Phosphorylation of the calcium-transporting adenosinetriphosphatase by lanthanum ATP: rapid phosphoryl transfer following a rate-limiting conformational change

The calcium-transport ATPase (CaATPase) of rabbit sarcoplasmic reticulum preincubated with 0.02 mM Ca2+ (cE.Ca2) is phosphorylated upon the addition of 0.25 mM LaCl3 and 0.3 mM [gamma-32P]ATP with an observed rate constant of 6.5 s-1 (40 mM MOPS, pH 7.0, 100 mM KCl, 25 degrees C). La.ATP binds to cE.Ca2 with a rate constant of 5 X 10(6) M-1 s-1, while ATP, Ca2+, and La3+ dissociate from cE.Ca2.La.ATP at less than or equal to 1 s-1. The reaction of ADP with phosphoenzyme (EP) formed from La.ATP is biphasic. An initial rapid loss of EP is followed by a slower first-order disappearance, which proceeds to an equilibrium mixture of EP.ADP and nonphosphorylated enzyme with bound ATP. The fraction of EP that reacts in the burst (alpha) and the first-order rate constant for the slow phase (kb) increase proportionally with increasing concentrations of ADP to give maximum values of 0.34 and 65 s-1, respectively, at saturating ADP (KADPS = 0.22 mM). The burst represents rapid phosphoryl transfer and demonstrates that ATP synthesis and hydrolysis on the enzyme are fast. The phosphorylation of cE.Ca2 by La.ATP at 6.5 s-1 and the kinetics for the reaction of EP with ADP are consistent with a rate-limiting conformational change in both directions. The conformational change converts cE.Ca2.La.ATP to the form of the enzyme that is activated for phosphoryl transfer, aE.Ca2.La.ATP, at 6.5 s-1; this is much slower than the analogous conformational change at 220 s-1 with Mg2+ as the catalytic ion [Petithory & Jencks (1986) Biochemistry 25, 4493]. The rate constant for the conversion of aE.Ca2.La.ATP to cE.Ca2.La.ATP is 170 s-1. ATP does not dissociate measurably from aE.Ca2.La.ATP. Labeled EP formed from cE.Ca2 and La.ATP with leaky vesicles undergoes hydrolysis at 0.06 s-1. It is concluded that the reaction mechanism of the CaATPase is remarkably similar with Mg.ATP and La.ATP; however, the strong binding of La.ATP slows both the conformational change that is rate limiting for EP formation and the dissociation of La.ATP. An interaction between La3+ at the catalytic site and the calcium transport sites decreases the rate of calcium dissociation by greater than 60-fold. When cE-Ca2 is mixed with 0.3 mM ATP and 1.0 mM Cacl2, the phosphoenzyme is formed with an observed rate constant of 3 s-1. The phosphoenzyme formed from Ca.ATP reacts with 2.0 mM ADP and labeled ATP with a rate constant of 30 s-1; there may be a small burst (alpha less than or equal to 0.05).     

Transient-state kinetics of phosphoenzyme transformation in the rabbit skeletal sarcoplasmic reticulum calcium-dependent adenosine triphosphatase reaction. Two distinct modes of ADP and K+ regulation

We have observed two modes each of ADP and K+ regulation of phosphoenzyme (EP) intermediates formed in the early phase of skeletal sarcoplasmic reticulum hydrolysis of ATP at 20 degrees C, using, for the first time, a five-syringe quench flow apparatus for transient-state kinetic measurements. The total acid-stable EP formed for 20.5 and 116 ms in the K+ medium appears to be composed of either two monomers in rapid equilibrium, E1P in equilibrium E'1P, or a dimer of the two subunits, PE1E'1P. The ADP-sensitive E1P may form an acid-labile ADP X E1P (or ATP X E1) complex rapidly, giving ATP as a consequence of acid quenching. The ADP may also induce decomposition of the ADP-reactive E'1P. Monomeric and dimeric mechanisms are introduced to account for the hyperbolic relation between the rate constant of the ADP-induced E'1P decomposition and [ADP], consistent with the fact that the E'1P may also give ATP in the presence of ADP. As to the K+ effects, the K+, which is bound to the unphosphorylated enzyme and possibly becomes occluded during EP formation, may either facilitate the one-to-one E1P in equilibrium E'1P equilibrium or maintain the dimeric functional unit. The subsequent forward transformation of the E'1P to the ADP-insensitive K+-sensitive E'2P, possibly the rate-determining step for the catalytic cycle, is found to be K+ independent. The major effect of the K+ in the medium is its catalytic cleavage of the E'2P, which is detected as the missing EP under these conditions. When K+ is not involved in the EP formation, the forward sequential transformation E1P----E'1P----E'2P----E2P or PE1E'1P----PE'2E2P is apparent in the time range from 20.5 to 116 ms after EP formation, and the E'2P may accumulate in the K+ devoid medium and be detected as the major component of the total acid-stable EP. The Mg2+-sensitive E2P represents the EP missing in the medium containing no ADP and K+.     

