Calcium and magnesium regulation of phosphorylation by ATP and ITP in sarcoplasmic reticulum vesicles.

Membrane phosphorylation and nucleoside triphosphatase activity of sarcoplasmic reticulum vesicles isolated from rabbit skeletal muscle were studied using ATP and ITP as substrates. The Ca2+ concentration was varied over a range large enough to saturate either the high affinity Ca2+-binding site or both high and low affinity binding sites. In intact vesicles, which are able to accumulate Ca2+, the steady state level of enzyme phosphorylated by either ATP or ITP is already high in 0.02 mM Ca2+ and does not vary as the Ca2+ concentration is increased to 10 mM. Essentially the same pattern of membrane phosphorylation by ATP is observed when leaky vesicles, which are unable to accumulate Ca2+, are used. However, for leaky vesicles, when ITP is used as substrate, the phosphoenzyme level increases 3- to 4-fold when the Ca2+ concentration is raised from 0.02 to 20 mM. When Mg2+ is omitted from the assay medum, the degree of membrane phosphorylation by ATP varies with Ca2+ in the same way as when ITP is used in the presence of Mg2+. Membrane phosphorylation of leaky vesicles by either ATP or ITP is observed in the absence of added Mg2+. When these vesicles are incubated in media containing ITP and 0.1 mM Ca2+, addition of Mg2+ up to 10 mM simultaneously decreases the steady state level of phosphoenzyme and increases the rate of ITP hydrolysis. When ATP is used, the addition of 10 mM Mg2+ increases both the steady state level of phosphoenzyme and the rate of ATP hydrolysis. When the Ca2+ concentration is raised to 10 or 20 mM, the degree of membrane phosphorylation by either ATP or ITP is maximal even in the absence of added Mg2+ and does not vary with the addition of 10 mM Mg2+. In these conditions the ATPase and ITPase activities are activated by Mg2+, although not to the level observed in 0.1 mM Ca2+. An excess of Mg2+ inhibits both the rate of hydrolysis and membrane phosphorylation by either ATP or ITP.     

(Na+ + K+)-ATPase : phosphorylation-dependent cross-linking of the alpha-subunits in the presence of Ca2+ and o-phenanthroline

In previous studies we had demonstrated that in the presence of 0.25 mM Cu2+ and 1.25 mM o-phenanthroline, cross-linking of the alpha-subunits of Na+ + K+)-dependent adenosine triphosphatase was induced by the addition of Na+ + ATP, and that the formation of the alpha,alpha-dimer was preceded by that of phosphoenzyme. The purpose of the present studies was the further evaluation of the role of phosphoenzyme in the process of cross-linking. Na+ + UTP did not induce cross-linking unless Mg2+ was also added. In contrast, Na+ + ATP-induced cross-linking did not require the addition of Mg2+. The different effects of ATP and UTP in the absence of added Mg2+ could be accounted for by the presence in the enzyme preparation of bound Mg2+ which supported enzyme phosphorylation by ATP but not by UTP. When the enzyme was phosphorylated by Pi, in the presence of Mg2 and ouabain, and the exposed to Cu2+ and o-phenanthroline, the alpha,alpha-dimer was obtained. Under these conditions, Na+ blocked both phosphorylation and cross-linking. These results indicate that it is the formation of phosphoenzyme per se that leads to conformational transitions favorable to cross-linking. They also suggest that Cu2+ and o-phenanthroline participate in the cross-linking reaction, but not in the phosphorylation reactions. In the digitonin-treated enzyme, Na+ and ATP induced the formation of phosphoenzyme, but not that of alpha,alpha-dimer. These findings indicate that in addition to phosphorylation, a proper orientation o alpha-subunits in an oligomer is also necessary for cross-linking.     

Na+ATPase of the mammalian erythrocyte membrane. Reversibility of phosphorylation at 0 degrees.

