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+.     

Role of Mg2+ in the Ca2+-Ca2+ exchange mediated by the membrane-bound (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles

Sarcoplasmic reticulum vesicles were preloaded with either 45Ca2+ or unlabeled Ca2+. The unidirectional Ca2+ efflux and influx, together with Ca2+-dependent ATP hydrolysis and phosphorylation of the membrane-bound (Ca2+, Mg2+)-ATPase, were determined in the presence of ATP and ADP. The Ca2+ efflux depended on ATP (or ADP or both). It also required the external Ca2+. The Ca2+ concentration dependence of the efflux was similar to the Ca2+ concentration dependences of Ca2+ influx, Ca2+-dependent ATP hydrolysis, and phosphoenzyme formation. The rate of the efflux was approximately in proportion to the concentration of the phosphoenzyme up to 10 microM Ca2+. These results and other findings indicate that the Ca2+ efflux represents the Ca2+-Ca2+ exchange (between the external medium and the internal medium) mediated by the phosphoenzyme. In the range of 0.6-5.2 microM Mg2+, no appreciable Ca2+-Ca2+ exchange was detected although phosphoenzyme formation occurred to a large extent. Elevation of Mg2+ in the range 5.2 microM-4.8 mM caused a remarkable activation of the exchange, whereas the amount of the phosphoenzyme only approximately doubled. The kinetic analysis shows that this activation results largely from the Mg2+-induced acceleration of an exchange between the bound Ca2+ of the phosphoenzyme and the free Ca2+ in the internal medium. It is concluded that Mg2+ is essential for the exposure of the bound Ca2+ of the phosphoenzyme to the internal medium.     

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.     

Transient state kinetic studies of phosphorylation by ATP and Pi of the calcium-dependent ATPase from sarcoplasmic reticulum.

The ATPase of the sarcoplasmic reticulum is phosphorylated by ATP in the presence of Ca2+. A rapid phosphorylation was observed when the enzyme was preincubated with Ca2+ prior to the addition of 0.1 or 1 mM ATP. The rate of phosphorylation was decreased when Ca2+ was omitted from the preincubation medium and added with ATP when the reaction was started. The rate of phosphorylation by ATP was further decreased when Pi was included in the preincubation medium without Ca2+. In this case, the enzyme was phosphorylated by Pi during the preincubation. When Ca2+ and ATP were added, a burst of phosphorylation by ATP was observed in the initial 16 ms. In the subsequent incubation intervals, the phosphorylation by ATP was synchronous with the hydrolysis of the phosphoenzyme formed by Pi. The rate of hydrolysis of the phosphoenzyme formed by Pi was measured when either the Pi concentration was decreased 10 fold, or when Ca2+, ATP or ATP plus Ca2+ was added to the medium. Upon the single addition of Ca2+, the time for half-maximal decay was in the range 500--1000 ms. In all other conditions it was in the range 70--90 ms.     

Induction by nucleotide triphosphate hydrolysis of a form of sarcoplasmic reticulum ATPase capable of medium phosphate-oxygen exchange in presence of calcium.

Sarcoplasmic reticulum vesicles rendered leaky by exposure to alkaline pH, like intact vesicles, catalyze a rapid Mg2+-dependent exchange of oxygens of medium Pi with water. The exchange with 10 mM Pi is strongly inhibited by 0.15 mM Ca2+. Upon addition and hydrolysis of ITP or ATP, a rapid phosphate-oxygen exchange is observed even with 0.15 mM Ca2+ present and a definite but smaller exchange at 8 mM Ca2+. Oxygen exchange per Pi formed is greater with ITP than with ATP. When no Pi is initially present, the extent of oxygen exchange is increased with time of incubation as Pi is formed. With 18O-labeled Pi present, ATP hydrolysis accelerates 18O loss. The results show that much of the oxygen exchange occurs as a result of reversible binding of medium Pi. Thus the binding and cleavage of ITP or ATP overcomes the Ca2+ inhibition of the medium Pi in equilibrium HOH exchange. Such findings support the concept that the cleavage cycle includes a transient conformational form which can reversibly react with Pi to give a phosphoryl enzyme and resultant oxygen exchange or in a rate-limiting step decay to a form with high Ca2+ and NTP affinity.     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum. The role of Mg2+ that activates hydrolysis of the phosphoenzyme

