Stimulatory and inhibitory effects of dimethylsulfoxide, propranolol and chlorpromazine on the partial reactions of ATPase of sarcoplasmic reticulum.

The effects of dimethylsulfoxide, propranolol and chlorpromazine on the partial reactions of the ATPase of sarcoplasmic reticulum were investigated. When analyzed according to a reaction scheme in which the ADP-sensitive (E1P) and ADP-insensitive (E2P) phosphoenzymes occur sequentially and P1 is derived from the latter, dimethylsulfoxide decreased the rate of E2P hydrolysis whereas it stimulated the rate of the E1P to E2P conversion. Propranolol increased the rate of E2P hydrolysis while it decreased the rate of the E1P to E2P conversion. Propranolol exerted an additional effect, presumably inhibition of the phosphoenzyme formation. These effects of dimethylsulfoxide and propranolol can account for both the stimulatory and inhibitory effects of these drugs on the overall rate of ATP hydrolysis observed in the presence and absence of added alkali metal salts. Chlorpromazine accelerated E2P hydrolysis whereas it appeared to inhibit the E1P to E2P conversion. These effects of chlorpromazine appear able to account for its stimulatory and inhibitory effects on the overall rate of ATP hydrolysis in the presence and absence of alkali metal salts. In the presence of chlorpromazine, however, the rate of Pi liberation during the steady state ATP hydrolysis was found to be greater than the hydrolysis rate of E2P. This finding suggests that under these conditions Pi is derived not only from E2P but also from source(s) other than E2P.     

Stimulatory and inhibitory effects of dimethylsulfoxide, propranolol and chlorpromazine on the partial reactions of ATPase of sarcoplasmic reticulum

The effects of dimethylsulfoxide, propranolol and chlorpromazine on the partial reactions of the ATPase of sarcoplasmic reticulum were investigated. When analyzed according to a reaction scheme in which the ADP-sensitive (E₁P) and ADP-insensitive (E₂P) phosphoenzymes occur sequentially and P_i is derived from the latter, dimethylsulfoxide decreased the rate of E₂P hydrolysis whereas it stimulated the rate of the E₁P to E₂P conversion. Propranolol increased the rate of E₂P hydrolysis while it decreased the rate of the E₁P to E₂P conversion. Propranolol exerted an additional effect, presumably inhibition of the phosphoenzyme formation. These effects of dimethylsulfoxide and propranolol can account for both the stimulatory and inhibitory effects of these drugs on the overall rate of ATP hydrolysis observed in the presence and absence of added alkali metal salts.

Chlorpromazine accelerated E₂P hydrolysis whereas it appeared to inhibit the E₁P to E₂P conversion. These effects of chlorpromazine appear able to account for its stimulatory and inhibitory effects on the overall rate of ATP hydrolysis in the presence and absence of alkali metal salts. In the presence of chlorpromazine, however, the rate of P_i liberation during the steady state ATP hydrolysis was found to be greater than the hydrolysis rate of E₂P. This finding suggests that under these conditions P_i is derived not only from E₂P but also from source(s) other than E₂P.     

Transient-state kinetics of the ADP-insensitive phosphoenzyme in sarcoplasmic reticulum: implications for transient-state calcium translocation

The kinetics of formation of the ADP-sensitive (EP) and ADP-insensitive (E*P) phosphoenzyme intermediates of the CaATPase in sarcoplasmic reticulum (SR) were investigated by means of the quenched-flow technique. At 21 degrees C, addition of saturating ADP to SR vesicles phosphorylated for 116 ms with 10 microM ATP gave a triphasic pattern of dephosphorylation in which EP and E*P accounted for 33% and 60% of the total phosphoenzyme, respectively. Inorganic phosphate (Pi) release was less than stoichiometric with respect to E*P decay and was not increased by preincubation with Ca2+ ionophore. The fraction of E*P present after only 6 ms of phosphoenzyme formation was similar to that at 116 ms, indicating that isomerization of EP to E*P occurs very rapidly. Comparison of the time course of E*P formation with intravesicular Ca2+ accumulation measured by quenching with ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid + ADP revealed that Ca2+ release on the inside of the vesicle was delayed with respect to E*P formation. Since Ca2+ should dissociate rapidly dissociation from the low-affinity transport sites, these results suggest that Ca2+ remains "occluded" after phosphoenzyme isomerization and that a subsequent slow transition controls the rate of Ca2+ release at the intravesicular membrane surface. Analysis of the forward and reverse rate constants for the EP to E*P transition gave an expected steady-state distribution of phosphoenzymes strongly favoring the ADP-insensitive form. In contrast, the observed ratio of EP to E*P was about 1:2. To account for this discrepancy, a mechanism is proposed in which stabilization of the ADP-sensitive phosphoenzyme is brought about by a conformational interaction between adjacent subunits in a dimer.     