Desensitization to Mg2+ of the phosphoenzyme in sarcoplasmic reticulum Ca2+-ATPase by the addition of a nonionic detergent and the restoration of sensitivity after its removal

Sarcoplasmic reticulum (SR) membranes from rabbit skeletal muscle were solubilized with a high concentration of dodecyl octaethyleneglycol monoether (C12E8) and the kinetic properties of the Ca2+,Mg2+-dependent ATPase [EC 3.6.1.3] were studied. The following results were obtained: 1. SR ATPase solubilized in C12E8 retains high ability to form phosphoenzyme ([EP] = 4--5 mol/10(6) g protein) for at least two days in the presence of 5 mM Ca2+, 0.5 M KCl, and 20% glycerol at pH 7.55. 2. The ATPase activity was dependent on both Mg2+ and Ca2+. However, the rate of E32P decay after the addition of unlabeled ATP was independent of Mg2+. 3. Most of the EP formed in the absence of Mg2+ was capable of reacting with ADP to form ATP in the backward reaction. However, in the presence of 5 mM Mg2+, the amount of ATP formed was markedly reduced without loss of the reactivity of the EP with ADP. 4. The removal of C12E8 from the ATPase by the use of Bio-Beads resulted in the full restoration of the Mg2+ dependency of the EP decomposition. 5. These results strongly suggest that in the case of SR solubilized with a high concentration of C12E8 the decomposition of phosphoenzyme is Mg2+ independent and ATP is mainly hydrolyzed through Mg2+-dependent decomposition of an enzyme-ATP complex, which is in equilibrium with phosphoenzyme and ADP.     

Bound adenosine 5'-triphosphate formation, bound adenosine 5'-diphosphate and inorganic phosphate retention, and inorganic phosphate oxygen exchange by chloroplast adenosinetriphosphatase in the presence of Ca2+ or Mg2+

When the heat-activated chloroplast F1 ATPase hydrolyzes [3H, gamma-32P]ATP, followed by the removal of medium ATP, ADP, and Pi, the enzyme has labeled ATP, ADP, and Pi bound to it in about equal amounts. The total of the bound [3H]ADP and [3H]ATP approaches 1 mol/mol of enzyme. Over a 30-min period, most of the bound [32P]Pi falls off, and the bound [3H]ATP is converted to bound [3H]ADP. Enzyme with such remaining tightly bound ADP will form bound ATP from relatively high concentrations of medium Pi with either Mg2+ or Ca2+ present. The tightly bound ADP is thus at a site that retains a catalytic capacity for slow single-site ATP hydrolysis (or synthesis) and is likely the site that participates in cooperative rapid net ATP hydrolysis. During hydrolysis of 50 microM [3H]ATP in the presence of either Mg2+ or Ca2+, the enzyme has a steady-state level of about one bound [3H]ADP per mole of enzyme. Because bound [3H]ATP is also present, the [3H]ADP is regarded as being present on two cooperating catalytic sites. The formation and levels of bound ATP, ADP, and Pi show that reversal of bound ATP hydrolysis can occur with either Ca2+ or Mg2+ present. They do not reveal why no phosphate oxygen exchange accompanies cleavage of low ATP concentrations with Ca2+ in contrast to Mg2+ with the heat-activated enzyme. Phosphate oxygen exchange does occur with either Mg2+ or Ca2+ present when low ATP concentrations are hydrolyzed with the octyl glucoside activated ATPase. Ligand binding properties of Ca2+ at the catalytic site rather than lack of reversible cleavage of bound ATP may underlie lack of oxygen exchange under some conditions.     

Kinetic studies on the ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase. Evidence for the K.S.T. mechanism with two enzyme-ATP complexes and two phosphorylated intermediates of high-energy type.

