Phosphatidylcholine makes specific activity of the purified Ca(2+)-ATPase from plasma membranes independent of enzyme concentration.

Ca(2+)-ATPase of plasma membranes (PMCA) was isolated from either human or pig red cells by calmodulin-affinity chromatography and supplemented with phosphatidylcholine (PC). The specific activity of the purified PMCA diluted in media with detergent (C(12)E(10)) was very low, and increased with the concentration of the enzyme along a curve that reached the maximum at 8 microg/ml with K(0.5)=1.2-2.5 microg/ml. Such behavior has been described and attributed to self-association of the enzyme (D. Kosk-Kosicka and T. Bzdega, J. Biol. Chem. 263 (1988) 18184-18189). After heat-inactivation, the PMCA was as effective an activator as the intact enzyme, increasing, to the maximum, the specific activity of diluted enzyme with K(0. 5)=2.2 microg/ml. The inactivated PMCA failed to increase the activity of concentrated enzyme, suggesting that activation did not depend on interaction of intact with denatured enzyme molecules. When enough PC was added to the reaction medium to make its final concentration 16-33 microg/ml, the specific activity of the PMCA was maximum and independent of enzyme concentration. Under these conditions, activation by calmodulin lowered to 10%. As a function of the concentration of pure PC, maximum specific activity was reached along a curve with K(0.5)=4 microg/ml. This curve was identical to that of activation at increasing enzyme concentration, suggesting that, in the latter case, activation could have depended on PC contributed to the assay medium by the enzyme. The results show that PC made the purified PMCA solubilized in detergent reach maximum activity at any concentration of the enzyme.

Replacement of Val674 by Pro increases the sensitivity of the plasma membrane Ca2+ pump to inhibition by Mg2+.

A cDNA encoding a plasma membrane Ca2+ pump mutant V674P(ct120) was constructed and expressed in COS-1 cells. Immunoblots of transfected COS-1 membranes showed that the V674P(ct120) and the wild-type hPMCA4b(ct120) proteins were expressed at similar levels. The change of Val674 to Pro reduced the activity of the hPMCA4b(ct120) to an extent similar to that observed previously in the full-length Ca2+ pump (Adamo et al. (1995) J. Biol. Chem. 270, 30111-30114). Despite its lower activity, the apparent affinity for Ca2+ of the V674P(ct120) enzyme was at least as high as that of hPMCA4b(ct120), indicating that substitution of Val674 by Pro did not impair the interaction of the enzyme with Ca2+. The sensitivity of the V674P(ct120) enzyme to inhibition by vanadate was not significantly different from that of the hPMCA4b(ct120), supporting the idea that the mutation did not alter the equilibrium between E2-E1. The study of the Mg2+ dependency of the Ca2+ transport showed that the V674P(ct120) mutant reached maximum activation at 100 microM Mg2+ in contrast with 500 microM in the hPMCA4b(ct120). Furthermore, while at 2 mM Mg2+ the hPMCA4b(ct120) showed no sign of inhibition, the activity of the mutant decreased to less than 50% of the maximum activity observed at 100 microM Mg2+. These results indicate that the decrease in the activity observed upon substitution of Val674 by Pro was due to a higher sensitivity to Mg2+ as inhibitor.

Pre-steady-state kinetic study of the mechanism of inhibition of the plasma membrane Ca(2+)-ATPase by lanthanum.