Occlusion of calcium in the ADP-sensitive phosphoenzyme of the adenosine triphosphatase of sarcoplasmic reticulum

In order to characterize the form of the phosphorylated Ca2+-ATPase of sarcoplasmic reticulum which occludes the calcium bound in the enzyme (Takisawa, H., and Makinose, M. (1981) Nature (Lond.) 290, 271-273), a kinetic method was developed allowing quantitation of the amount of ADP-sensitive and ADP-insensitive phosphoenzyme. The relationships between occluded Ca2+ in the enzyme and the two forms of phosphoenzyme were studied at various concentrations of CaCl2 and MgCl2. The amount of tightly bound Ca2+ in the phosphoenzyme increases concordantly with the increase in the amount of ADP-sensitive phosphoenzyme, suggesting that occlusion of Ca2+ occurs in the ADP-sensitive phosphoenzyme. These results suggest that 1 mol of ADP-sensitive phosphoenzyme occludes 2 mol of Ca2+. Ca2+ is released from the enzyme under conditions which favor the formation of the ADP-insensitive phosphoenzyme (e.g. 5 mM MgCl2 and 50 microM CaCl2). Ca2+ release correlates approximately with the formation of the ADP-insensitive phosphoenzyme. The simulated time course of Ca2+ release, based on the Ca2+-binding properties of the two forms of phosphoenzyme, shows a good fit with the Ca2+ release curves observed, indicating that the ADP-insensitive phosphoenzyme binds no Ca2+ under these conditions.     

Coincidence of H+ binding and Ca2+ dissociation in the sarcoplasmic reticulum Ca-ATPase during ATP hydrolysis

H+ and Ca2+ concentration changes in the reaction medium following MgATP addition at pH 6.0 were determined with the partially purified Ca-ATPase from sarcoplasmic reticulum vesicles in the presence of 25-50 microM CaCl2 and 5 mM MgCl2 at 4 degrees C. Previously, we showed a sequential occurrence of H+ binding and H+ dissociation in the Ca-ATPase during ATP hydrolysis and further suggested that the H+ binding takes place inside the vesicles (Yamaguchi, M., and Kanazawa, T. (1984) J. Biol. Chem. 259, 9526-9531). The present results demonstrate that the H+ binding occurred coincidently with Ca2+ dissociation from the enzyme upon conversion of the phosphoenzyme (EP) intermediate from the ADP-sensitive form to the ADP-insensitive form in the catalytic cycle of ATP hydrolysis. As KCl decreased in the medium, the extent of the H+ binding increased almost proportionately with the extent of either the Ca2+ dissociation or the accumulation of ADP-insensitive EP. Both the H+ binding and the Ca2+ dissociation were prevented by a modification of the specific SH group of the enzyme essential for the conversion of ADP-sensitive EP to ADP-insensitive EP. In the late stage of the reaction, H+ dissociation from the enzyme occurred coincidently with Ca2+ binding to the dephosphoenzyme which was formed by EP decomposition. These results are consistent with the possibility that the H+ ejection during the Ca2+ uptake with the intact vesicles previously shown by several investigators takes place through a Ca2+/H+ exchange directly mediated by the membrane-bound Ca-ATPase.     

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.     