The role of Mg2+ in the activation of phosphoenzyme hydrolysis has been investigated with the (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. The enzyme of the native and solubilized vesicles was phosphorylated with ATP at 0 degrees C, pH 7.0, in the presence of Ca2+ and Mg2+. When Ca2+ and Mg2+ in the medium were chelated, phosphoenzyme hydrolysis continued for about 15 s and then ceased. The extent of this hydrolysis increased with increasing concentrations of Mg2+ added before the start of phosphorylation. This shows that the hydrolysis was activated by the Mg2+ added. The Mg2+ which activated phosphoenzyme hydrolysis was distinct from Mg2+ derived from MgATP bound to the substrate site. The Mg2+ site at which Mg2+ combined to activate phosphoenzyme hydrolysis was located on the outer surface of the vesicular membranes. During the catalytic cycle, Mg2+ combined with the Mg2+ site before Ca2+ dissociated from the Ca2+ transport site of the ADP-sensitive phosphoenzyme with bound Ca2+. This Mg2+ did not activate hydrolysis of the ADP-sensitive phosphoenzyme with bound Ca2+, but markedly activated hydrolysis of the ADP-insensitive phosphoenzyme without bound Ca2+. It is concluded that during the catalytic cycle, Mg2+ activates phosphoenzyme hydrolysis only after Ca2+ has dissociated from the Ca2+ transport site of phosphoenzyme.     

Ca2+ translocation and catalytic activity of the sarcoplasmic reticulum ATPase. Modulation by ATP, Ca2+, and Pi

The ratio between Ca2+ uptake and Ca(2+)-dependent ATP hydrolysis measured in sarcoplasmic reticulum vesicles of rabbit skeletal muscle was found to vary greatly depending on the concentrations of oxalate or Pi used. In the presence of 5 mM oxalate, 20 mM Pi, and 1 mM Pi, the ratios found were in the range of 1.4-2.3, 0.6-0.8, and 0.01-0.10, respectively. The rates of Ca2+ exchange and ATP synthesis were measured at the steady state by adding trace amounts of 45Ca and 32Pi, after the vesicles had been loaded with Ca2+. In the presence of 1 mM Pi, 10 mM MgCl2, and 0.2 mM CaCl2, the ratio between Ca2+ exchange and ATP synthesis varied from 9 to 14. This ratio approached two when Ca2+ in the medium was reduced to a very low level, or when in the presence of Ca2+, dimethyl sulfoxide was added to the assay medium, or when the Pi concentration was raised from 1 to 20 mM. A ratio of two was also measured when the steady state was attained using ITP instead of ATP. In all the conditions that led to a ratio close to two, there was an increase in the fraction of enzyme phosphorylated by Pi. It is proposed that the coupling between Ca2+ translocation and ATP hydrolysis or synthesis is modulated by the phosphorylation of the ATPase by Pi.     

Transient state in the phosphorylation of sodium- and potassium- transport adenosine triphosphatase by adenosine triphosphate

The rate of phosphorylation of sodium and potassium ion-transport adenosine triphosphatase by 10 microM [gamma-32P]ATP was much slower with Ca2+ than with Mg2+ (0.13-10 mM) in the presence of 16 to 960 mM Na+ at 0 degrees C and pH 7.4. In the presence of a fixed concentration of Mg2+ or Ca2+, the rate became slower with increasing Na+ concentration. When the Na+ concentration was fixed, the rate became slower with decreasing divalent cation concentration. Sodium ions appear to antagonize the divalent cation in the phosphorylation to slow its rate. In the presence of 1 mM Ca2+ and 126 or 270 mM Na+, the rate was slow enough to permit the manual addition of a chasing solution at various times before the phosphorylation reached the steady state. Therefore, we studied the time-dependent change of the sensitivity to ADP or to K+ of the phosphoenzyme by a chase with unlabeled ATP containing ADP or K+ during the time range from the transient to the steady state of the phosphorylation. The ADP sensitivity decreased and the K+ sensitivity increased with the progress of the phosphorylation. With 270 mM Na+, the phosphoenzyme found at 1 s, when its amount was 5.5% of the maximum level, was virtually completely sensitive to ADP. Under these conditions, it was concluded that the form of the phosphoenzyme initially produced from the enzyme.ATP complex has ADP sensitivity and that the phosphoenzyme acquires K+ sensitivity later. The initially produced ADP-sensitive phosphoenzyme partially lost its normal instability and sensitivity upon adding a chelating agent, probably because of dissociation of a divalent cation from the phosphoenzyme.     

