activity of the sarcolemmal Ca2+ pump in vascular smooth muscle is regulated through its direct phosphorylation by PKC.     

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.     

Characterization of a calcium-dependent ATPase in Entamoeba invadens

A high-affinity calcium-dependent ATPase (Ca2+-ATPase) was identified in a crude plasma membrane fraction from Entamoeba invadens (IP-1 strain). The Ca2+-ATPase activity was solubilized from the membrane by utilizing the non-ionic detergent octylglucoside. The activity had an apparent half maximal saturation constant of 0.4 +/- 0.05 microM for free calcium. The calcium activation of ATPase activity followed a cooperative mechanism (Hill number of 2.3 +/- 0.13) which suggests that two interacting sites were involved. The high-affinity Ca2+-ATPase appeared to be magnesium-independent, since by lowering contaminant free magnesium with trans-cyclohexane-1,2-diamine-N,N,N',N'-tetraacetic acid did not modify the activity observed with Ca2+. The apparent Km of the enzyme for ATP was 31 microM. The observed activity had an optimum pH of 8.8. The enzyme was insensitive to various agents such as Na+, K+, ouabain, dicyclohexylcarbodiimide, KCN, NaN3, mersalyl, quercetin, ruthenium red and vanadate. Only lanthanum (0.5 mM) inhibited 100% the enzymatic activity. Calmodulin and trifluoperazine at the concentrations tested did not modify the Ca2+-ATPase activity.     

The functional unit of sarcoplasmic reticulum Ca2+-ATPase. Active site titration and fluorescence measurements

The properties of sarcoplasmic reticulum Ca2+-ATPase have been studied after modification of the ATP high affinity binding site with fluorescein isothiocyanate, both in the membranous state and after solubilization with the nonionic detergent, octaethyleneglycol monododecyl ether. Total inactivation of both membrane-bound and solubilized Ca2+-ATPase requires covalent attachment of 1 mol of fluorescein/mol of enzyme (115,000 g of protein) or per binding site for ATP. Sedimentation velocity studies of soluble enzyme showed that both unlabeled and fluorescein-labeled Ca2+-ATPase were present in a predominantly monomeric form. The phosphorylation level of unlabeled Ca2+-ATPase was unchanged by solubilization. Dephosphorylation measurements at 0 degree C indicated that the phosphorylation is an intermediate in the ATPase reaction catalyzed by solubilized Ca2+-ATPase. Fluorescein labeling of half of the Ca2+-ATPase in the membrane did not influence the enzyme kinetics of the remaining unmodified Ca2+-ATPase. Measurements of both fluorescein and tryptophan fluorescence indicated that the soluble monomer of Ca2+-ATPase like the membrane-bound enzyme exists in a Ca2+-dependent equilibrium between two principal conformations (E and E). E (absence of Ca2+) is unstable in the soluble form, but the pCa dependence of the E - E equilibrium is identical with that of the membranous Ca2+-ATPase (pCa0.5 = 6.7 and Hill coefficient 2). These results suggest that the Ca2+-ATPase polypeptides function with a high degree of independence in the membrane.     

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

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

Characterization of a putative Ca2(+)-transporting Ca2(+)-ATPase in the pellicles of Paramecium tetraurelia

In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.     

Inhibition of phosphoenzyme formation from phosphate and sarcoplasmic reticulum Ca(2+)-ATPase by vanadate binding to high- or low-affinity site on the enzyme.

In the preceding paper, we suggested that 1 mol Ca(2+)-ATPase of sarcoplasmic reticulum (SR) contains 0.5 ml of high-affinity vanadate binding sites as well as 0.5 ml of low-affinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (Pi). When vanadate was added to the reaction medium in which the Ca(2+)-ATPase had been phosphorylated by Pi in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17 microM, and reached maximum at 50 microM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of E2P was competitively prevented by Pi with a dissociation constant of 7.4 mM, which was very close to Km for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with Pi at a phosphorylation site on the Ca(2+)-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca(2+)-ATPase.(ABSTRACT TRUNCATED AT 250 WORDS)     

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.     