Lanthanides are known to be effective inhibitors of the PMCa(2+)-ATPase. The effects of LaCl3 on the partial reactions that take place during ATP hydrolysis by the calcium-dependent ATPase from plasma membrane (PMCa(2+)-ATPase) were studied at 37 degrees C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. LaCl3 added before phosphorylation (K0.5 = 2.8 +/- 0.2 microM) raised the kapp of the E2-->E1 transition from 14 +/- 2 to 23 +/- 4 s-1. The effect was independent of Ca2+ and Mg2+, as if La3+ substituted for Mg2+ and/or Ca2+ in accelerating the formation of E1 with higher efficiency. At non-limiting conditions, LaCl3 doubled the apparent concentration of E1 in the enzyme at rest with Ca2+ and Mg2+. LaCl3 during phosphorylation (K0.5 near 20 microM) lowered the vo of the reaction from 300 +/- 20 to 60 +/- 7 pmol/mg of protein/s, a close rate to that in the absence of Mg2+. This effect was reversed by Mg2+ (and not by Ca2+), and the K0.5 for Mg2+ as activator of the phosphorylation reaction increased linearly with the concentration of LaCl3, suggesting that La3+ slowed phosphorylation by displacing Mg2+ from the activation site(s). If added before phosphorylation, LaCl3 lowered the kapp for decomposition of EP to 0.8 +/- 0.1 s-1, a value which is characteristic of phosphoenzyme without Mg2+. The K0.5 for this effect was 0.9 +/- 0.5 microM LaCl3 and increased linearly with the concentration of Mg2+. If added after phosphorylation, LaCl3 did not change the kapp of 90 +/- 7 s-1 of decomposition of EP, suggesting that La3+ displaced Mg2+ from the site whose occupation accelerates the shifting of E1P to E2P. In medium with 0.5 mM MgCl2, 2 microM LaCl3 lowered rapidly the rate of steady-state hydrolysis of ATP by the PMCa(2+)-ATPase to a value close to the rate of decomposition of EP made in medium with LaCl3. Increasing MgCl2 to 10 mM protected the PMCa(2+)-ATPase against inhibition during the first 10 min of incubation. Results show that combination of La3+ to the Mg2+ (and Ca2+) site(s) in the unphosphorylated PMCa(2+)-ATPase accelerates the E2-->E1 transition and inhibits the shifting E1P--> E2P. Since with less apparent affinity La3+ slowed but did not impede phosphorylation, it seems that the sharp slowing of the rate of transformation of E1P into E2P by displacement of Mg2+ was the cause of the high-affinity inhibition of the PMCa(2+)-ATPase by La3+.

Deletion of amino acid residues 18-75 inactivates the plasma membrane Ca2+ pump.

A mutant of the plasma membrane Ca2+ pump hPMCA4b(d18-75)(ct120) containing a deletion of the N-terminal amino acid residues 18-75 and lacking the C-terminal 120 amino acid residues was expressed in COS-1 cells. The deletion in the N-terminal region did not significantly affect the level of expression of the Ca2+ pump. Tryptic digestion of the hPMCA4b(d18-75)(ct120) mutant resulted in the appearance of the same fragments obtained by proteolysis of the hPMCA4b(ct120) enzyme, suggesting that deletion of residues 18-75 neither impeded the insertion in the membrane nor extensively affected the folding of the mutant protein. The functional competence of the hPMCA4b(d18-75)(ct120) enzyme was examined by measuring the Ca2+ transport and the Ca2+ ATPase activity of COS-1 cell microsomes expressing the mutant protein. Both tests proved the mutant to be inactive. Under conditions in which hPMCA4b(ct120) becomes phosphorylated, hPMCA4b(d18-75)(ct120) was incapable of reacting with ATP and Ca2+ to form the phosphoenzyme. Taken together these results suggest that the segment of amino acids 18-75 is essential for the activity of the plasma membrane Ca2+ pump.

Pre-steady-state kinetic study of the effects of K+ on the partial reactions of the catalytic cycle of the plasma membrane Ca(2+)-ATPase.

The effects of 100 mM K+ on the partial reactions that take place during ATP hydrolysis on the calcium ion-dependent ATPase from plasma membrane (PM-Ca(2+)-ATPase) were studied at 37 degrees C on fragmented intact membranes from pig red cells by means of a rapid chemical quenching technique. At 10 microM [gamma-32P]ATP plus non-limiting concentrations of Ca2+ and Mg2+, K+ increased the k(app) of formation by 140% to 84 11 s-1 and the steady-state level of phosphoenzyme (EP) by 25% to 3.4 0.17 pmol/mg of protein. If added together with [gamma-32P]ATP at the beginning of phosphorylation, K+ was much less effective than if added earlier, indicating that it did not act on the phosphorylation reaction. Measurements of the E2 --> E1 transition by phosphorylation showed that in medium with Ca2+ and Mg2+, K+ increased the k(app) of the transition by 55% to 14 3 s-1 and the apparent concentration of E1 by 45%, suggesting that this may be the cause of the increased rate of phosphorylation observed in enzyme preincubated with K+. The presence of K+ did not change the slow decay of EP without Mg2+ but activated the decay of EP made with Mg2+, increasing its k(app) by 60% to 91 12 s-1. In contrast with observations made during phosphorylation, if added at the beginning of dephosphorylation K+ was fully effective in favouring decomposition of EP made in medium containing no K+. In the presence of either 3mM ATP or 3 mM ATP plus calmodulin, which activate hydrolysis of CaE2P, the effect of K+ on dephosphorylation was conserved. Because the sites for K+ are intracellular and the concentration of K+ in normal red cells is above 100 mM, the effects described here must be taken into account to describe the catalytic cycle of the PM-Ca(2+)-ATPase under physiological conditions.