The substitution of calcium for magnesium in H+,K+-ATPase catalytic cycle. Evidence for two actions of divalent cations

In order to determine the role of divalent cations in the reaction mechanism of the H+,K+-ATPase, we have substituted calcium for magnesium, which is required by the H+,K+-ATPase for phosphorylation from ATP and from PO4. Calcium was chosen over other divalent cations assayed (barium and manganese) because in the absence of magnesium, calcium activated ATP hydrolysis, generated sufficiently high levels of phosphoenzyme (573 +/- 51 pmol.mg-1) from [gamma-32P]ATP to study dephosphorylation, and inhibited K+-stimulated ATP hydrolysis. The Ca2+-ATPase activity of the H+,K+-ATPase was 40% of the basal Mg2+-ATPase activity. However, the Ca2+,K+-ATPase activity (minus the Ca2+ basal activity) was only 0.7% of the Mg2+,K+-ATPase, indicating that calcium could partially substitute for Mg2+ in activating ATP hydrolysis but not in K+ stimulation of ATP hydrolysis. Approximately 0.1 mM calcium inhibited 50% of the Mg2+-ATPase or Mg2+,K+-ATPase activities. Inhibition of Mg2+,K+-ATPase activity was not competitive with respect to K+. Inhibition by calcium of Mg2+,K+ activity p-nitrophenyl phosphatase activity was competitive with respect to Mg2+ with an apparent Ki of 0.27 mM. Proton transport measured by acridine orange uptake was not detected in the presence of Ca2+ and K+. In the presence of Mg2+ and K+, Ca2+ inhibited proton transport with an apparent affinity similar to the inhibition of the Mg2+, K+-ATPase activity. The site of calcium inhibition was on the exterior of the vesicle. These results suggest that calcium activates basal turnover and inhibits K+ stimulation of the H+,K+-ATPase by binding at a cytosolic divalent cation site. The pseudo-first order rate constant for phosphoenzyme formation from 5 microM [gamma-32P]ATP was at least 22 times slower in the presence of calcium (0.015 s-1) than magnesium (greater than 0.310 s-1). The Ca.EP (phosphoenzyme formed in the presence of Ca2+) formed dephosphorylated four to five times more slowly that the Mg.EP (phosphoenzyme formed in the presence of Mg2+) in the presence of 8 mm trans-1,2-diaminocyclohexane-N,N,N',N'-tetraacetic acid (CDTA) or 250 microM ATP. Approximately 10% of the Ca.EP formed was sensitive to a 100 mM KCl chase compared with greater than 85% of the Mg.EP. By comparing the transient kinetics of the phosphoenzyme formed in the presence of magnesium (Mg.EP) and calcium (Ca.EP), we found two actions of divalent cations on dephosphorylation.(ABSTRACT TRUNCATED AT 400 WORDS)     

Lanthanum inhibits steady-state turnover of the sarcoplasmic reticulum calcium ATPase by replacing magnesium as the catalytic ion