Direct evidence for an ADP-sensitive phosphointermediate of (K+ + H+)-ATPase

Direct evidence for the occurrence of an ADP-sensitive phosphoenzyme of (K+ + H+)-ATPase, the proton-pumping system of the gastric parietal cell is presented. The enzyme is phosphorylated with 5 microM [gamma-32P]ATP in 50 mM imidazole-HCl (pH 7.0) and in the presence of 7-15 microM Mg2+. Addition of 5 mM ADP to this preparation greatly accelerates its hydrolysis. We have been able to establish this by stopping the phosphorylation with radioactive ATP, by adding 1 mM non-radioactive ATP, which leads to a slow monoexponential process of dephosphorylation of 32P-labeled enzyme. The relative proportion of the ADP-sensitive phosphoenzyme is 22% of the total phosphoenzyme. Values for the rate constants of breakdown and interconversion of the two phosphoenzyme forms have been determined.     

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.     

ADP- and K+-sensitive phosphorylated intermediate of Na,K-ATPase

In the phosphoenzyme (EP) of the electric eel Na,K-ATPase, the sum of the ADP-sensitive EP and the K+-sensitive EP exceeds 150% of EP in the presence of 100 mM Na+. This unusual phenomenon can be explained by the formation of three phosphoenzymes: ADP-sensitive K+-insensitive (E1P), K+-sensitive ADP-insensitive (E2P), and ADP- and K+-sensitive (E*P) phosphoenzymes, as proposed by N?rby et al. (N?rby, J. G., Klodos, I., and Christiansen, N. O. (1983) J. Gen. Physiol. 82, 725-757). By applying a simple approximation method for the assay of E1P, E*P, and E2P, it was found that the phosphorylation of the enzyme was much faster than the conversion among each EP and the phosphoenzyme changed as E1NaATP----E1P----E*P----E2P. In the fragmental eel enzyme, the step of E*P to E2P was much slower than the step of E1P to E*P. In the steady state, the E1P was predominant above 400 mM Na+, whereas E*P and E2P were predominant between 60 and 300 mM Na+ and below 60 mM Na+, respectively. The characteristic difference of the eel enzyme from the beef brain enzyme and probably from the kidney enzyme seems to be that the dissociation constant of Na+ on the E1P-E*P equilibrium is higher than that on the E*P-E2P. The E*P and E1P both interacted with ADP to form ATP without formation of inorganic phosphate in the absence of free Mg2+. In the Na,K-ATPase proteoliposomes, the vesicle membrane interfered with the conversion of E1P to E2P, especially the change of E1P to E*P, and furthermore, the E1P content increased. This barrier effect was partially counteracted by monensin or carbonyl cyanide m-chlorophenylhydrazone. Oligomycin reacted with E1P and probably with E*P, therefore inhibiting their conversion to E2P and interaction with K+.     

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.     

Effect of divalent cation bound to the ATPase of sarcoplasmic reticulum. Activation of phosphoenzyme hydrolysis by Mg2+

In order to study the mechanism for activation of ATP hydrolysis by Mg2+, the stoichiometry of the high affinity calcium-binding sites with respect to each form of reaction intermediate of sarcoplasmic reticulum ATPase was determined at 0 degrees C and pH 7.0 in the presence and absence of added Mg2+ using the purified ATPase preparation. High affinity calcium binding to the enzyme-ATP complex and to ADP-sensitive (E1P) and ADP-insensitive (E2P) phosphoenzymes occurred with stoichiometric ratios of 2, 2, and 0, and 3, 3, and 1 in the presence and absence of added Mg2+, respectively. The results were interpreted to indicate that in addition to 2 mol of calcium bound to the transport sites of the ATPase, 1 mol of divalent cation, which is derived from the metal component of the substrate, the metal-ATP complex, remains bound to each mole of the enzyme at least until E2P is hydrolyzed. As activation of phosphoenzyme hydrolysis by Mg2+ was blocked by the low concentrations of Ca2+ used in the calcium binding experiments, it was concluded that it is the magnesium derived from MgATP that is responsible for rapid hydrolysis of the phosphoenzyme intermediate.     

