Cyclopiazonic acid is a specific inhibitor of the Ca2+-ATPase of sarcoplasmic reticulum

The mycotoxin, cyclopiazonic acid (CPA), inhibits the Ca2+-stimulated ATPase (EC 3.6.1.38) and Ca2+ transport activity of sarcoplasmic reticulum (Goeger, D. E., Riley, R. T., Dorner, J. W., and Cole, R. J. (1988) Biochem. Pharmacol. 37, 978-981). We found that at low ATP concentrations (0.5-2 microM) the inhibition of ATPase activity was essentially complete at a CPA concentration of 6-8 nmol/mg protein, indicating stoichiometric reaction of CPA with the Ca2+-ATPase. Cyclopiazonic acid caused similar inhibition of the Ca2+-stimulated ATP hydrolysis in intact sarcoplasmic reticulum and in a purified preparation of Ca2+-ATPase. Cyclopiazonic acid also inhibited the Ca2+-dependent acetylphosphate, p-nitrophenylphosphate and carbamylphosphate hydrolysis by sarcoplasmic reticulum. ATP protected the enzyme in a competitive manner against inhibition by CPA, while a 10(5)-fold change in free Ca2+ concentration had only moderate effect on the extent of inhibition. CPA did not influence the crystallization of Ca2+-ATPase by vanadate or the reaction of fluorescein-5'-isothiocyanate with the Ca2+-ATPase, but it completely blocked at concentrations as low as 1-2 mol of CPA/mol of ATPase the fluorescence changes induced by Ca2+ and [ethylenebis(oxyethylenenitrilo)]tetraacetic acid (EGTA) in FITC-labeled sarcoplasmic reticulum and inhibited the cleavage of Ca2+-ATPase by trypsin at the T2 cleavage site in the presence of EGTA. These observations suggest that CPA interferes with the ATP-induced conformational changes related to Ca2+ transport. The effect of CPA on the sarcoplasmic reticulum Ca2+-ATPase appears to be fairly specific, since the kidney and brain Na+,K+-ATPase (EC 3.6.1.37), the gastric H+,K+-ATPase (EC 3.6.1.36), the mitochondrial F1-ATPase (EC 3.6.1.34), the Ca2+-ATPase of erythrocytes, and the Mg2+-activated ATPase of T-tubules and surface membranes of rat skeletal muscle were not inhibited by CPA, even at concentrations as high as 1000 nmol/mg protein.     

Reversal of the sarcoplasmic reticulum ATPase cycle by substituting various cations for magnesium. Phosphorylation and ATP synthesis when Ca2+ replaces Mg2+

Reversal of the cycle of sarcoplasmic reticulum ATPase starts from ATPase phosphorylation by Pi, in the presence of Mg2+, and leads to ATP synthesis. We show here that ATP can also be synthesized when Ca2+ replaces Mg2+. In the absence of a calcium gradient and in the presence of dimethyl sulfoxide, ATPase phosphorylation from Pi and Ca2+ led to the formation of an unstable phosphoenzyme. This instability was due to a competition between the phosphorylation reaction induced by Pi and Ca2+ and the transition induced by Ca2+ binding to the transport sites, which led to a conformation that could not be phosphorylated from Pi. Dimethyl sulfoxide and low temperature stabilized the calcium phosphoenzyme, which under appropriate conditions, subsequently reacted with ADP to synthesize ATP. Substitution of Co2+, Mn2+, Cd2+, or Ni2+ for Mg2+ induced ATPase phosphorylation from Pi, giving phosphoenzymes of various stabilities. However, substitution of Ba2+, Sr2+, or Cr3+ produced no detectable phosphoenzymes, under the same experimental conditions. Our results show that ATPase phosphorylation from Pi, like its phosphorylation from ATP, does not have a strict specificity for magnesium.     