When human erythrocyte membranes are phosphorylated with a very low concentration of [gamma-32P]ATP (0.02 muM) at 0 degrees, and then EDTA is added, rapid disappearance of the phosphoenzyme intermediate of Na+ATPase is observed. The initial rapid phase of phosphoenzyme disappearance is, for the most part, not associated with P1 release and its rate constant, kD, is severalfold greater than the ratio of Na+ATPase activity to phosphoenzyme intermediate, v:EP, at steady state. It is concluded that this rapid disappearance of phosphoenzyme is due to resynthesis of ATP via reversal of phosphorylation. In contrast, rapid reversal is not observed when excess nonradioactive ATP is added to reduce E32P formation, provided Mg2+ is present; however, K+ added with the ATP stimulates reversal. Rapid reversal following EDTA addition is unlikely also when higher ATP concentrations (greater than or equal to 10(-6) M) are used to phosphorylate the enzyme since, at higher ATP, kD congruent to v:EP. The results are compatible with the concept that the Na+ATPase enzyme is composed of two or more catalytic subunits, in which ATP at one catalytic site can regulate the reactivity at another site.     

Phosphorylation and dephosphorylation kinetics of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa.

Partial reactions of potassium-stimulated ATP phosphohydrolase from hog gastric mucosa were studied by means of a rapid-mixing apparatus. At 21 degrees C, in the presence of 2 mM MgCl2 and 5 microM [gamma-32P]ATP there was a rapid phosphorylation of the enzyme with a pseudofirst order rate constant of 1400 min-1. Addition of the ATP about 120 ms before the MgCl2 increased this rate constant to 4400 min-1. In the absence of MgCl2 there was no phosphorylation. Addition of 4 or 10 mM KCl to the phosphoenzyme which had been formed in the absence of KCl produced a rapid initial rate of dephosphorylation (k = 2600 and 3200 min-1 respectively). An additional slow component of dephosphorylation was observed when unlabeled ATP was added together with the KCl (k = 700 to 900 min-1). At a 4 mM concentration, KCl stimulated the ATPase activity about 9-fold. At higher concentrations, the activity was reduced in parallel with a reduction of the steady state level of phosphoenzyme. Addition of KCl to the enzyme before the addition of ATP plus MgCl2 resulted in a low rate and extent of phosphorylation. KCl appeared to inhibit the phosphorylation at a level preceeding the E.ATP complex.     

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.     

Pathway for ATP synthesis by sarcoplasmic reticulum ATPase

Millisecond mixing and quenching experiments were performed in order to study the rate of phosphorylation by P_i of the Ca²⁺-dependent ATPase of sarcoplasmic reticulum vesicles. A rapid phosphoenzyme formation was observed when the vesicles were preincubated in the absence of Ca²⁺ prior to the addition of P_i and Mg²⁺ to the medium, the half-time being in the range of 6 to 10 ms. A lag phase and a 5- to 10-fold slower rate of phosphoenzyme formation were observed when the enzyme was preincubated with Ca²⁺ prior to the addition to the reaction mixture of P_i, Mg²⁺, and an excess of ethylene glycol bis(β-aminoethyl ether)N,N′-tetraacetic acid. The rate of phosphoenzyme hydrolysis was measured either by the addition of Ca²⁺ or, in the absence of Ca²⁺, by tracing the hydrolysis of radioactive phosphoenzyme upon the addition of nonradioactive P_i. In the presence of Ca²⁺, the rate of phosphoenzyme hydrolysis was found to be one order of magnitude slower than the rate of hydrolysis measured in the absence of Ca²⁺. Different rates of phosphoenzyme formation and cleavage were found depending on whether sarcoplasmic reticulum vesicles or purified Ca²⁺-dependent ATPase were used. A transient phosphorylation by P_i was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, Mg²⁺, and excess of Ca²⁺. The enzyme was phosphorylated during the initial 100 ms, the phosphoenzyme formed being slowly hydrolyzed in the subsequent incubation intervals. In these conditions ATP synthesis was observed if ADP was added to the mixture 100 ms after starting the reaction. No transient phosphorylation by P_i was observed when the enzyme was preincubated with Ca²⁺. Synthesis of a small but significant amount of ATP was observed when the enzyme was preincubated in the absence of Ca²⁺ and then added to a medium containing P_i, ADP, Mg²⁺, and 20 mm CaCl₂. This was not observed when the enzyme was preincubated in the presence of Ca²⁺.     