LaATP is shown to be an effective inhibitor of the calcium ATPase of sarcoplasmic reticulum because the binding of LaATP to cE.Ca2 results in the formation of lanthanum phosphoenzyme, which decays slowly. Steady-state activity of the calcium ATPase in leaky sarcoplasmic reticulum vesicles is inhibited 50% by 0.16 microM LaCl3 (15 nM free La3+, 21 nM LaATP) in the presence of 25 microM Ca2+ and 49 microM MgATP (5 mM MgSO4, 100 mM KCl, 40 mM 4-morpholinepropanesulfonic acid, pH 7.0, 25 degrees C). However, 50% inhibition of the uptake of 45Ca and phosphorylation by [gamma-32P]ATP in a single turnover experiment requires 100 microM LaCl3 (28 microM free La3+) in the presence of 25 microM Ca2+; this inhibition is reversed by calcium but inhibition of steady-state turnover is not. Therefore, binding of La3+ to the cytoplasmic calcium transport site is not responsible for the inhibition of steady-state ATPase activity. The addition of 6.7 microM LaCl3 (1.1 microM free La3+) has no effect on the rate of dephosphorylation of phosphoenzyme formed from MgATP and enzyme in leaky vesicles, while 6.7 mM CaCl2 slows the rate of phosphoenzyme hydrolysis as expected; 6.7 microM LaCl3 and 6.7 mM CaCl2 cause 95 and 98% inhibition of steady-state ATPase activity, respectively. This shows that inhibition of ATPase activity in the steady state is not caused by binding of La3+ to the intravesicular calcium transport site of the phosphoenzyme. Inhibition of ATPase activity by 2 microM LaCl3 (0.16 microM free La3+, 0.31 microM LaATP) requires greater than 5 s, which corresponds to approximately 50 turnovers, to reach a steady-state level of greater than or equal to 80% inhibition. Inhibition by La3+ is fully reversed by the addition of 0.55 mM CaCl2 and 0.50 mM EGTA; this reactivation is slow with t1/2 approximately 9 s. Two forms of phosphoenzyme are present in reactions that are partially inhibited by La3+: phosphoenzyme with Mg2+ at the catalytic site and phosphoenzyme with La3+ at the catalytic site, which undergo hydrolysis with observed rate constants of greater than 4 and 0.05 s-1, respectively. We conclude, therefore, that La3+ inhibits steady-state ATPase activity under these conditions by replacing Mg2+ as the catalytic ion for phosphoryl transfer. The slow development of inhibition corresponds to the accumulation of lanthanum phosphoenzyme. Initially, most of the enzyme catalyzes MgATP hydrolysis, but the fraction of enzyme with La3+ bound to the catalytic site gradually increases because lanthanum phosphoenzyme undergoes hydrolysis much more slowly than does magnesium phosphoenzyme.(ABSTRACT TRUNCATED AT 400 WORDS)     

Substrate regulation of the sarcoplasmic reticulum ATPase. Transient kinetic studies.

The rate of phosphorylation of the Ca2+-dependent ATPase of sarcoplasmic reticulum vesicles by ITP and ATP was studied using a millisecond mixing and quenching device. The rate of phosphorylation was slower when the vesicles were preincubated in a Ca2+-free medium than when preincubated with Ca2+, regardless of the substrate used and of the pH of the medium. When the vesicles were preincubated with Ca2+ at pH 7.4 an overshoot of phosphorylation was observed in the presence of ITP. The overshoot was abolished when the pH of the medium was decreased to 6.0 or when the vesicles were preincubated in a Ca2+-free medium. Using vesicles preincubated with Ca2+ the apparent Km for ITP found was 2.5 mM at pH 6.0 and 1.0 mM at pH 7.4. The Vmax observed (77 mumol g-1 s-1) did not change with the pH of the medium. Both at pH 6.0 and 7.4 the apparent Km for ATP was 3 microM when preincubated in a Ca2+-free medium. At pH 6.0 the Vmax for ATP varied from 96 to 33 mumol g-1 s-1 depending on whether the vesicles were preincubated in the presence or absence of Ca2+. At pH 7.4 the Vmax for ATP was 90 mumol g-1 s-1 in both conditions. The rate of phosphorylation of the vesicles was dependent on the relative Ca2+ and Mg2+ concentrations of the reaction medium regardless of the substrate used.     