Quercetin interaction with the (Ca2+ + Mg2+)-ATPase of sarcoplasmic reticulum

In sarcoplasmic reticulum vesicles or in the (Ca2+ + Mg2+)-ATPase purified from sarcoplasmic reticulum, quercetin inhibited ATP hydrolysis, Ca2+ uptake, ATP-Pi exchange, ATP synthesis coupled to Ca2+ efflux, ATP-ADP exchange, and steady state phosphorylation of the ATPase by inorganic phosphate. Steady state phosphorylation of the ATPase by ATP was not inhibited. Quercetin also inhibited ATP and ADP binding but not the binding of Ca2+. The inhibition of ATP-dependent Ca2+ transport by quercetin was reversible, and ATP, Ca2+, and dithiothreitol did not affect the inhibitory action of quercetin.     

Modulation of the sarcoplasmic reticulum (Ca2+ + Mg2+)-ATPase by pentobarbital

The dependence of the (Ca2+ + Mg2+)-ATPase activity of sarcoplasmic reticulum vesicles upon the concentration of pentobarbital shows a biphasic pattern. Concentrations of pentobarbital ranging from 2 to 8 mM produce a slight stimulation, approximately 20-30%, of the ATPase activity of sarcoplasmic reticulum vesicles made leaky to Ca2+, whereas pentobarbital concentrations above 10 mM strongly inhibit the activity. The purified ATPase shows a higher sensitivity to pentobarbital, namely 3-4-fold shift towards lower values of the K0.5 value of inhibition by this drug. These effects of pentobarbital are observed over a wide range of ATP concentrations. In addition, this drug shifts the Ca2+ dependence of the (Ca2+ + Mg2+)-ATPase activity towards higher values of free Ca2+ concentrations and increases several-fold the passive permeability to Ca2+ of the sarcoplasmic reticulum membranes. At the concentrations of pentobarbital that inhibit this enzyme in the sarcoplasmic reticulum membrane, pentobarbital does not significantly alter the order parameter of these membranes as monitored with diphenylhexatriene, whereas the temperature of denaturation of the (Ca2+ + Mg2+)-ATPase is decreased by 4-5 C degrees, thus, indicating that the conformation of the ATPase is altered. The effects of pentobarbital on the intensity of the fluorescence of fluorescein-labeled (Ca2+ + Mg2+)-ATPase in sarcoplasmic reticulum also support the hypothesis of a conformational change in the enzyme induced by millimolar concentrations of this drug. It is concluded that the inhibition of the sarcoplasmic reticulum ATPase by pentobarbital is a consequence of its binding to hydrophobic binding sites in this enzyme.     

Mg2+ and ATP effects on K+ activation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum.

ATP and the divalent cations Mg2+ and Ca2+ regulated K+ stimulation of the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum vesicles. Millimolar concentrations of total ATP increased the K+-stimulated ATPase activity of the Ca2+ pump by two mechanisms. First, ATP chelated free Mg2+ and, at low ionized Mg2+ concentrations, K+ was shown to be a potent activator of ATP hydrolysis. In the absence of K+ ionized Mg2+ activated the enzyme half-maximally at approximately 1 mM, whereas in the presence of K+ the concentration of ionized Mg2+ required for half-maximal activation was reduced at least 20-fold. Second MgATP apparently interacted directly with the enzyme at a low affinity nucleotide site to facilitate K+-stimulation. With a saturating concentration of ionized Mg2+, stimulation by K+ was 2-fold, but only when the MgATP concentration was greater than 2 mM. Hill plots showed that K+ increased the concentration of MgATP required for half-maximal enzymic activation approx. 3-fold. Activation of K+-stimulated ATPase activity by Ca2+ was maximal at an ionized Ca2+ concentration of approx. 1 microM. At very high concentrations of either Ca2+ or Mg2+, basal Ca2+-dependent ATPase activity persisted, but the enzymic response to K+ was completely inhibited. The results provide further evidence that the Ca2+-transport ATPase of cardiac sarcoplasmic reticulum has distinct sites for monovalent cations, which in turn interact allosterically with other regulatory sites on the enzyme.     