Insight into the uncoupling mechanism of sarcoplasmic reticulum ATPase using the phosphorylating substrate UTP

Ca(2+) transport and UTP hydrolysis catalyzed by sarcoplasmic reticulum Ca(2+)-ATPase from skeletal muscle was studied. A passive Ca(2+) load inside microsomal vesicles clearly decreased the net uptake rate and the final accumulation of Ca(2+) but not the UTP hydrolysis rate, causing energy uncoupling. In the absence of passive leak, the Ca(2+)/P(i) coupling ratio was 0.7-0.8. UTP hydrolysis did not maintain a rapid component of Ca(2+) exchange between the cytoplasmic and lumenal compartments as occurs with ATP. The uncoupling process in the presence of UTP is associated with: (i) the absence of a steady state accumulation of ADP-insensitive phosphoenzyme; (ii) the cytoplasmic dissociation of Ca(2+) bound to the ADP-sensitive phosphoenzyme; and (iii) the absence of enzyme inhibition by cyclopiazonic acid. All these characteristics confirm the lack of enzyme conformations with low Ca(2+) affinity and point to the existence of an uncoupling mechanism mediated by a phosphorylated form of the enzyme. Suboptimal coupling values can be explained in molecular terms by the proposed functional model.     

Inhibition of (Na+, K+)adenosine triphosphatase and its partial reactions by quercetin.

The bioflavonoid, quercetin, inhibited the (Na+, K+)adenosine triphosphatase purified from the electric organ of electric eel (Electrophorus electricus) or from lamb kidney. An analysis of its mode of action revealed that the formation of phosphoenzyme from Pi but not from ATP was inhibited. Quercetin increased the amount of ADP-sensitive phosphoenzyme (E1--P), indicating an inhibition of the conversion of E1--P to the ADP-insensitive form (E2--P). The rate of dephosphorylation of the phosphoenzyme formed from ATP was slowed by quercetin. These results suggest that quercetin inhibits the formation of E2--P from either Pi or E1-P as well as the hydrolysis of the phosphoenzyme. Its mode of action is therefore different from that of ouabain and other inhibitors of the Na+, K+)adenosine triphosphatase.     

Inhibition of sarcoplasmic reticulum Ca2+-ATPase by Mg2+ at high pH

Steady state turnover of Ca2+-ATPase of sarcoplasmic reticulum has generally been reported to have a bell-shaped pH profile, with an optimum near pH 7.0. While a free [Mg2+] of 2 mM is optimal for activity at pH 7.0, it was found that this level was markedly inhibitory (K1/2 = 2 mM) at pH 8.0, thus accounting for the generally observed low activity at high pH. High activity was restored at pH 8.0 using an optimum free [Mg2+] of 0.2 mM. The mechanism of the Mg2+-dependent inhibition at pH 8.0 was probed. Inhibition was not due to Mg2+ competition with Ca2+ for cytoplasmic transport sites nor to inhibition of formation of steady state phosphoenzyme from ATP. Mg2+ inhibited (K1/2 = 1.8 mM) decay of steady state phosphoenzyme; thus, the locus of inhibition was one of the phosphoenzyme interconversion steps. Transient kinetic experiments showed that Mg2+ competitively inhibited (Ki = 0.7 mM) binding of Ca2+ to lumenal transport sites, blocking the ability of Ca2+ to reverse the catalytic cycle to form ADP-sensitive, from ADP-insensitive, phosphoenzyme. The data were consistent with a hypothesis in which Mg2+ binds lumenal Ca2+ transport sites with progressively higher affinity at higher pH to form a dead-end complex; its dissociation would then be rate-limiting during steady state turnover.     