Calcium and proton dependence of sarcoplasmic reticulum ATPase

The influence of Ca2+ and H+ concentrations on the sequential reactions of the ATPase cycle was studied by a series of pre-steady state and steady state experiments with sarcoplasmic reticulum vesicles. It is shown that H+ competition with calcium binding results in a reduced population of activated enzyme, which is manifested by a lower level of phosphorylated enzyme intermediate following addition of ATP. Further effects of Ca2+ and H+ are demonstrated on the progression of the phosphoenzyme through the reaction cycle and on the final hydrolytic cleavage of Pi. The overall dependence of steady state ATP flux on Ca2+ and H+ concentrations in leaky vesicles is expressed by a series of curves showing that as the H+ concentration is raised higher Ca2+ concentrations are required to obtain half-maximal ATP fluxes. At saturating Ca2+, maximal ATP fluxes are observed at an intermediate H+ concentration (pH 7.2), while lower levels are obtained as the H+ concentration is reduced (to pH 8) or increased (to pH 6). A preliminary model is then proposed based on the presence of two interacting domains permitting competitive binding of Ca2+ or H+, per each catalytic site undergoing phosphorylation by ATP. The model considers three main states and thirteen substates (depending on the occupancy of the binding sites in each state by Ca2+, H+, or neither) in the progression of the ATP cycle, coupled to transport of Ca2+ and counter transport of H+ in leaky vesicles. Considering the preliminary nature of the model and the experimental scatter, a rather satisfactory agreement is noted between a family of curves generated by theoretical analysis and the ATP flux curves obtained experimentally.     

Side-specific effects of sodium on (Na,K)-ATPase. Studies with inside-out red cell membrane vesicles.

Using inside-out vesicles of human red cell membranes, the side-specific effects of Na+ on phosphorylation of (Na,K)-ATPase have been studied using low concentrations of [gamma-32P]ATP (less than or equal to 0.1 microM). Phosphorylation is stimulated by Na+ at the cytoplasmic membrane surface (extravesicular Na+) alone and not by Na+ at the external surface (intravesicular Na+). At 37 degrees C, external Na+ (less than or equal to 10 mM) does, however, increase the steady state level (approximately 2 1/2-fold) of phosphoenzyme above that observed with cytoplasmic Na+ alone; hydrolysis is increased to only a small extent. Little stimulation by external Na+ is observed at 0 degrees C. As Na+ at the cytoplasmic side is decreased to very low levels (less than or equal to 0.2 mM) several kinetic changes are observed: (i) the apparent turnover of phosphoenzyme (ratio Na+-ATP-ase/phosphoenzyme level) is markedly increased (approximately 3-fold, (ii) Rbext sensitivity (inhibition of (Na)-ATPase at low ATP levels) is reduced, and (iii) the ratio of Na+ ions transported per molecule of ATP hydrolyzed is decreased. These results are compatible with a reaction pathway involving a transition from one form of phosphoenzyme, E1-P, to another, E2-P of which the hydrolysis is decreased by moderate levels of external Na+. It is suggested also that an alternate reaction pathway for Na+-ATPase occurs at very low cytoplasmic Na+, one via hydrolysis of E1-P and not associated with Na+ translocation.     

The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of calcium

The hydrolytic cycle of sarcoplasmic reticulum Ca2+-ATPase in the absence of Ca2+ was studied. At pH 6.0, 10 degrees C and in the absence of K+, the enzyme displays a very low velocity of ATP hydrolysis. Addition of up to 15% dimethyl sulfoxide increased this velocity severalfold (from 5-18 nmol of Pi X mg of protein-1 X h-1) and then decreased at higher solvent concentrations. Dimethyl sulfoxide increased both enzyme phosphorylation from ATP and the affinity for this substrate. Maximal levels of 1.0-1.2 nmol of EP X mg of protein-1 and apparent KM for ATP of 5 X 10(-6) M were obtained at a concentration of 30% dimethyl sulfoxide. The same preparation under optimal conditions (pH 7.5, 10 microM CaCl2, 100 mM KCl and no dimethyl sulfoxide at 37 degrees C) displays a velocity of ATP hydrolysis between 8 and 12 X 10(5) nmol of Pi X mg of protein-1 X h-1 while the phosphoenzyme levels varied between 3.5 and 4.0 nmol of EP X mg of protein-1. Enzyme phosphorylation from ATP in the absence of Ca2+ always preceded Pi liberation into the assay media. Two different phosphoenzyme species were formed which were kinetically distinguished by their decomposition rates. The observed steady-state velocity of ATP hydrolysis could be accounted for either by the decay of the fast component or by the simultaneous decomposition of both phosphoenzyme species. The hydrolysis of the phosphoenzyme formed in the absence of Ca2+ was KCl-stimulated and ADP-independent. The rate constant of breakdown was equal to that observed for the phosphoenzyme formed in the presence of Ca2+. It is suggested that the rapidly decaying phosphoenzyme (and possibly both rapidly and slowly decaying species) are intermediates in the reaction cycle of Mg2+-dependent ATP hydrolysis of sarcoplasmic reticulum Ca2+-ATPase and may represent a bypass of Ca2+ activation by dimethyl sulfoxide.     