Effects of arsenate on the Ca2+ ATPase of sarcoplasmic reticulum

The effect of arsenate on the partial reactions of the catalytic cycle of the Ca2+ ATPase of skeletal muscle of sarcoplasmic reticulum was studied. With the use of native vesicles it was found that arsenate accelerates the rate of ITP hydrolysis and inhibits both Ca2+ or Sr2+ uptake. These effects were not observed when ATP was used as substrate or, with the use of ITP, when leaky vesicles were assayed. Activation of ITP hydrolysis is related to an increase of the enzyme's apparent affinity for ITP. Arsenate increases the steady-state level of the phosphoenzyme formed from ITP. This depends on the concentration of both Pi and Ca2+, in the medium. Ca2+ and Sr2+ efflux were accelerated by arsenate. The fast Ca2+ efflux promoted by arsenate is impaired by external Ca2+. Arsenate competes with Pi for the phosphorylating site of the enzyme.     

Effects of p-nitrophenylphosphate on Ca2+ transport in inside-out vesicles from human red-cell membranes

Ca2+-ATPase activity and Ca2+ uptake in inside-out vesicles from human red cell membranes are changed in parallel by p-nitrophenylphosphate. This indicates that, unlike the Ca2+ pump of sarcoplasmic reticulum, the Ca2+ pump of the red cell membrane does not utilize p-nitrophenylphosphate hydrolysis to drive Ca2+ transport.     

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).     

Effects of nonsolubilizing and solubilizing concentrations of Triton X-100 on Ca2+ binding and Ca2+-ATPase activity of sarcoplasmic reticulum

The effect of low concentrations of Triton X-100, below that required for solubilization, on the properties of the Ca2+-ATPase of sarcoplasmic reticulum has been investigated. The changes observed have been compared with the changes produced on solubilization of the vesicles at higher concentrations of detergent. In the range 0.02-0.05% (w/v) Triton X-100, concentrations which did not solubilize the vesicles but completely inhibit ATP-mediated Ca2+ accumulation, 8-16 mol of detergent/mol of ATPase was associated with the vesicles. This amount of Triton X-100 altered equilibrium Ca2+ binding and Ca2+ activation of p-nitrophenyl phosphate and of ATP hydrolysis in a manner which lowered the apparent Ca2+ cooperatively (nH = 1 or less), and which increased the K0.5(Ca) value 20-fold. These changes in Ca2+ binding and activation parameters were associated with a 90% lower Ca2+-induced change in fluorescence of fluorescein isothiocyanate modified enzyme. The rates of p-nitrophenyl phosphate and of ATP hydrolysis, at saturating Ca2+ concentrations, were about half that of detergent-free vesicles. The rate constant for phosphoenzyme hydrolysis in the absence of Ca2+, calculated from medium Pi in equilibrium HOH exchange and phosphoenzyme measurements, was lowered from 38 to 11 s-1. The steady-state level of phosphoenzyme formed from Pi in the absence of Ca2+ was slightly increased up to 0.02% Triton X-100 and then decreased about half at 0.05%. The synthesis of ATP in single turnover type experiments was not affected by detergent binding. Pi in equilibrium ATP exchange was inhibited 65%.(ABSTRACT TRUNCATED AT 250 WORDS)     

Fe2+ and other divalent metal ions uncouple Ca2+ transport from (Ca2+-Mg2+)-ATPase in rat liver plasma membranes

The addition of nanomolar concentrations of free Fe2+, Mn2+, or Co2+ to rat liver plasma membranes resulted in an activation of ATP hydrolysis by these membranes which was not additive with the Ca2+-stimulated ATPase activity coupled to the Ca2+ pump. Detailed analysis showed that, if fact, (i) as for the stimulation of (Ca2+-Mg2+)-ATPase by Ca2+, activation of ATP hydrolysis by Fe2+, Mn3+, or Co2+ followed a cooperative mechanism involving two ions; (ii) two interacting sites for ATP were involved in the activation of both Fe2+- and Ca2+-stimulated ATPase activities; (iii) micromolar concentrations of magnesium caused the same dramatic inhibition of both activities; and (iv) the subcellular distribution of Fe2+-activated ATP hydrolysis activity corresponded to that of plasma membrane markers. This suggests that the (Ca2+-Mg2+)-ATPase might be stimulated not only by Ca2+, but also by Fe2+, Mn2+, or Co2+. However, interaction of (Ca2+-Mg2+)-ATPase with Fe2+, Mn2+, or Co2+ inhibited the Ca2+ pump activity. Furthermore, neither the formation of the phosphorylated intermediate of (Ca2+-Mg2+)-ATPase, nor ATP-dependent (59Fe) uptake could be detected in the presence of Fe2+ concentrations which stimulated ATP hydrolysis. We conclude that: (i) under the influence of certain metal ions, the Ca2+ pump in the liver plasma membrane may be switched to an uncoupled state which displays ATP hydrolysis activity, but does not insure ion transport; (ii) therefore the Ca2+ pump in liver plasma membranes specifically insures Ca2+ transport.     