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum

The interaction of vanadate ions with the Ca-ATPase from sarcoplasmic reticulum vesicles was studied in a native and a fluorescein-labeled ATPase preparation (Pick, U., and Karlish, S. J. D. (1980) Biochim. Biophys. Acta 626, 255-261). Vanadate induced a fluorescence enhancement in a fluorescein-labeled enzyme, indicating that it shifts the equilibrium between the two conformational states of the enzyme by forming a stable E2-Mg-vanadate complex (E2 is the low affinity Ca2+ binding conformational state of the sarcoplasmic reticulum Ca-ATPase). Indications for tight binding of vanadate to the enzyme (K1/2 = 10 microM) in the absence of Ca2+ and for a slow dissociation of vanadate from the enzyme in the presence of Ca2+ are presented. The enzyme-vanadate complex was identified by the appearance of a time lag in the onset of Ca2+ uptake and by a slowing of the fluorescence quenching response to Ca2+. Ca2+ prevented the binding of vanadate to the enzyme. Pyrophosphate (Kd = 2 mM) and ATP (Kd = 25 microM) competitively inhibited the binding of vanadate, indicating that vanadate binds to the low affinity ATP binding site. Binding of vanadate inhibited the high affinity Ca2+ binding to the enzyme at 4 degrees C. Vanadate also inhibited the phosphorylation reaction by inorganic phosphate (Ki = 10 microM) but had no effect on the phosphorylation by ATP. It is suggested that vanadate binds to a special region in the low affinity ATP binding site which is exposed only in the E2 conformation of the enzyme in the absence of Ca2+ and which controls the rate of the conformation transition in the dephosphorylated enzyme. The implications of these results to the role of the low affinity ATP binding sites are discussed.     

Simultaneous binding of calcium and vanadate to the Ca2+-ATPase of sarcoplasmic reticulum

The interaction of vanadate with the Ca2+-ATPase of sarcoplasmic reticulum vesicles has been studied by making use of the ATPase activity as a measure of uncomplexed enzyme. The binding/dissociation is slow, so that initial rates can be used to study the equilibrium binding. The results indicate that in addition to a Ca2+-free complex E.Van (KV = 0.4 microM), there must also be a Ca2+-enzyme-vanadate complex (K'V = 7 microM). This observation is confirmed by the difference between the kinetics of decay of activity on vanadate addition, and on addition of ATP to enzyme preincubated with vanadate and Ca2+, which requires two enzyme-vanadate complexes. ATP increases the apparent affinity of the enzyme for vanadate by inducing calcium release. Upper limits for the kinetic parameters for vanadate binding and dissociation are estimated.     

Calcium transport and phosphorylated intermediate of (Ca2+ + Mg2+)-ATPase in plasma membranes of rat liver

We have identified and characterized calcium transport and the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in plasma membrane vesicles prepared from rat liver. The calcium transport did not absolutely require the presence of oxalate and was completely inhibited by 1 microM of ionophore A23187. Oxalate, which serves as a trapping agent in calcium uptake of skeletal muscle and liver microsomes, was not absolutely required to maintain the net accumulation of calcium. The Vmax and Km for calcium uptake were 35.2 +/- 10.1 pmol of calcium/mg of protein/min, and 17.6 +/- 2.5 nM of free calcium, respectively. Ten mM magnesium was required for the maximal accumulation of calcium. Substitution of 5 and 10 mM ADP, CTP, GTP, and UTP for ATP could not support calcium uptake. The calcium uptake was not affected by 0.5 mM ouabain, 20 mM azide, or 2 micrograms/ml of oligomycin but was inhibited in a dose-dependent fashion by vanadate, with a Ki of approximately 20 microM for vanadate. The substrate affinities and specificities of this calcium-transport activity suggest that it is closely associated with the (Ca2+ + Mg2+)-ATPase reported in the plasma membranes of liver (Lotersztajn, S., Hanoune, J., and Pecker, F. (1981) J. Biol. Chem. 256, 11209-11215). A calcium-stimulated and magnesium-dependent phosphoprotein was also demonstrated in the same membrane vesicles. The free calcium concentration at which its phosphorylation was half-maximal was 15.5 +/- 5.6 nM. Sodium fluoride, ouabain, sodium azide, oligomycin, adriamycin, and N,N'-dicyclohexylcarbodiimide did not affect its formation while vanadate at 100 microM inhibited the calcium-dependent phosphorylation by approximately 60%. The properties of this phosphoprotein suggest that it may be the phosphorylated intermediate of the (Ca2+ + Mg2+)-ATPase in the plasma membranes of rat liver.     