The kinetic properties of the [3H]ADP-ATP exchange reaction catalyzed by Na+, K+-dependent ATPase [EC 3.6.1,3] were investigated, using NaI-treated microsomes from bovine brain, and the following results were obtained. 1. The rates of the Na+-dependent exchange reaction in the steady state were measured in a solution containing 45 micronM free Mg2+, 100 mMNaCl, 80 micronM ATP, and 160 micronM ADP at pH 6.5 and 4-5 degrees. The rate and amount of decrease in phosphorylated intermediate on adding ADP, i.e., the amount of ADP-sensitive EP, were measured while varying one of the reaction parameters and fixing the others mentioned above. Plots of the exchange rate and the amount of ADP-sensitive EP against the logarithm of free Mg2+ concentration gave bell-shaped curves with maximum values at 50-60 micronM free Mg2+. Plots of the exchange rate and the amount of ADP-sensitive EP against pH also gave bell-shaped curves with maximum values at pH 6.9-7. They both increased with increase in the concentration of NaCl to maximum values at 150-200 mM NaCl, and then decreased rapidly with increase in the NaCl concentration above 200 mM. The dependences of the exchange rate and the amount of ADP-sensitive EP on the concentration of ADP followed the Michaelis-Menten equation, and the Michaelis constants Km, for both were 43 micronM. The dependence of the exchange rate on the ATP concentration also followed the Michaelis-Menten equation, and the Km value was 30 micronM. The amount of ADP-sensitive EP increased with increase in the ATP concentration, and reached a maximum value at about 5 micronM ATP. 2. The N+-dependent [3H]ADP-ATP exchange reaction was started by adding [3H]ADP to EP at low Mg2+-concentration. The reaction consisted of a rapid initial phase and a slow steady phase. The amount of [3H]ATP formed during the rapid initial phase, i.e. the size of the ATP burst, was equal to that of ADP-sensitive EP, and was proportional to the rate in the steady state. At high Mg2+ concentration, the rate of Na+-dependent exchange in the steady state was almost zero, and EP did not show any ADP sensitivity. However, rapid formation of [3H]ATP was observed in the pre-steady state, and the size of the ATP burst increased with increase in the KCl concentration. From these findings, we concluded that an enzyme-ATP complex (E2ATP) formed at low Mg2+ concentration is in equilibrium with EP + ADP, that the rate-limiting step for the exchange reaction is the release of ATP from the enzyme-ATP complex, that the ADP-insensitive EP (formula: see text) produced at high Mg2+ concentration is in equilibrium with the enzyme-ATP complex, and that the equilibrium shifts towards the enzyme-ATP complex on adding KCl. Actually, the ratio of the size of the ATP burst to the amount of EP was equal to the reciprocal of the equilibrium constant of step (formula: see text), determined by a method previously reported by us.     

Phosphorylation of the plasma membrane calcium pump at high ATP concentration. On the mechanism of ATP hydrolysis

The plasma membrane calcium ATPase (PMCA) reacts with ATP to form acid-stable phosphorylated intermediates (EP) that can be measured using (gamma-32P)ATP. However, the steady-state level of EP at [ATP] higher than 100 microM has not yet been studied due to methodological problems. Using a microscale method and a purified preparation of PMCA from human red blood cells, we measured the steady-state concentration of EP as a function of [ATP] up to 2 mM at different concentrations of Mg2+, both at 4 and 25 degrees C. We have measured the Ca2+-ATPase activity (v) under the same conditions as those used for phosphorylation experiments. While the curves of ATPase activity vs [ATP] were well described by the Michaelis-Menten equation, the corresponding curves of EP required more complex fitting equations, exhibiting at least a high- and a low-affinity component. Mg2+ increases the apparent affinity for ATP of this latter component, but it shows no significant effect on its high-affinity one or on the Ca2+-ATPase activity. We calculated the turnover of EP (k(pEP)) as the ratio v/EP. At 1 mM Mg2+, k(pEP) increases hyperbolically with [ATP], while at 8 microM Mg2+, it exhibits a behavior that cannot be explained by the currently accepted mechanism for ATP hydrolysis. These results, together with measurements of the rate of dephosphorylation at 4 degrees C, suggest that ATP is acting in additional steps involving the interconversion of phosphorylated intermediates during the hydrolysis of the nucleotide.     

Properties of the conversion of an enzyme-ATP complex to a phosphorylated intermediate in the reaction of Na+-K+-dependent ATPase1.