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+.     

ATP inactivates hydrolysis of the K+-sensitive phosphoenzyme of kidney Na+,K+-transport ATPase and activates that of muscle sarcoplasmic reticulum Ca2+-transport ATPase

In order to characterize low affinity ATP-binding sites of renal (Na+,K+) ATPase and sarcoplasmic reticulum (Ca2+)ATPase, the effects of ATP on the splitting of the K+-sensitive phosphoenzymes were compared. ATP inactivated the dephosphorylation in the case of (Na+,K+)ATPase at relatively high concentrations, while activating it in the case of (Ca2+)ATPase. When various nucleotides were tested in place of ATP, inactivators of (Na+,K+)ATPase were found to be activators in (Ca2+)ATPase, with a few exceptions. In the absence of Mg2+, the half-maximum concentration of ATP for the inhibition or for the activation was about 0.35 mM or 0.25 mM, respectively. These values are comparable to the previously reported Km or the dissociation constant of the low affinity ATP site estimated from the steady-state kinetics of the stimulation of ATP hydrolysis or from binding measurements. By increasing the concentration of Mg2+, but not Na+, the effect of ATP on the phosphoenzyme of (Na+,K+)ATPase was reduced. On the other hand, Mg2+ did not modify the effect of ATP on the phosphoenzyme of (Ca2+)ATPase. During (Na+,K+)ATPase turnover, the low affinity ATP site appeared to be exposed in the phosphorylated form of the enzyme, but the magnesium-complexed ATP interacted poorly with the reactive K+-sensitive phosphoenzyme, which has a tightly bound magnesium, probably because of interaction between the divalent cations. In the presence of physiological levels of Mg2+ and K+, ATP appeared to bind to the (Na+,K+)ATPase only after the dephosphorylation, while it binds to the (Ca2+)-ATPase before the dephosphorylation to activate the turnover.     

Inhibition of oxidative phosphorylation by Ca2+ or Sr2+: a competition with Mg2+ for the formation of adenine nucleotide complexes

Intramitochondrial Sr2+, similar to Ca2+, inhibits oxidative phosphorylation in intact rat-liver mitochondria. Both Ca2+ and Sr2+ also inhibit the hydrolytic activity of the ATPase in submitochondrial particles. Half-maximal inhibition of ATPase activity was attained at a concentration of 2.5 mM Ca2+ or 5.0 mM Sr2+ when the concentration of Mg2+ in the medium was 1.0 mM. The inhibition of ATPase activity by both cations was strongly decreased by increasing the Mg2+ concentration in the reaction medium. In addition, kinetical data and the determination of the concentration of MgATP, the substrate of the ATPase, in the presence of different concentrations of Ca2+ or Sr2+ strongly indicate that these cations inhibit ATP hydrolysis by competing with Mg2+ for the formation of MgATP. On the basis of a good agreement between these results with submitochondrial particles and the results of titrations of oxidative phosphorylation with carboxyatractyloside or oligomycin in mitochondria loaded with Sr2+ it can be concluded that intramitochondrial Ca2+ or Sr2+ inhibits oxidative phosphorylation in intact mitochondria by decreasing the availability of adenine nucleotides to both the ADP/ATP carrier and the ATP synthase.     