Interdependence of Ca2+ occlusion sites in the unphosphorylated sarcoplasmic reticulum Ca(2+)-ATPase complex with CrATP.

The beta, gamma-bidentate chromium(III) complex of ATP (CrATP) was used as a substrate analog to stabilize a form of the Ca(2+)-ATPase of the sarcoplasmic reticulum containing both of the bound calcium ions in an occluded state without enzyme phosphorylation. The kinetics of dissociation of Ca2+ from the occlusion sites in the CrATP-enzyme complex were consistent with the existence of two nonequivalent and interdependent Ca2+ occlusion sites, both in the membranous Ca(2+)-ATPase and in a detergent-solubilized monomeric Ca(2+)-ATPase preparation. The rate constant for release of the first calcium ion was k1 = 0.99 h-1, whereas the second calcium ion was released with a rate constant of k2 = 0.25 h-1 when the first site was empty and with a rate constant of k3 = 0.13 h-1 when the first site was occupied by Ca2+. Ca2+ binding at the first site occurred with a rate constant of k-1 = 0.96 microM-1 h-1 (apparent Kd = 1.0 microM). The Ca(2+)-occluded state was further stabilized by ADP, binding in exchange with ATP with an apparent Kd of 8.6 microM. Two kinetic classes of CrATP-binding sites were observed, each with a stoichiometry of 3-4 nmol/mg of protein; but only the fast phase of CrATP binding was associated with Ca2+ occlusion. Derivatization of the Ca(2+)-ATPase with N-cyclohexyl-N'-(4-dimethylamino-1-naphthyl)carbodimide resulted in inactivation of phosphorylation of the enzyme from MgATP, whereas the ability to occlude Ca2+ in the presence of CrATP was retained, albeit with a reduced apparent affinity for Ca2+.     

Effect of solubilization on adenosine 5'-triphosphate induced calcium release from purified sarcoplasmic reticulum calcium adenosinetriphosphatase

ATP-induced Ca2+ release from the purified sarcoplasmic reticulum Ca2+-ATPase has been monitored in several different ATPase environments. Arsenazo III was used as a Ca2+ indicator in stopped-flow experiments and was shown to detect the early burst in Ca2+ transport, slower steady-state transport, and release of Ca2+ from fragmented sarcoplasmic reticulum. ATP-induced rapid release of Ca2+ followed by a slower rebinding step could be demonstrated for purified Ca2+-ATPase in leaky vesicles if the reaction was slowed by lowering the pH to 6.1 and by including dimethyl sulfoxide in the reaction medium. At a dodecyl octaoxyethylene glycol monoether (C12E8) to protein weight ratio of 0.2, a detergent concentration too low for solubilization to occur, ATP-induced Ca2+ release occurred more rapidly than for native leaky membranes, whereas the rebinding step was slower. In contrast, no Ca2+ release was observed for any soluble preparation. The kinetics of Ca2+ release was studied under conditions where the ATPase was monomeric or aggregated, and also in the presence of added phospholipid. The ATPase was shown to be monomeric by sedimentation equilibrium measurements in the presence of Ca2+, ADP, and beta, gamma-methylene-ATP at a C12E8 to protein weight ratio of 2.0. It is concluded that solubilization of the Ca2+-ATPase may result in uncoupling of ATP hydrolysis from ATP-induced Ca2+ release.     