Reaction mechanism of Ca2+-dependent adenosine triphosphatase of sarcoplasmic reticulum. ATP hydrolysis with CaATP as a substrate and role of divalent cation

ATP hydrolysis with CaATP as a substrate was characterized at 0 degrees C and pH 7.0 using purified ATPase preparations of sarcoplasmic reticulum and compared with that with MgATP as a substrate. The maximal rate of enzyme phosphorylation and the Km value for the phosphorylation were 8 to 10 times less for CaATP than for MgATP. Each substrate appeared to act as a competitive inhibitor with respect to the other in enzyme phosphorylation. The phosphoenzyme formed from CaATP turned over slowly because the conversion rate of the ADP-sensitive (E1P) to ADP-insensitive (E2P) phosphoenzyme was very slow. E2Ps, formed from both CaATP and MgATP, were similar in that KCl, MgCl2, or ATP accelerated their decomposition. Their sensitivity to KCl and/or ATP was retained even after a long incubation with excess EDTA. When the enzyme had been phosphorylated from CaATP, calcium remained bound to the enzyme even in the presence of excess EDTA. The observed parallelism between the amount and behavior of the enzyme-bound calcium and those of E2P strongly suggests that 1 mol of E2P has 1 mol of tightly bound calcium. During steady state ATP hydrolysis with CaATP as a substrate, a significant amount of the enzyme-ATP complex accumulated as a reaction intermediate because of slow dissociation of CaATP from the CaATP-enzyme complex and slow enzyme phosphorylation from the CaATP-enzyme complex. These results indicate that Mg2+ is not essential for the turnover of the calcium pump ATPase. It was proposed that the metal component of the substrate basically determines affinity of the substrate to the enzyme and the catalytic mechanism of subsequent reaction steps.     

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+.     

Plasma membrane ATPase of red beet forms a phosphorylated intermediate

When a plasma membrane-enriched fraction isolated from red beet (Beta vulgaris L.) was incubated in the presence of 40 micromolar [γ-³²P] ATP, 40 micromolar MgSO₄ at pH 6.5, a rapidly turning over phosphorylated protein was formed. Phosphorylation of the protein was substrate-specific for ATP, sensitive to diethylstilbestrol and vanadate, but insensitive to azide. When the dephosphorylation reaction was specifically studied, KCl was found to increase the turnover of the phosphorylated protein consistent with its stimulatory effect upon plasma membrane ATPase. The protein-bound phosphate was found to be most stable at a pH between 2 and 3 and under cold temperature, suggesting that the protein phosphate bond was an acyl-phosphate. When the phosphorylated protein was analyzed with lithium dodecyl sulfate gel electrophoresis, a labeled polypeptide with a molecular weight of about 100,000 daltons was observed. Phosphorylation of this polypeptide was rapidly turning over and Mg-dependent. It is concluded that the phosphorylation observed represents a reaction intermediate of the red beet plasma membrane ATPase.     

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.     

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.     

Studies on the heat-precipitated phosphoenzyme complex of eel electric organ (Na + K).ATPase.

When the hydrolytic reaction between eel electric organ (Na + K) · ATPase and [γ-³²P]ATP is terminated at neutral pH by heat precipitation, a phosphoenzyme complex is formed which reaches maximal levels in the simultaneous presence of Mg, Na, and K. After formation of a steady-state level of phosphoenzyme in the presence of Mg and Na, a pulse of K increases the level of the heat-precipitated phosphoenzyme (while decreasing the level of the acid-precipitated phosphoenzyme). The formation of the heat-precipitated phosphoenzyme is clearly inhibited by ouabain only when the phosphoenzyme is formed in the presence of Mg, Na, and K. Inorganic phosphate decreases the level of the heat-precipitated phosphoenzyme, but not that of the acid-precipitated phosphoenzyme (in the presence of Mg and Na or in the presence of Mg, Na, and K). Moreover, a heat-precipitated, ouabain-sensitive phosphoenzyme forms in the reaction between the eel (Na + K) · ATPase and ³²P_i with or without ATP. The pH stability of the heat-precipitated phosphoenzyme complex is maximal at pH 6 to 8, and this complex shows little or no reactivity with neutral hydroxylamine, suggesting that the phosphate is not bound to an acyl residue of the protein. These experiments indicate that both heat-resistant and acid-resistant phosphoenzymes are formed during the (Na + K) · ATPase reaction at pH 7.4.     