Effects of magnesium plus vanadate on partial reactions of the Ca(2+)-ATPase from human red cell membranes.

Under conditions in which pretreatment with Mg2+ plus vanadate activate the Ca(2+)-ATPase, the initial rate of phosphorylation of the enzyme increased from 141 to 259 pmol/mg protein per s while the steady-state level of phosphoenzyme lowered from 1.9 to 1.1 pmol/mg protein. The drop in phosphoenzyme level was caused by incubation and washing during treatment rather than by vanadate. The data allowed to estimate a turnover number for the enzyme that raised by 170% after pretreatment. The results show that the activation of the Ca(2+)-ATPase by Mg2+ plus vanadate is due to changes in the kinetic properties of the enzyme.

The dephosphorylation reaction of the Ca(2+)-ATPase from plasma membranes.

The breakdown of phosphoenzyme (EP) of the Ca(2+)-ATPase from pig red blood cell membranes was studied at 37 degrees C by means of a rapid chemical quenching technique. When the enzyme was phosphorylated with [gamma-32P]ATP in media without added MgCl2, all the EP formed disappeared along two single exponential curves, a rapid one with k(app) = 90 +/- 10 s-1 and a slow one with k(app) = 0.7 +/- 0.3 s-1. The amount of EP involved in each reaction was close to 50% of the EP present at the beginning. Only EP of rapid breakdown could account for the steady-state hydrolysis of ATP observed under the same experimental conditions. ADP accelerated the slow reaction 45-fold (k(app) = 31 +/- 9 s-1) with K0.5 = 740 +/- 120 microM as if this reaction represented the decay of CaE1P, which donated its phosphate to water slowly in the forward direction and rapidly to ADP in the reverse direction of the cycle. Combination of Mg2+ with K0.5 = 26.3 +/- 5.0 microM at a single class of site in E1 before phosphorylation increased EP of rapid breakdown at the expense of ADP-sensitive EP so that, at nonlimiting concentrations of Mg2+ in the phosphorylation media, all EP decomposed at high rate. Rapid decomposition was observed even with enough CDTA to chelate most of the Mg2+ remaining from phosphorylation, suggesting that the role of Mg2+ during dephosphorylation was to accelerate the transition CaE1P-->CaE2P, preparing EP for hydrolysis. The combination of ATP at a single class of site with Km = 845 +/- 231 microM accelerated the hydrolysis of CaE2P. Calmodulin alone had no effects on dephosphorylation but enhanced acceleration of hydrolysis of CaE2P by ATP making the decay of EP under these conditions the fastest among those measured. Comparison of the rates of dephosphorylation of EP made in the presence of Mg2+ with those of steady-state Ca(2+)-ATPase activity with and without calmodulin showed that the transition CaE1P-->CaE2P and decomposition of CaE2P by hydrolysis are compatible with their role as obligatory intermediate reactions in the cycle of hydrolysis of ATP by the Ca(2+)-ATPase.

Low affinity acceleration of the phosphorylation reaction of the Na,K-ATPase by ATP.

The maximum rate of phosphorylation (rm) of a highly purified Na,K-ATPase from red outer medulla of pig kidney was measured at 25 degrees C as a function of ATP concentration in media with Mg2+, Na+, and no K+. When rm was plotted as a function of the concentration of ATP a biphasic response was observed with a hyperbolic component of high affinity (Km = 15.7 +/- 2.6 microM) and low velocity ((rm)max = 460 +/- 40 nmol of Pi/(mg of protein.s)) plus a parabolic component which showed no saturation up to 1000 microM ATP, concentration at which rm was 1768.1 +/- 429.6 nmol Pi/(mg protein.s) (mean +/- S.E.; n = 3). This low affinity effect of ATP on the rate of phosphorylation disappeared when the Na,K-ATPase underwent turnover in medium without K+ suggesting that, like superphosphorylation (Peluffo, R. D., Garrahan, P. J., and Rega, A. F. (1992) J. Biol. Chem. 267, 6596-6601), it required the enzyme to be at rest. This property of the Na,K-ATPase was not predicted by the Albers-Post reaction scheme. The observed behavior of the enzyme could be simulated by a scheme that involves a resting enzyme (Er) functionally different from E1 or E2, which is able to bind three molecules of ATP, one with high and two with low affinity, and that after phosphorylation is converted into the phosphointermediates that are generally considered to participate in the reaction cycle described by Albers and Post.