Previously, we proposed the following reaction machanism for the transport ATPase (EC 3.6.1.3) reaction in the presence of high concentrations of Mg2+ and Na+:(see article). Some kinetic and thermodynamic properties of steps 3 and 4 were investigated, and the following results were obtained. 1. When the reaction was started by adding ATP to the enzyme in the presence of 50 mM Na+ and 0.5 mM K+ or in the presence of 50mM Na+ and 0.5mM Rb+, the amount of E ADP P increased with time and maintained a constant level after reaching a maximum. We could not observe the initial burst of EP formation, which was observed by Post er al. in the presence of 8 mM Na+ and 0.01 mM Rb+. 2. The existence of quasi-equilibrium between E2ATP and E ADP P in the presence of low concentrations of Na+ was suggested by the fact that the values of the reciprocal of the equilibrium constant, K3 of step 3 obtained by the following three methods were almost the same. a) The value of 1+K3 was estimated from the ratio of vo/[EP] to kd, where vo is the rate of ATP hydrolysis in the steady state, [EP] the concentration of EP, and kd the first-order rate constant of EP disappearance after stopping EP formation. b) This value was also calculated from the ratio of the amount of P1 liberated to that of decrease in EP after stopping EP formation. c) The value of K3 was also calculated from the initial rapid decrease in EP on adding K+ and EDTA, assuming that the rapid decrease was due to a shift of the equilibrium toward E2ATP on adding K+. For example, the value of K3 with 10mM NaCL and 0.5mM KCL was 7--11. Although ATP formation due to a shift of the equilibrium toward E2ATP by a K+ jump in the presence of a low concentration of Na+ was observed at 0 degrees, the amount of ATP formed by a K+ jump at 15 degrees was less than the value expected from the shift of the equilibrium. 3. The values of delta H degrees and delta S degrees of step 3 were estimated in the presence of a sufficient amount of Na+ and in the absence of K+. They were +4--+5 kcal mole minus 1 and +15--+16 entropy units mole minus1, respectively. On the basis of kinetic studies of the elementary steps and the overall reaction of Na+-K+-dependent ATPase [EC 3.6.1.3], we (1--4) showed that a phosphorylated intermediate, EP, is formed via two kinds of enzyme-substrate complex, E1ATP and E2ATP, that the EP is in K+-dependent quasi-equilibrium with E2ATP, and that in the presence of high concentration of Mg2+, EP is in a high-energy state and contains bound ADP, E ADP P.(see article).     

Changes in affinity for calcium ions with the formation of two kinds of phosphoenzyme in the Ca2+,Mg2+-dependent ATPase of sarcoplasmic reticulum

The amount of Ca2+ bound to the Ca2+,Mg2+-dependent ATPase of deoxycholic acid-treated sarcoplasmic reticulum was measured during ATP hydrolysis by the double-membrane filtration method [Yamaguchi, M. & Tonomura, Y. (1979), J. Biochem. 86, 509--523]. The maximal amount of phosphorylated intermediate (EP) was adopted as the amount of active site of the ATPase. In the absence of ATP, 2 mol of Ca2+ bound cooperatively to 1 mol of active site with high affinity and were removed rapidly by addition of EGTA. AMPPNP did not affect the Ca2+ binding to the ATPase in the presence of MgCl2. Under the conditions where most EP and ADP sensitive at steady state (58 microM Ca2+, 50 microM EGTA, and 20 mM MgCl2 at pH 7.0 and 0 degrees C), bound Ca2+ increased by 0.6--0.7 mol per mol active site upon addition of ATP. The time course of decrease in the amount of bound 45Ca2+ on addition of unlabeled Ca2+ + EGTA was biphasic, and 70% of bound 45Ca2+ was slowly displaced with a rate constant similar to that of EP decomposition. Similar results were obtained for the enzyme treated with N-ethylmaleimide, which inhibits the step of conversion of ADP-sensitive EP to the ADP-insensitive one. Under the conditions where most EP was ADP insensitive at steady state (58 microM Ca2+, 30 microM EGTA, and 20 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ increased slightly, then decreased slowly by 1 mol per mol of EP formed after addition of ATP. Under the conditions where about a half of EP was ADP sensitive (58 microM Ca2+, 25 microM EGTA, and 1 mM MgCl2 at pH 8.8 and 0 degrees C), the amount of bound Ca2+ did not change upon addition of ATP. These findings suggest that the Ca2+ bound to the enzyme becomes unremovable by EGTA upon formation of ADP-sensitive EP and is released upon its conversion to ADP-insensitive EP.     