Energy interconversion in sarcoplasmic reticulum vesicles in the presence of Ca2+ and Sr2+ gradients

Sarcoplasmic reticulum vesicles of rabbit skeletal muscle are able to accumulate Ca2+ or Sr2+ at the expense of ATP hydrolysis. Depending on the conditions used, vesicles loaded with Ca2+ can catalyze either an ATP in equilibrium Pi exchange or the synthesis of ATP from ADP and Pi. Both reactions are impaired in vesicles loaded with Sr2+. The Sr2+ concentration required for half-maximal ATPase activity increases from 2 microM to 60-70 microM when the Mg2+ concentration is raised from 0.5 to 50 mM. The enzyme is phosphorylated by ATP in the presence of Sr2+. The steady state level of phosphoenzyme varies depending on both the Sr2+ and Mg2+ concentrations in the medium. Phosphorylation of the enzyme by Pi is inhibited by both Ca2+ and Sr2+. In the presence of 2 and 20 mM Mg2+, half-maximal inhibition is attained in the presence of 4 and 8 microM Ca2+ or in the presence of 0.24 mM and more than 2 mM Sr2+, respectively. After the addition of Sr2+, the phosphoenzyme is cleaved with two different rate constants, 0.5-1.5 s-1 and 10-18 s-1. The fraction of phosphoenzyme cleaved at a slow rate is smaller the higher the Sr2+ concentration in the medium. Ca2+ inhibition of enzyme phosphorylation by Pi is overcome by the addition of ITP. This is not observed when Ca2+ is replaced by Sr2+.     

Activation of calcium transport in skeletal muscle sarcoplasmic reticulum by monovalent cations.

The rates of calcium transport and Ca2+-dependent ATP hydrolysis by rabbit skeletal muscle sarcoplasmic reticulum were stimulated by monovalent cations. The rate of decomposition of phosphoprotein intermediate of the Ca2+-dependent ATPase of sarcoplasmic reticulum was also increased by these ions to an extent that is sufficient to account for the stimulation of calcium transport and Ca2+-dependent ATPase activity. The order of effectiveness of monovalent cations tested at saturating concentrations in increasing rate of phosphoprotein decomposition is: K+, Na+ greater than Rb+, NH4+ greater than Cs+ greater than Li+, choline+, Tris+.     

(Ca2+ + Mg2+)-ATPase activity associated with the maintenance of a Ca2+ gradient by sarcoplasmic reticulum at submicromolar external [Ca2+]. The effect of hypothyroidism

The formation and maintenance of Ca2+-filling levels by sarcoplasmic reticulum vesicles from euthyroid (control) and hypothyroid skeletal muscle were investigated using the Ca2+-indicator quin-2, at [Ca2+] in the medium [( Cao2+]) of 0.05-0.3 microM. Rapid ATP-dependent Ca2+ uptake resulted in a steady-state Ca2+-filling level, Cai2+, within one minute. This Ca2+ gradient was maintained for at least three minutes, during which less than 20% of the ATP was consumed. Cai2+ was maximal (120 nmol/mg) for [Cao2+] greater than 0.3 microM and decreased to 40 nmol/mg at [Cao2+] of 0.05 microM. Preparations from both experimental groups showed qualitatively and quantitatively the same relationship between Cai2+ and [Cao2+] at steady state, despite a significantly lower Ca2+-pump content of hypothyroid sarcoplasmic reticulum, which resulted in a 25% lower maximal (Ca2+ + Mg2+)-ATPase activity. Maintenance of the steady state, at all levels of Cai2+, was associated with net ATP consumption by the Ca2+ pump and cycling of Ca2+, which processes were 30% slower in the hypothyroid group as compared to the control group. Determination of the passive efflux of Ca2+, as well as the fraction of leaky or unsealed sarcoplasmic reticulum fragments, excluded either of these possibilities as an explanation for the relatively high (Ca2+ + Mg2+)-ATPase rates at steady state. On the basis of these and previously reported results, it is concluded that the maintenance of a Ca2+ gradient by sarcoplasmic reticulum under physiological conditions with respect to external [Ca2+] and the concentrations of ATP, ADP and Pi, is associated with the cycling of Ca2+ coupled to net ATP hydrolysis. Using the obtained data it is calculated that the sarcoplasmic reticulum may account for 20% of the resting metabolic rate in skeletal muscle. Consequently, together with the previously reported lower sarcoplasmic reticulum content of skeletal muscle in hypothyroidism, we calculate that about one third of the decrease in basal metabolic rate in this thyroid state can be related to the alterations of the sarcoplasmic reticulum.     