Toward a general method to observe the phosphate groups of phosphoenzymes with infrared spectroscopy

A general method to study the phosphate group of phosphoenzymes with infrared difference spectroscopy by helper enzyme-induced isotope exchange was developed. This allows the selective monitoring of the phosphate P-O vibrations in large proteins, which provides detailed information on several band parameters. Here, isotopic exchange was achieved at the oxygen atoms of the catalytically important phosphate group that transiently binds to the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a). [gamma-(18)O(3)]ATP phosphorylated the ATPase, which produced phosphoenzyme that was initially isotopically labeled. The helper enzyme adenylate kinase regenerated the substrate ATP from ADP (added or generated upon ATP hydrolysis) with different isotopic composition than used initially. With time this produced the unlabeled phosphoenzyme. The method was tested on the ADP-insensitive phosphoenzyme state of the Ca(2+)-ATPase for which the vibrational frequencies of the phosphate group are known, and it was established that the helper enzyme is effective in mediating the isotope exchange process.     

Phosphoenzyme formation from ATP in the ATPase of sarcoplasmic reticulum. Effect of KCl or ATP and slow dissociation of ATP from precursor enzyme-ATP complex

The ATP-dependent phosphoenzyme formation and its reversal were studied at 0 degrees C and pH 7.0 in the ATPase of sarcoplasmic reticulum. Addition of KCl or several other salts (approximately 100 mM) decreased the maximum rate of ADP-induced dephosphorylation of phosphoenzyme as well as the apparent affinity of the phosphoenzyme toward ADP. High ATP had a similar effect on the latter, whereas it had little effect on the former. In contrast, high KCl or a considerable change in the ionic strength had little effect on the initial rate of phosphoenzyme formation at saturating ATP concentrations. During steady state phosphorylation at 1.0 mM MgCl2 and 5.0 mM CaCl2 in the absence of added KCl, a significant amount of [gamma-32P]ATP remained bound to the enzyme even when the enzyme concentration was much in excess over that of [gamma-32P]ATP. Evidence is presented that this enzyme-ATP complex represents a precursor to the phosphoenzyme. ATP dissociated slowly (0.20 s-1) from this enzyme-ATP complex and addition of high KCl or other salts accelerated its dissociation. In contrast, when the enzyme was complexed with adenyl-5'-yl (beta, gamma-methylene)diphosphonate in the absence of added KCl under these conditions, dissociation of the nucleotide from the complex as estimated in the displacement experiment with [gamma-32P]ATP, was found to be much faster than that of ATP.     

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.     

Selective inhibition by lasalocid of hydrolysis of the ADP-insensitive phosphoenzyme in the catalytic cycle of sarcoplasmic reticulum Ca2(+)-ATPase

The effect of a carboxylic ionophore (lasalocid) on the sarcoplasmic reticulum Ca2(+)-ATPase was investigated. The purified enzyme was preincubated with lasalocid in the presence of Ca2+ and the absence of K+ at pH 7.0 and 0 degrees C for 2 h. The Ca2(+)-dependent ATPase activity was strongly inhibited by this preincubation, whereas the activity of the contaminant Mg2(+)-ATPase was unaffected. The steady-state level of the phosphoenzyme (EP) intermediate remained constant over the wide range of lasalocid concentrations. The Ca2(+)-induced enzyme activation was unaffected. The kinetics of phosphorylation of the Ca2(+)-activated enzyme by ATP as well as the rate of conversion of ADP-sensitive EP to ADP-insensitive EP were also unaffected. Accumulation of ADP-insensitive EP was greatly enhanced, and almost all of the EP accumulating at steady state was ADP-insensitive. Hydrolysis of ADP-insensitive EP was strongly inhibited. A similar strong inhibition of the Ca2(+)-dependent ATPase activity by lasalocid was found with sarcoplasmic reticulum vesicles. To examine the effect of lasalocid on the conformational change in each reaction step, the Ca2(+)-ATPase of sarcoplasmic reticulum vesicles was labeled with a fluorescent probe (N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine) without a loss of catalytic activity and then preincubated with lasalocid as described above. The conformational changes involved in hydrolysis of ADP-insensitive EP and in the reversal of this hydrolysis were appreciably retarded by lasalocid. The conformational changes involved in other reaction steps were unaffected. These results demonstrate that hydrolysis of ADP-insensitive EP in the catalytic cycle of this enzyme is selectively inhibited by lasalocid.     

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.     

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

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