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)     

A fluorescence probe study of the phosphorylation reaction of the calcium ATPase of sarcoplasmic reticulum

The fluorescent thiol reagent N-(1-anilinonaphthyl-4)maleimide (ANM) reacts covalently with the Ca2+ ATPase moiety of fragmented sarcoplasmic reticulum in two phases as determined by the increase of fluorescence intensity and optical density at 350 nm. In the rapid phase, 5.5 nmol of ANM reacts with 1 mg of fragmented sarcoplasmic reticulum protein. Assuming that 55% of the total membrane protein is the Ca2+ ATPase, this is equivalent to 1 mol of SH/10(5) g of ATPase, designated as SH1-ANM. ANM reacts with the second SH (SH2-ANM) at a much slower rate. Reaction of ANM with both SH1-ANM and SH2-ANM produces no inhibition of phosphoenzyme (EP) formation. Upon addition of Mg . ATP in the micromolar range, at [Ca2+] = 1 microM there is an increase in the fluorescence intensity of ANM attached to SH2-ANM, while the ANM attached to SH1-ANM does not respond to Mg . ATP. Under conditions in which there is no EP formation, there is no fluorescence change. Furthermore, the enhancement of ANM fluorescence produced by Mg . ATP is reversed by ADP as it reacts with EP to form ATP. Thus, it appears that the Mg . ATP-induced fluorescence increase reflects changes of enzyme conformation produced by EP formation.     

Characterization of the phosphoenzyme that is involved in the Ca2+ -Ca2+ exchange catalyzed by the Ca2+ -ATPase of sarcoplasmic reticulum vesicles

The rate of Ca2+ efflux was determined with 45Ca2+ -loaded sarcoplasmic reticulum vesicles (mainly with the light fraction of vesicles) at pH 6.5 and 0 degrees C. The efflux depended on external Ca2+, Mg2+, ATP and ADP, but it was not activated by AMP. The results indicate that the efflux is derived from Ca2+ -Ca2+ exchange mediated by the phosphoenzyme (EP) of membrane-bound Ca2+ -ATPase. EP was formed with Ca2+ -loaded vesicles (light fraction) under similar conditions without added ADP. The subsequent addition of EGTA and ADP induced triphasic EP dephosphorylation. Three species of EP (EP1, EP2, and EP3) were distinguished on the basis of this dephosphorylation kinetics, EP1, EP2, and EP3, corresponding to the first, second, and third phases of the dephosphorylation. Dephosphorylation of EP1 and EP2 resulted in stoichiometric ATP formation, while dephosphorylation of EP3 led to stoichiometric Pi liberation. The rate of Ca2+ efflux was compatible with that of EP2 dephosphorylation, whereas it was much lower than the rate of EP1 dephosphorylation and much higher than the rate of EP3 dephosphorylation. The intravesicular Ca2+ concentration dependence of the rate of EP2 dephosphorylation agreed with that of the rate of Ca2+ efflux. The results suggest that isomerization between EP1 and EP2 is the rate-limiting process in the Ca2+ -Ca2+ exchange and that EP3 is not involved in this exchange.     

Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+

To determine the biochemical events of Na+ transport, we studied the interactions of Na+, Tris+, and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain. The enzyme was phosphorylated by incubation at 0 degrees C with 1 mM Mg2+, 25 microM [32P]ATP, and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+, and (4) 2.5 mM ADP plus Na+ up to 600 mM. In dephosphorylation types 2-4, the curves were bi- or multiphasic. "ADP-sensitive EP" and "K+-sensitive EP" were determined by extrapolation of the slow phase of the curves to the ordinate and their sum was always larger than Etotal. These results required a minimal model consisting of three consecutive EP pools, A, B, and C, where A was ADP sensitive and both B and C were K+ sensitive. At high [Na+], B was converted rapidly to A (type 4 experiment). The seven rate coefficients were dependent on [Na+], [Tris+], and [K+], and to explain this we developed a comprehensive model for cation interaction with EP. The model has the following features: A, B, and C are equilibrium mixtures of EP forms; EP in A has two to three Na ions bound at high-affinity (internal) sites, pool B has three, and pool C has two to three low-affinity (external) sites. The putative high-affinity outside Na+ site may be on E2P in pool C. The A leads to B conversion is blocked by K+ (and Tris+). We conclude that pool A can be an intermediate only in the Na-ATPase reaction and not in the normal operation of the Na,K pump.     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. I. Phosphoenzyme with bound Ca2+ which is exposed to the external medium