Low affinity superphosphorylation of the Na,K-ATPase by ATP.

Pre-steady-state phosphorylation of purified Na,K-ATPase from red outer medulla of pig kidney was studied at 25 degrees C and an ample range of [tau-32P]ATP concentrations. At 10 microM ATP phosphorylation followed simple exponential kinetics reaching after 40 ms a steady level of 0.76 +/- 0.04 nmol of P/mg of protein with kapp = 73.0 +/- 6.5 s-1. At 500 microM ATP the time course of phosphorylation changed drastically, since the phosphoenzyme reached a level two to four times higher at a much higher rate (kapp greater than or equal to 370 s-1) and in about 40 ms dropped to the same steady level as with 10 microM ATP. This superphosphorylation was not observed in Na,K-ATPase undergoing turnover in a medium with Mg2+, Na+, and ATP, suggesting that it required the enzyme to be at rest. Superphosphorylation depended on Mg2+ and Na+ and was fully inhibited by ouabain and FITC. After denaturation the phosphoenzyme made by superphosphorylation had the electrophoretic mobility of the alpha-subunit of the Na,K-ATPase, and its hydrolysis was accelerated by hydroxylamine. On a molar basis, the stoichiometry of phosphate per ouabain bound was 2.40 +/- 0.60 after phosphorylation with 1000 microM ATP. The results are consistent with the idea that under proper conditions every functional Na,K-ATPase unit can accept two, or more, phosphates of rapid turnover from ATP.

Does calmodulin regulate the affinity of the human red cell Ca2+ pump for ATP?

(1) We have reexamined the effects of calmodulin and of the calmodulin antagonist, compound 48/80 on the interaction of ATP at its low-affinity site in the Ca2(+)-ATPase from human red cells. (2) At variance with our earlier proposal (Biochim. Biophys. Acta (1985) 816, 379-386) calmodulin increased the maximum effect of ATP without changing the apparent affinity for ATP at the low-affinity site. Accordingly, ATP increased the maximum activation by calmodulin without altering the apparent affinity of the Ca2(+)-ATPase for calmodulin. (3) Confirming our previous observation (Biochim. Biophys. Acta (1985) 816, 379-386) compound 48/80 lowered the apparent affinity of the Ca2(+)-ATPase for ATP at the low-affinity site. This has to be attributed to a direct effect of this compound on the enzyme rather than to its effect as calmodulin antagonist.

Magnesium-ions accelerate the formation of the phosphoenzyme of the (Ca2+ + Mg2+)-activated ATPase from plasma membranes by acting on the phosphorylation reaction.

Magnesium ions in the reaction medium at 37 degrees C increased up to 222 s-1 the kapp for phosphorylation by ATP of the Ca2(+)-ATPase of pig red cell membranes. This effect was observed after partial proteolysis with trypsin which makes the enzyme behave like the E1 conformer during phosphorylation. These findings lead to the conclusion that Mg2+ increased the rate of phosphorylation of the Ca2(+)-ATPase by acting directly on this reaction. The apparent dissociation constant of Mg2+ for this effect was 44 microM whereas the apparent dissociation constant for Mg2+ to accelerate the shift E2----E1 between conformers measured on the intact enzyme was 50 microM. This suggests that Mg2+ accelerated both reactions from a single class of site.

The E2 in equilibrium E1 transition of the Ca2(+)-ATPase from plasma membranes studied by phosphorylation.

The relative abundance of the two conformers (E1 and E2) of the Ca2(+)-ATPase of plasma membranes and the rates of their interconversion were estimated measuring the initial velocity of phosphorylation of the Ca2(+)-ATPase in pig red cell membranes at 37 degrees C. This was based on the hypothesis that only E1 catalyzes phosphorylation from ATP. In the absence of ligands near 90% of the Ca2(+)-ATPase was in the E2 conformation. Ca2+ shifted the equilibrium toward E1. The K0.5 of Ca2+ for this effect was 15 microM, suggesting that it acted at the transport site. The conversion of E2 into E1 was slow (t1/2 = 911 s) while the conversion of E1 into E2 was faster (t1/2 less than or equal to 60 s). Mg2+ accelerated the E2----E1 reaction lowering its t1/2 to 0.25 s. In the presence of 4 mM Ca2+ t1/2 was 7.8 s, as if at this concentration to some extent Ca2+ replaced Mg2+ in accelerating the E2----E1 reaction. Results suggest that in intact membranes at 37 degrees C Ca2+ stabilized E1 and that the Ca2(+)-induced E2----E1 transition was strongly accelerated by Mg2+. Both cations were effective at near physiological concentrations and in the absence of other ligands like ATP or calmodulin that could also modify the reaction. After partial proteolysis with trypsin the Ca2(+)-ATPase behaved during phosphorylation as if it were E1.