Bovine brain Na+,K+-stimulated ATP phosphohydrolase studied by a rapid-mixing technique. K+-stimulated liberation of [32P] orthophosphate from [32P] phosphoenzyme and resolution of the dephosphorylation into two phases.

Dephosphorylation of [32P]phosphoenzyme of bovine brain Na+,K+-stimulated ATP phosphohydrolase (EC 3.6.1.3), labelled by [gamma-32P]ATP, was investigated at 21 degrees C by means of a rapid-mixing technique. On addition of a high concentration of KCl (10 mM) to [32P]phosphoenzyme at steady state in the presence of Mg2+ and Na+, very rapid dephosphorylation was obtained. Simultaneously, the amount of [32P]orthophosphate increased at about the same rate. It was concluded that this K+-stimulated dephosphorylation and liberation of [32P]orthophosphate from the [32P]phosphoenzyme was rapid enough to participate in the Na+,K+-stimulated hydrolysis of ATP. In order to study the dephosphorylation in absence of continuing 32P-labelling, excess unlabelled ATP or a chelator of Mg2+ was added. Simultaneous addition of a high concentration of KCl to the [32P]phosphoenzyme formed in the presence of Mg2+ and Na+ but in the absence of K+, resulted in an initial very rapid phase and a subsequent slower phase of dephosphorylation. With KCl also initially present in the incubation medium, only the slow phase was observed. The slow phase of dephosphorylation also seemed to be sufficiently rapid to participate in the Na+, K+-stimulated ATPase reaction. On addition of a high concentration of ADP (5 mM) to [32P]phosphoenzyme formed in the presence of Mg2+ and Na+, an initial comparatively rapid, and later slow phase of dephosphorylation were detected. This gave further support for different forms of phosphoenzyme. Approximate concentrations of these forms, in the absence and presence of KCl, were estimated by extrapolation and the turnover of these forms was calculated. The nature of the kinetically different components of phosphoenzyme and their role in the Na+, K+-stimulated ATPase reaction is discussed.     

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.     

Intermediate reaction states of the red beet plasma membrane ATPase

The phosphorylation reaction for the plasma membrane ATPase of red beet (Beta vulgaris L.) was examined in order to further understand the mechanism of this enzyme. The level of steady-state phosphorylation had a pH optimum of about 6.0 while ATPase activity (32Pi production) measured under identical conditions had a pH optimum of 7.0. Phosphoenzyme decomposition was accelerated as both the pH and temperature were increased. The former effect may account for the observed difference between the pH optimum for phosphorylation and ATPase. Although the kinetics of K+ stimulation of ATP hydrolysis have been observed to be complex, the kinetics of K+ stimulation of phosphoenzyme turnover were observed to be simple Michaelis-Menten. An antagonism was observed between MgATP and K+ for the stimulation of phosphoenzyme turnover. Increased MgATP concentration reduced the degree of K+ stimulation of phosphoenzyme turnover and ATPase activity. These effects could be explained by the observation that two forms of phosphoenzyme occur during ATP hydrolysis. One form is discharged by ADP while the other form is ADP insensitive. Potassium stimulation of phosphoenzyme breakdown occurs primarily because of effects on the ADP-insensitive phosphoenzyme form. These results are consistent with a mechanism of ATP hydrolysis involving interconversions of conformational states.     

The interaction of magnesium ions with the calcium pump of sarcoplasmic reticulum.

1. In the presence of Ca2+, ATP phosphorylates the Ca2+ pump of sarcoplasmic reticulum at the same site and to the same extent regardless of whether Mg2+ is added or not to the incubation media, the main effect of added Mg2+ being to increase the rate of phosphorylation. 2. When phosphoenzyme is made in Mg2+-containing media it dephosphorylates about 30-times faster than when it is made in the absence of added Mg2+. Addition of Mg2+ after phosphorylation is uneffective in accelerating the hydrolysis of phosphoenzyme even in solubilized enzyme, suggesting that phosphorylation of the Ca2+ pump results in occlusion of the site at which Mg2+ combines to accelerate the release of phosphate. 3. Occlusion of the site for Mg2+ can be partially reversed by trans-1,2-diaminocyclohexonetetraacetic acid (CDTA). Use was made of this property to demonstrate that for the rapid release of phosphate to occur Mg2+ has to be bound to the enzyme. 4. Results seem to indicate that Mg2+ combines with the Ca2+ pump prior to phosphorylation.     