Sarcoplasmic reticulum vesicles were phosphorylated with [gamma-32P]ATP in the presence of external Ca2+ without added Mg2+. The phosphoenzyme (EP) formed had tightly bound Ca2+ and was dephosphorylated by ADP. When the external Ca2+ was chelated after phosphorylation, Ca2+ dissociated from the EP and ADP addition no longer induced dephosphorylation. Subsequent addition of CaCl2 caused rapid recombination of Ca2+ and restoration of the ADP sensitivity. These findings show that the dissociation and recombination of Ca2+ took place on the outer surface of the membranes, indicating the existence of EP with bound Ca2+ which was exposed to the external medium (Caout.EP). The Ca2+ affinity of the Ca2+ binding site in Caout.EP was comparable to that of the high affinity Ca2+ binding site in the dephosphoenzyme (E). This shows that phosphorylation is not accompanied by an appreciable reduction in the Ca2+ affinity of the Ca2+ binding site, provided this site is exposed to the external medium. The transition from ADP-sensitive EP to ADP-insensitive induced by Ca2+ chelation was unaffected by Mg2+ in the medium. Mg2+ did not activate hydrolysis of the ADP-sensitive EP with bound Ca2+, whereas it markedly accelerated hydrolysis of the ADP-insensitive EP without bound Ca2+.     

Mechanism of the calcium pump in the endoplasmic reticulum of liver: phosphoproteins as reaction intermediates

Microsomal fractions, highly enriched with endoplasmic reticulum of rat and human liver exhibit Ca2+ uptake catalyzed by a Ca2+-pumping ATPase. The mechanism of Ca2+-translocation involves: (i) reversible Ca2+-dependent formation of an acyl-phosphoenzyme intermediate (Mr 116,000 to 118,000) with bound Ca2+, which in the reversed reaction can transphosphorylate its Pi to ADP to re-synthesize ATP; (ii) reversible transition of the ADP-reactive phosphoenzyme into an isomer without bound Ca2+, not further reactive to ADP; (iii) hydrolytic cleavage, stimulated by Mg2+, K+, and ATP of the ADP-unreactive phosphoenzyme with liberation of Pi. By analogy to a mechanism proposed for the Ca2+ pump of sarcoplasmic reticulum, the translocation of Ca2+ to and dissociation from the inner side of the membrane is suggested to occur by a conformational change, coupled with a decrease in Ca2+-affinity of the phosphoenzyme during its transition into the ADP-unreactive isomer. With CaATP as the effective substrate the reactions proceed normally but at a considerably slower rate.     

Reaction mechanism of (Ca2+, Mg2+)-ATPase of sarcoplasmic reticulum vesicles. II. (ATP, ADP)-dependent Ca2+-Ca2+ exchange across the membranes

Sarcoplasmic reticulum vesicles were preloaded with unlabeled CaCl2, and 45Ca2+ incorporation into the vesicles was determined by adding 45CaCl2 to the external medium in the presence of ATP and ADP. In the absence of added MgCl2, the steady state rate of the (ATP, ADP)-dependent 45Ca2+ incorporation was extremely low, being in good agreement with that of the Ca2+-dependent ATP hydrolysis which was catalyzed by the membrane-bound (Ca2+, Mg2+)-ATPase. In contrast, it was greatly increased by addition of MgCl2 and became much higher than the steady state rate of the Ca2+-dependent ATP hydrolysis. The kinetic analysis of the results gave support to the probability that the MgCl2 addition markedly shifted the equilibrium of the reaction of Caout . EP and Cain . EP represent phosphoenzymes with bound Ca2+ which is exposed to the external medium and to the internal medium, respectively).     