Binding of divalent cation to phosphoenzyme of sodium- and potassium-transport adenosine triphosphatase.

In order to study the action of the divalent cation which is essential for phosphorylation of sodium- and potassium-transport adenosine triphosphatase, magnesium ion, the normal ligand, was replaced with calcium ion, which had properties diffeerent from those of Mg2+, Mn2+, Fe2+, Co2+, Ni2+, or Zn2+. Phosphorylation of the enzyme from ATP at pH 7.4 in the presence of Na+ and Ca2+ yielded a Ca.phosphoenzyme (60% of the maximal level) with a normal rate of dephosphorylation following a chase with unlabeled Ca.ATP (PK = 0.092S-1 at 0 degrees C). In contrast, after a chase by a chelator, namely ethylenediaminetetraacetic acid, 1,2-cyclohexylenedinitrilotetraacetic acid, or ethylene glycol bis-(beta-aminoethyl ether)N,N'-tetraacetic acid, dephosphorylation slowed within 5 s and half of the initial phosphoenzyme remained with a stability about 5-fold greater than normal. Three states of the phosphoenzyme were distinguished according to their relative sensitivity to ADP or to K+ added during a chase. Normally prepared Mg.phosphoenzyme was sensitive to K+ but not to ADP; Ca.phosphoenzyme was sensitive either to ADP or to K+; and the stabilized phosphoenzyme prepared from Ca.phosphoenzyme by addition of a chelator was sensitive neither to ADP nor to K+ nor to both together. Addition of Ca2+ to the stabilized phosphoenzyme restored the reactivity to that of Ca.phosphoenzyme. Addition of Mg2+ to the stabilized phosphoenzyme changed the reactivity to that of Mg.phosphoenzyme. Therefore, this unreactive, stabilized state of the phosphoenzyme appeared to be a divalent cation-free phosphoenzyme. With respect to sensitivity to ouabain, Ca.phosphoenzyme was as sensitive as Mg.phosphoenzyme but calcium-free phosphoenzyme was much less sensitive. It was concluded that the divalent cation required for phosphorylation normally remains tightly bound to the phosphoenzyme and is required for normal reactivity. Calcium ion was almost unique in dissociating relatively easily from the phosphoenzyme. Strontium ion appeared to act similarly to Ca2+.     

Acetyl phosphate as a substrate for the calcium ATPase of sarcoplasmic reticulum

Acetyl phosphate is hydrolyzed by the calcium ATPase of leaky sarcoplasmic reticulum vesicles from rabbit skeletal muscle with Km = 6.5 mM and kcat = 7.9 s-1 in the presence of 100 microM calcium (180 mM K+, 5 mM MgSO4, pH 7.0, 25 degrees C). In the absence of calcium, hydrolysis is 6% of the calcium-dependent rate at low and 24% at saturating concentrations of acetyl phosphate. Values of K0.5 for calcium are 3.5 and 2.2 microM (n = 1.6) in the presence of 1 and 50 mM acetyl phosphate, respectively; inhibition by calcium follows K0.5 = 1.6 mM (n approximately 1.1) with 50 mM acetyl phosphate and K0.5 = 0.5 mM (n approximately 1.3) with 1.5 mM ATP. The calcium-dependent rate of phosphoenzyme formation from acetyl phosphate is consistent with Km = 43 mM and kf = 32 s-1 at saturation; decomposition of the phosphoenzyme occurs with kt = 16 s-1. The maximum fraction of phosphoenzyme formed in the steady state at saturating acetyl phosphate concentrations is 43-46%. These results are consistent with kc congruent to 30 s-1 for binding of Ca2+ to E at saturating [Ca2+], to give cE.Ca2, in the absence of activation by ATP. Phosphoenzyme formed from ATP and from acetyl phosphate shows the same biphasic reaction with ADP, rate constants for decomposition that are the same within experimental error, and similar or identical activation of decomposition by ATP. It is concluded that the reaction pathways for acetyl phosphate and ATP in the presence of Ca2+ are the same, with the exception of calcium binding and phosphorylation; an alternative, faster route that avoids the kc step is available in the presence of ATP. The existence of three different regions of dependence on ATP concentration for steady state turnover is confirmed; activation of hydrolysis at high ATP concentrations involves an ATP-induced increase in kt.     