A study to see whether phosphatidylserine, partial proteolysis and EGTA substitute for calmodulin during activation of the Ca2+-ATPase from red cell membranes by ATP.

(1) The effects of treatments that mimic calmodulin in increasing the apparent affinity for Ca2+ were tested to see whether, like calmodulin, they also change the activation of the Ca2+-ATPase from human red cell membranes by ATP at the low-affinity site. (2) Short incubations with either trypsin or acidic phospholipids such as phosphatidylserine increased the apparent affinity for ATP at the low-affinity site. (3) Under conditions in which it increased the apparent affinity of the Ca2+-ATPase for Ca2+, EGTA failed to change the activation by ATP. (4) As in calmodulin-bound Ca2+-ATPase, compound 48/80 inhibited the activity of the enzyme in the presence of phosphatidylserine by lowering the apparent affinity for ATP at the low-affinity site, leaving the maximum velocity of the enzyme unaltered. (5) Compound 48/80 also inhibited the Ca2+-ATPase after partial proteolysis, but in this case it lowered the maximum activity, leaving the apparent affinity of the enzyme for ATP at the low-affinity site unaltered. (6) Inhibition of the Ca2+-ATPase by compound 48/80 in the absence of calmodulin suggests that the inhibitor can act directly on the enzyme.

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.

Comparison between plasma membrane Ca2+ and Na,K-ATPases: short review.

1. This paper is a short review of the comparative biochemistry of the Na,K-ATPase and Ca2+-ATPase of plasma membranes. 2. The two ATPases share the same biphasic activation by ATP. Ca2+-ATPase activation by ATP is strongly affected by calmodulin. 3. The possibility of Mg2+ occlusion is proposed in connection with low-affinity activation by ATP. 4. Both ATPases are activated by alkaline earth metal ions and display phosphatase activity toward p-nitrophenylphosphate for which Ca2+-ATPase is strongly dependent on K+ and regulated by calmodulin. 5. The requirements for ligands of the phosphatase activity of both ATPases are strikingly similar except for the effect of calmodulin. 6. Both ATPases are inhibited by vanadate and for both the effect of vanadate is modulated by Mg2+ and K+ in the same way. 7. These similarities indicate that, although Ca2+-ATPase and Na,K-ATPase are different enzymes, their mechanisms of action may have more features in common than previously thought.

Comparasion between plasma membrane Ca2 + and Na, K-ATPases: short review

This paper is a short review of the comparative biochemistry of the Na, K-ATPase and Ca2+ -ATPase of plasma membranes. The two ATPases share the same biphasic activation by ATP. Ca2+ - ATPase activation by ATP is strongly affected by calmodulin. The possibility of Mg2+ occlusion is proposed in connection with low-affinity activation by ATP. Both ATPase are activated by alkaline earth metal ions and display phosphatase activity toward p-nitrophenylphosphat for which Ca2 + - ATPase is strongly dependent on K + and regulated by calmodulin. The requirements for ligands of the phosphatase activity of both ATPase are strikingly similar except for the effect of calmodulin. Both ATPases are inhibited by vanadat and for both the effect of vanadate is modulated by Mg2+ and K + in the same way. These similarities indicate that, although Ca2 + - ATPase and Na, K-ATPase are different enzymes, their mechanisms of action may have more feactures in common than previously thought

Gramicidin S inhibition of the Ca2+-ATPase of human red blood cells.

The cationic amphiphilic polypeptide gramicidin S inhibits the Ca2+-ATPase of human red-cell membranes by lowering the maximum velocity of the high-affinity component and the apparent affinity of the low-affinity component of the velocity-versus-ATP concentration curve of the enzyme. Gramicidin S does not alter the apparent affinity of the Ca2+-ATPase for Ca2+. Calmodulin is not essential for the inhibition, but increases the sensitivity of the enzyme to the inhibitor. The effects of gramicidin S on the Ca2+-ATPase can be reversed with phosphatidylcholine vesicles but not with buffer solutions, suggesting that gramicidin S acts from the lipid phase of the membrane.