The effect of di- and trivalent cations on the phosphorylation of the Ca2+-ATPase in sarcoplasmic reticulum vesicles

The steady-state level of phosphorylated intermediate (EP) of (Mg2+ + Ca2+)-ATPase is influenced by magnesium and calcium concentration in the Ca2+-transporting system of sarcoplasmic reticulum vesicles. At micromolar [Ca2+], the level of EP is increased by Mg2+, depending on its concentration. The effect of Mg2+ is less pronounced at lower Ca2+ concentration. At low [Mg2+], the EP formation increases at millimolar concentrations of Ca2+, suggesting, in accordance with earlier results, that the substrate may also be CaATP instead of MgATP. LaCl3 (1 mM) enhanced the EP formation at low Mg2+ concentration. Surprisingly, 10 microM LaCl3 caused a marked decrease in EP formation at high [Mg2+] and had little or no effect on the level of EP at low Mg2+ concentration. The inducing effect of 1 mM LaCl3 on the EP formation at low [Mg2+] and the inhibitory effect of 10 microM LaCl3 at high Mg2+ concentration draw attention to the involvement of divalent cation-binding sites with different affinity in phosphorylation and to the particular role of Mg2+ in the EP formation and EP decomposition.     

Ca2+,Mg2+-ATPase of microsomal membranes from bovine aortic smooth muscle. Identification and characterization of an acid-stable phosphorylated intermediate of the Ca2+,Mg2+-ATPase

An acid-stable phosphoprotein was formed in a microsomal membrane fraction isolated from bovine aortic smooth muscle in the presence of Mg2+ + ATP and Ca2+. The microsomes also showed Ca2+ uptake activity. The Ca2+ dependence of phosphoprotein formation and of Ca2+ uptake occurred over the same range of Ca2+ concentration (1-10 microM), and resembled similar findings from rabbit skeletal microsomes. The molecular weight of the phosphorylated protein, estimated by SDS-gel electrophoresis, was approximately 105,000. The phosphoprotein was labile at alkaline pH, and its decomposition was accelerated by hydroxylamine. Half-maximum incorporation of 32P in the presence of 10 microM Ca2+ occurred at 60 nM ATP. The calcium-dependent phosphoprotein formation was not affected by 5 mM NaN3, but was inhibited in a dose-dependent fashion by ADP with a 50% inhibition occurring at 180 microM. Fifty mM MgCl2 was required for the maximal phosphorylation. The rate of phosphoprotein decomposition after adding 2 mM EGTA was accelerated by varying the Mg2+ concentration from 10 microM to 3 mM. Alkaline pH (9.0) slowed the rate of phosphoprotein decay. Optimal Ca2+-dependent phosphoprotein occurred at 15 degrees C over a broad pH range (6.4 to 9.0). The activation energy of EGTA-induced phosphoprotein decomposition was 25.6 kcal/mol between 0 and 16 degrees C and 14.6 kcal/mol between 16 and 30 degrees C. The phosphoprotein formed by aortic microsomes was thus quite similar to the acid-stable phosphorylated intermediate of the Ca2+-transport ATPase of sarcoplasmic reticulum from skeletal and cardiac muscle. These data suggest that the Ca2+-dependent phosphoprotein is a reaction intermediate of the Ca2+,Mg2+-ATPase of the aortic microsomes.     

Studies on conformational transitions of Ca2+,Mg2+-adenosine triphosphatase of sarcoplasmic reticulum. II. Conformational characteristics of stabilized reaction intermediates as revealed by fluorescent and paramagnetic probes

Various reaction intermediates of sarcoplasmic reticulum Ca2+,Mg2+-ATPase were stabilized and accumulated by modifying a specific SH group or by using nucleotide analogs. Conformational changes of the Ca2+,Mg2+-ATPase during the catalytic cycle were studied in the stabilized intermediates by the use of fluorescent and spin probes, which were introduced at specific SH groups of ATPase, namely one highly reactive but functionally nonessential (SHN) and one essential for the decomposition of the E-P intermediate (SHD) [Kawakita, M., et al. (1980) J. Biochem. 87, 609-617]. The fluorescence intensity of N-(7-dimethylamino-4-methyl-3-coumarinyl)maleimide attached to SHD decreased by 2.5% upon addition of 10 microM AMP-P(NH)P provided that Ca2+ was also present. The AMP-P(NH)P-induced fluorescence change could also be detected by using other fluorescent probes such as N-[p-(2-benzimidazolyl)phenyl]maleimide and N-(1-anilinonaphthyl-4)maleimide. Moreover, labeling at SHN gave similar results. When SHN was labeled with N-[p-(2-benzimidazolyl)phenyl]maleimide, the fluorescence intensity also decreased by 2.5% upon addition of ATP only in the presence of Ca2+, where E-P formation took place. A conformational difference between ECa1-P X ADP and ECa1-P was suggested from saturation transfer ESR measurement of spin-labeled ATPase by using ADP beta S as an ADP analog to cause accumulation of ECa1-P X ADP beta S complex. Possible structural similarities among some of the intermediates are discussed based on these findings.     