Pre-steady-state phosphorylation of the human red cell Ca2+-ATPase

The pre-steady-state kinetics of phosphorylation of the Ca2+-ATPase by ATP was studied at 37 degrees C and in intact red cell membranes to approach physiological conditions. ATP and Ca2+ activate with K0.5 of 4.9 and 26.4 microM, respectively. Preincubation with Ca2+ did not change the K0.5 for ATP. Preincubation with ATP did not alter the initial velocity of phosphorylation suggesting that binding of ATP was not rate-limiting. Mg2+ added at the start of the reaction increased the initial rate of phosphorylation from 4 to 8 pmol/mg/s. With 30 microM Ca2+, the K0.5 for Mg2+ was 60 microM. Mg2+ and Ca2+ added together beforehand accelerated phosphorylation to 70 pmol/mg/s. Phosphorylation of calmodulin-bound membranes was the fastest (280 pmol/mg/s), and its time course showed a neat overshoot before steady state. The results suggest that either preincubation with Ca2+ plus Mg2+ or calmodulin accelerated phosphorylation shifting toward E1 the equilibrium between the E1 and E2 conformers of the enzyme. K+ had no effect on the initial rate of phosphorylation and lowered by 40% the steady-state level of phosphoenzyme in the absence of Mg2+. Phosphorylation is not rate-limiting for the overall reaction since its initial rate was always higher than ATPase activity. In the absence of K+, the turnover of the phosphoenzyme was 2000 min-1, which is close to the values for other transport ATPases.     

Magnesium permeability of sarcoplasmic reticulum. Mg2+ is not countertransported during ATP-dependent Ca2+ uptake by sarcoplasmic reticulum

Magnesium transport across sarcoplasmic reticulum (SR) vesicles was investigated in reaction mixtures of various composition using antipyrylazo III or arsenazo I to monitor extravesicular free Mg2+. The half-time of passive Mg2+ efflux from Mg2+-loaded SR was 100 s in 100 mM KCl, 150 S in 100 mM K gluconate, and 370 S in either 100 mM Tris methanesulfonate or 200 mM sucrose solutions. The concentration and time course of Mg2+ released into the medium was also measured during ATP-dependent Ca2+ uptake by SR. In reaction mixtures containing up to 3 mM Mg2+, small changes in free magnesium of 10 microM or less were accurately detected without interference from changes in free Ca2+ of up to 100 microM. Three experimental protocols were used to determine whether the increase of free [Mg2+] in the medium after an addition of ATP was due to Mg2+ dissociated from ATP following ATP hydrolysis or to Mg2+ translocation from inside to outside of the vesicles. 1) In the presence of ATP-regenerating systems which maintained constant ATP to ADP ratios and normal rates of active Ca2+ uptake, the increase of Mg2+ in the medium was negligible. 2) Mg2+ released during ATP-dependent Ca2+ uptake by SR was similar to that observed during ATP hydrolysis catalyzed by apyrase, in the absence of SR. 3) In SR lysed with Triton X-100 such that Ca2+ transport was uncoupled from ATPase activity, the rate and amount of Mg2+ release was greater than that observed during ATP-dependent Ca2+ uptake by intact vesicles. Taken together, the results indicate that passive fluxes of Mg2+ across SR membranes are 10 times faster than those of Ca2+ and that Mg2+ is not counter-transported during active Ca2+ accumulation by SR even in reaction mixtures containing minimal concentrations of membrane permeable ions that could be rapidly exchanged or cotransported with Ca2+ (e.g. K+ or Cl-).     