Existence of ADP- and KCl-insensitive phosphoenzyme intermediate of Na+,K(+)-ATPase at alkaline Ph

The Na(+)-ATPase activity of Na+,K(+)-ATPase in the absence of K+ was least dependent on the sodium concentration when the pH was 9.5. Around 40% of the phosphoenzyme formed from ATP in the presence of 0.5 mM MgCl2 at alkaline pH was insensitive to both KCl and ADP. High-Na+ chase reversed this insensitivity, i.e., the phosphoenzyme became sensitive to KCl or ADP. On the other hand, phosphorylation at 0.1 mM MgCl2 instead of 0.5 mM showed at least 95% sensitivity to KCl. These observations suggest that ADP- and KCl-insensitive phosphoenzyme was formed when excess Mg++ was present during phosphorylation at alkaline pH. This phosphoenzyme might be an intermediate in the process of ATP hydrolysis.     

Slow transition of phosphoenzyme from ADP-sensitive to ADP-insensitive forms in solubilized Ca2+, Mg2+-ATPase of sarcoplasmic reticulum: evidence for retarded dissociation of Ca2+ from the phosphoenzyme.

Solubilized Ca²⁺, Mg²⁺-ATPase of sarcoplasmic reticulum was phosphorylated with ATP without added MgCl₂. The phosphoenzyme formed was ADP-sensitive. Ca²⁺ in the medium was chelated after phosphorylation. This induced a slow transition of the phosphoenzyme from ADP-sensitive to ADP-insensitive forms. The ADP-sensitivity was restored by subsequent addition of CaCl₂. These results showed that the transition was caused by dissociation of Ca²⁺ bound to the phosphoenzyme. Further observations indicated that, when Ca²⁺ in the medium was chelated, Ca²⁺ bound to the phosphoenzyme was dissociated much more slowly than Ca²⁺ bound to the dephosphoenzyme. This suggests a possible formation of the occluded form of the Ca²⁺-binding site in the phosphoenzyme.     

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.     

Effect of phospholipid, detergent and protein-protein interaction on stability and phosphoenzyme isomerization of soluble sarcoplasmic reticulum Ca-ATPase

The purpose of the present study was to elucidate the separate roles of lipid, detergent and protein-protein interaction for stability and catalytic properties of sarcoplasmic reticulum Ca-ATPase solubilized in the non-ionic detergent octa(ethylene glycol) monododecyl ether (C12E8). The use of large-zone high-performance liquid chromatography permitted us to define the self-association state of Ca-ATPase peptide at various detergent, phospholipid and protein concentrations, and also during enzymatic turnover with ATP. Conditions were established for monomerization of Ca-ATPase in the presence of a high concentration of phospholipid relative to detergent. The lipid-saturated monomeric preparation was relatively resistant to inactivation in the absence of Ca2+, whereas delipidated enzyme in monomeric or in oligomeric form was prone to inactivation. Kinetics of phosphoenzyme turnover were examined in the presence and absence of Mg2+. Dephosphorylation rates were sensitive to Mg2+, irrespective of whether the peptide was present in soluble monomeric form or was membrane-bound. C12E8-solubilized monomer without added phospholipid was, however, characterized by a fast initial phase of dephosphorylation in the absence of Mg2+. This was not observed with monomer saturated with phospholipid or with monomer solubilized in myristoylglycerophosphocholine or deoxycholate. The mechanism underlying this difference was shown to be a C12E8-induced acceleration of conversion of ADP-sensitive phosphoenzyme (E1P) to ADP-insensitive phosphoenzyme (E2P). The phosphoenzyme isomerization rate was also found to be enhanced by low-affinity binding of ATP. This was demonstrated both in membrane-bound and in soluble monomeric Ca-ATPase. Our results indicate that a single peptide chain constitutes the target for modulation of phosphoenzyme turnover by Mg2+ and ATP, and that detergent effects, distinct from those arising from disruption of protein-protein contacts, are the major determinants of kinetic differences between C12E8-solubilized and membrane-bound enzyme preparations.