Characterization of a High-Affinity Mg2+-Independent Ca2+-ATPase from Rat Brain Synaptosomal Membranes

A high-affinity Mg2+-independent Ca2+-ATPase (Ca2+-ATPase) has been differentiated from the Mg2+-dependent, Ca2+-stimulated ATPase (Ca2+,Mg2+-ATPase) in rat brain synaptosomal membranes. Using ATP as a substrate, the K0.5 of Ca2+ for Ca2+-ATPase was found to be 1.33 microM with a Km for ATP of 19 microM and a Vmax of 33 nmol/mg/min. Using Ca-ATP as a substrate, the Km for Ca-ATP was found to be 0.22 microM. Unlike Ca2+,Mg2+-ATPase, Ca2+-ATPase was not inhibited by N-ethylmaleimide, trifluoperazine, lanthanum, zinc, or vanadate. La3+ and Zn2+, in contrast, stimulated the enzyme activity. Unlike Ca2+, Mg2+-ATPase activity, ATP-dependent Ca2+ uptake was negligible in the absence of added Mg2+, indicating that the Ca2+ transport into synaptosomal endoplasmic reticulum may not be a function of the Ca2+-ATPase described. Ca2+-ATPase activity was not stimulated by the monovalent cations Na+ or K+. Ca2+, Mg2+-ATPase demonstrated a substrate preference for ATP and ADP, but not GTP, whereas Ca2+-ATPase hydrolyzed ATP and GTP, and to a lesser extent ADP. The results presented here suggest the high-affinity Mg2+-independent Ca2+-ATPase may be a separate form from Ca2+,Mg2+-ATPase. The capacity of Mg2+-independent Ca2+-ATPase to hydrolyze GTP suggests this protein may be involved in GTP-dependent activities within the cell.     

Vanadyl and vanadate inhibit Ca2+ transport systems of the adipocyte plasma membrane and endoplasmic reticulum

Vanadate and vanadyl have many insulin-mimetic effects on cellular metabolism and also have been shown to alter cellular Ca2+ fluxes. In this report, vanadate and vanadyl, like insulin, are shown to inhibit the plasma membrane (Ca2+ + Mg2+)-ATPase/Ca2+ transport system as well as Ca2+ transport by endoplasmic reticulum from rat adipocytes. Ca2+ transport by the endoplasmic reticulum was inhibited half-maximally (I50) by vanadate and vanadyl at concentrations of 30 and 33 microM, respectively. Inhibition of the plasma membrane Ca2+ transport by vanadate and vanadyl was less sensitive, with I50 values of 144 and 92 microM, respectively. These I50 values for plasma membrane Ca2+ transport were similar when measured under conditions of calmodulin-stimulated and non-calmodulin-stimulated Ca2+ transport. The predominant effect of both ions on the kinetic parameters of Ca2+ transport was a substantial decrease in the Vmax by 43-46% for both transport systems. An increase in intracellular Ca2+ following the inhibition of the (Ca2+ + Mg2+)-ATPase/Ca2+ pump in the plasma membrane and endoplasmic reticulum by these vanadium ions may result, at least in part, in the observed insulin-mimetic alterations in cellular metabolism.     

Stimulatory effect of regucalcin on ATP-dependent Ca(2+) uptake activity in rat liver mitochondria

The effect of Ca(2+)-binding protein regucalcin on Ca(2+)-ATPase activity in isolated rat liver mitochondria was investigated. The presence of regucalcin (0.1, 0.25, and 0.5 microM) in the enzyme reaction mixture led to a significant increase in Ca(2+)-ATPase activity. Regucalcin significantly stimulated ATP-dependent (45)Ca(2+) uptake by the mitochondria. Ruthenium red (10(-5) M) or lanthanum chloride (10(-4) M), an inhibitor of mitochondrial Ca(2+) uptake, completely inhibited regucalcin (0.25 microM)-increased mitochondrial Ca(2+)-ATPase activity and (45)Ca(2+) uptake. The effect of regucalcin (0.25 microM) in increasing Ca(2+)-ATPase activity was completely inhibited by the presence of digitonin (10(-2)%), a solubilizing reagent of membranous lipids, or vanadate (10(-5) M), an inhibitor of phosphorylation of ATPase. The activatory effect of regucalcin (0.25 microM) on Ca(2+)-ATPase activity was not further enhanced in the presence of dithiothreitol (2.5 mM), a protecting reagent of the sulfhydryl (SH) group of the enzyme, or calmodulin (0.60 microM), a modulator protein of Ca(2+) action that could increase mitochondrial Ca(2+)-ATPase activity. The present study demonstrates that regucalcin can stimulate Ca(2+) pump activity in rat liver mitochondria, and that the protein may act on an active site (SH group)-related to phosphorylation of mitochondrial Ca(2+)-ATPase.