Interaction between free fatty acids and Ca2+-dependent ATPase from sarcoplasmic reticulum membranes

The interaction between free fatty acids and Ca2+-dependent ATPase, an intrinsic protein of sarcoplasmic reticulum membranes, was studied with relevance to the changes in membrane permeability induced by free fatty acids. It was found that only unsaturated fatty acids increase the permeability of reticulum membranes for Ca2+, this effect being completely reversible. The increase in the membrane permeability by fatty acids is coupled to a generation of a channel for Ca2+ efflux under effect of Ca2+-dependent ATPase. The interaction between fatty acids and Ca2+-dependent ATPase was demonstrated by the protein fluorescence and electron paramagnetic resonance methods, using spin-labelled fatty acid derivatives. A model demonstrating the increase of sarcoplasmic reticulum membrane permeability for Ca2+ in the presence of the fatty acid-Ca2+-dependent ATPase complex is proposed.     

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.     

Chemical modification of sarcoplasmic reticulum with methylbenzimidate. Stimulation of Ca2+ efflux.

Treatment of sarcoplasmic reticulum membranes with 12 mM-methylbenzimidate (MBI) for 5 min, in the presence of 5 mM-ATP at pH 8.5, resulted in a 2-3-fold stimulation of ATP hydrolysis and over 90% inhibition of Ca2+ accumulation. This phenomenon was strictly dependent upon the presence of nucleotides with the following order of effectiveness: adenosine 5'-[beta, gamma-imido]triphosphate greater than or equal to ATP greater than UTP greater than ADP greater than AMP. Divalent cations such as Ca2+, Mg2+ and Mn2+, when present during the MBI treatment, prevented both the stimulation of ATPase activity and the inhibition of Ca2+ accumulation. Modification with MBI had no effect on E-P formation from ATP, ADP-ATP exchange, Ca2+ binding or ATP-Pi exchange catalysed by the membranes. Membranes modified with MBI in the presence of ATP and then passively loaded with Ca2+ released about 80% of their Ca2+ content within 3 s. Control membranes released only 3% of their Ca2+ during the same time period. MBI modification inhibited Ca2+ accumulation by proteoliposomes reconstituted with the partially purified ATPase but not with the purified ATPase fraction. These results suggest that MBI in the presence of ATP stimulates Ca2+ release by modifying a protein factor(s) other than the (Ca2+ + Mg2+)-ATPase.     

Transient kinetics of a Ca2+-induced fluorescence change from membrane-associated and solubilized sarcoplasmic reticulum Ca2+-ATPase

The kinetics and extent of the fluorescence change induced by Ca2+ interaction with the Ca2+-ATPase from sarcoplasmic reticulum have been compared by stopped flow fluorimetry for three preparations: sarcoplasmic reticulum; purified ATPase in membrane vesicles; and solubilized, delipidated ATPase. The kinetics of Ca2+ release and binding for both purified preparations could be described by a single exponential as has been observed for sarcoplasmic reticulum. The rate and extent of the fluorescence change for the solubilized and membrane-associated preparations are shown to be quite similar to those of the sarcoplasmic reticulum. From these results, it is concluded that all of the Ca2+-induced fluoescence change in sarcoplasmic reticulum originates from the Ca2+-ATPase. In addition, since the change in fluorescence is probably result of a conformational change in the ATPase during the Ca2+ pumping cycle, the results provide additional evidence that monomeric Ca2+-ATPase may be capable of Ca2+ transport since the delipidated preparation is monomeric under the conditions used for these experiments. Finally, it is concluded that phospholipid bilayer is not essential for this conformational change.