Nucleotide-dependent and dicyclohexylcarbodiimide-sensitive conformational changes in the epsilon subunit of Escherichia coli ATP synthase.

The rate of trypsin cleavage of the epsilon subunit of Escherichia coli F1F0 (ECF1F0) is shown to be ligand-dependent as measured by Western analysis using monoclonal antibodies. The cleavage of the epsilon subunit was rapid in the presence of ADP alone, ATP + EDTA, or AMP-PNP + Mg2+, but slow when Pi was added along with ADP + Mg2+ or when ATP + Mg2+ was added to generate ADP + Pi (+Mg2+) in the catalytic site. Trypsin treatment of ECF1Fo was also shown to increase enzymic activity on a time scale corresponding to that of the cleavage of the epsilon subunit, indicating that the epsilon subunit inhibits ATPase activity in ECF1Fo. The ligand-dependent conformational changes in the epsilon subunit were also examined in cross-linking experiments using the water-soluble carbodiimide 1-ethyl-3-[3-(dimethylamino)propyl]-carbodiimide (EDC). In the presence of ATP + Mg2+ or ADP + Pi + Mg2+, the epsilon subunit cross-linked product was much reduced. Prior reaction of ECF1Fo with dicyclohexylcarbodiimide (DCCD), under conditions in which only the Fo part was modified, blocked the conformational changes induced by ligand binding. When the enzyme complex was reacted with DCCD in ATP + EDTA, the cleavage of the epsilon subunit was rapid and yield of cross-linking of beta to epsilon subunit low, whether trypsin cleavage was conducted in ATP + EDTA or ATP + Mg2+. When enzyme was reacted with DCCD in ATP + Mg2+, cleavage of the epsilon subunit was slow and yield of cross-linking of beta to epsilon high, under all nucleotide conditions for proteolysis.(ABSTRACT TRUNCATED AT 250 WORDS)     

The role of Mg2+ and K+ in the phosphorylation of Na+,K(+)-ATPase by ATP in the presence of dimethylsulfoxide but in the absence of Na+.

We have previously demonstrated that Na+,K(+)-ATPase can be phosphorylated by 100 microM ATP and 5 mM Mg2+ and in the absence of Na+, provided that 40% dimethylsulfoxide (Me2SO) is present. Phosphorylation was stimulated by K+ up to a steady-state level of about 50% of Etot (Barrabin et al. (1990) Biochim. Biophys. Acta 1023, 266-273). Here we describe the time-course of phosphointermediate (EP) formation and of dephosphorylation of EP at concentrations of Mg2+ from 0.1 to 5000 microM and of K+ from 0.01 to 100 mM. The results were simulated by a simplified version of the commonly accepted Albers-Post model, i.e. a 3-step reaction scheme with a phosphorylation, a dephosphorylation and an isomerization/deocclusion step. Furthermore it was necessary to include an a priori, Mg(2+)- and K(+)-independent, equilibration between two enzyme forms, only one of which (constituting 14% of Etot) reacted directly with ATP. The role of Mg(2+) was two-fold: At low Mg2+, phosphorylation was stimulated by Mg2+ due to formation of the substrate MgATP, whereas at higher concentrations it acted as an inhibitor at all three steps. The affinity for the inhibitory Mg(2+)-binding was increased several-fold, relative to that in aqueous media, by dimethylsulfoxide. K+ stimulated dephosphorylation at all Mg(2+)-concentrations, but at high, inhibitory [Mg2+], K+ also stimulated the phosphorylation reaction, increasing both the rate coefficient and the steady-state level of EP. Generally, the presence of Me2SO seems to inhibit the dephosphorylation step, the isomerization/deocclusion step, and to a lesser extent (if at all) the phosphorylation reaction, and we discuss whether this reflects that Me2SO stabilizes occluded conformations of the enzyme even in the absence of monovalent cations. The results confirm and elucidate the stimulating effect of K+ on EP formation from ATP in the absence of Na+, but they leave open the question of the molecular mechanism by which Me2SO, inhibitory Mg2+ and stimulating K+ interact with the Na+,K(+)-ATPase.