ATP regulation of calcium transport in back-inhibited sarcoplasmic reticulum vesicles

At high concentrations of ATP, ATP hydrolysis and Ca2+ transport by the (Ca2+ + MG2+)-ATPase of intact sarcoplasmic reticulum vesicles exhibit a secondary activation that varies with the extent of back-inhibition by Ca2+ accumulated within the vesicles. When the internal ionized Ca2+ is clamped at low and intermediate levels by the use of Ca-precipitating anions, the apparent Km values for activation by ATP are lower than in fully back-inhibited vesicles (high internal Ca2+). In leaky vesicles unable to accumulate Ca2+, raising Ca2+ in the assay medium from 20-30 microM to 5 mM abolishes the activation of hydrolysis by high concentrations of ATP. The level of [32P]phosphoenzyme formed during ATP hydrolysis from [32P]phosphate added to the medium also varies with the extent of back-inhibition; it is highest when Ca2+ is raised to a level that saturates the internal, low-affinity Ca2+ binding sites. In intact vesicles, increasing the ATP concentration from 10 to 400 microM competitively inhibits the reaction of inorganic phosphate with the enzyme but does not change the rate of hydrolysis. In a previous report (De Meis, L., Gomez-Puyou, M.T. and Gomez-Puyou, A. (1988) Eur. J. Biochem. 171, 343-349), it has been shown that the hydrophobic molecules trifluoperazine and iron bathophenanthroline compete for the catalytic site of the Pi-reactive form of the enzyme. Here it is shown that inhibition of ATP hydrolysis by these compounds is reduced or abolished when Ca2+ binds to the low-affinity Ca2+ binding sites of the enzyme. Since inhibition by these agents is indifferent to activation of hydrolysis by high concentrations of ATP, it is suggested that the second Km for ATP and the inhibition by hydrophobic molecules involve two different Ca-free forms of the enzyme.     

Ca2+-stimulated, Mg2+-dependent ATPase activity in neutrophil plasma membrane vesicles. Coupling to Ca2+ transport

Low concentrations of free Ca2+ stimulated the hydrolysis of ATP by plasma membrane vesicles purified from guinea pig neutrophils and incubated in 100 mM HEPES/triethanolamine, pH 7.25. In the absence of exogenous magnesium, apparent values obtained were 320 nM (EC50 for free Ca2+), 17.7 nmol of Pi/mg X min (Vmax), and 26 microM (Km for total ATP). Studies using trans- 1,2-diaminocyclohexane- N,N,N',N',-tetraacetic acid as a chelator showed this activity was dependent on 13 microM magnesium, endogenous to the medium plus membranes. Without added Mg2+, Ca2+ stimulated the hydrolysis of several other nucleotides: ATP congruent to GTP congruent to CTP congruent to ITP greater than UTP, but Ca2+-stimulated ATPase was not coupled to uptake of Ca2+, even in the presence of 5 mM oxalate. When 1 mM MgCl2 was added, the vesicles demonstrated oxalate and ATP-dependent calcium uptake at approximately 8 nmol of Ca2+/mg X min (based on total membrane protein). Ca2+ uptake increased to a maximum of approximately 17-20 nmol of Ca2+/mg X min when KCl replaced HEPES/triethanolamine in the buffer. In the presence of both KCl and MgCl2, Ca2+ stimulated the hydrolysis of ATP selectively over other nucleotides. Apparent values obtained for the Ca2+-stimulated ATPase were 440 nM (EC50 for free Ca2+), 17.5 nmol Pi/mg X min (Vmax) and 100 microM (Km for total ATP). Similar values were found for Ca2+ uptake which was coupled efficiently to Ca2+-stimulated ATPase with a molar ratio of 2.1 +/- 0.1. Exogenous calmodulin had no effect on the Vmax or EC50 for free Ca2+ of the Ca2+-stimulated ATPase, either in the presence or absence of added Mg2+, with or without an ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N',-tetraacetic acid pretreatment of the vesicles. The data demonstrate that calcium stimulates ATP hydrolysis by neutrophil plasma membranes that is coupled optimally to transport of Ca2+ in the presence of concentrations of K+ and Mg2+ that appear to mimic intracellular levels.