Phosphoenzyme Decomposition Research Articles

Inhibition mechanism of the intracellular transporter Ca2+-pump from sarco-endoplasmic reticulum by the antitumor agent dimethyl-celecoxib.

Dimethyl-celecoxib is a celecoxib analog that lacks the capacity as cyclo-oxygenase-2 inhibitor and therefore the life-threatening effects but retains the antineoplastic properties. The action mechanism at the molecular level is unclear. Our in vitro assays using a sarcoplasmic reticulum preparation from rabbit skeletal muscle demonstrate that dimethyl-celecoxib inhibits Ca2+-ATPase activity and ATP-dependent Ca2+ transport in a concentration-dependent manner. Celecoxib was a more potent inhibitor of Ca2+-ATPase activity than dimethyl-celecoxib, as deduced from the half-maximum effect but dimethyl-celecoxib exhibited higher inhibition potency when Ca2+ transport was evaluated. Since Ca2+ transport was more sensitive to inhibition than Ca2+-ATPase activity the drugs under study caused Ca2+/Pi uncoupling. Dimethyl-celecoxib provoked greater uncoupling and the effect was dependent on drug concentration but independent of Ca2+-pump functioning. Dimethyl-celecoxib prevented Ca2+ binding by stabilizing the inactive Ca2+-free conformation of the pump. The effect on the kinetics of phosphoenzyme accumulation and the dependence of the phosphoenzyme level on dimethyl-celecoxib concentration were independent of whether or not the Ca2+–pump was exposed to the drug in the presence of Ca2+ before phosphorylation. This provided evidence of non-preferential interaction with the Ca2+-free conformation. Likewise, the decreased phosphoenzyme level in the presence of dimethyl-celecoxib that was partially relieved by increasing Ca2+ was consistent with the mentioned effect on Ca2+ binding. The kinetics of phosphoenzyme decomposition under turnover conditions was not altered by dimethyl-celecoxib. The dual effect of the drug involves Ca2+-pump inhibition and membrane permeabilization activity. The reported data can explain the cytotoxic and anti-proliferative effects that have been attributed to the celecoxib analog. Ligand docking simulation predicts interaction of celecoxib and dimethyl-celecoxib with the intracellular Ca2+ transporter at the inhibition site of hydroquinones.

Characterization of the palytoxin effect on Ca 2+-ATPase from sarcoplasmic reticulum (SERCA)

The effect of palytoxin was studied in a microsomal fraction enriched in longitudinal tubules of the sarcoplasmic reticulum membrane. Half-maximal effect of palytoxin on Ca 2+-ATPase activity yielded an apparent inhibition constant of approx. 0.4 μM. The inhibition process exhibited the following characteristics: (i) the degree of inhibition was dependent on membrane protein concentration; (ii) no protection was observed when the ATP concentration was raised; (iii) dependence on Ca 2+ concentration with a decreased maximum catalytic rate; (iv) it occurred in the absence of Ca 2+ ionophoric activity. Likewise, the inhibition mechanism was linked to: (i) rapid enzyme phosphorylation from ATP in the presence of Ca 2+ but lower steady-state levels of phosphoenzyme; (ii) more drastic effect on phosphoenzyme levels when the toxin was added to the enzyme in the absence of Ca 2+; (iii) decreased phosphoenzyme levels at saturating Ca 2+ concentrations; (iv) no effect on kinetics of phosphoenzyme decomposition. The palytoxin effect is related with lock of the enzyme in the Ca 2+-free conformation so that progression of the catalytic cycle is impeded.

Drug Action of Ritodrine on the Sarcoplasmic-Reticulum Ca2+-ATPase from Skeletal Muscle

Ritodrine inhibits the steady-state Ca2+-ATPase activity of isolated sarcoplasmic reticulum vesicles in a dose-dependent manner, The observed K0.5 value for inhibition (∼3 mM) gives proof of a low-affinity interaction. Ritodrine also interferes with the steady-state Ca2+ transport ability decreasing the maximal rate without modification of the Ca2+ or ATP affinity for the enzyme, This is consistent with an absence of competition for the transport and the catalytic sites. Analysis of the catalytic and transport cycle shows that: (i) ritodrine inhibits the phosphorylation partial reaction, This is supported by pre-steady-state kinetic experiments of Ca2+ transport and also by the temperature dependence of the phosphoenzyme level. (ii) At high ritodrine concentrations the dephosphorylation step becomes rate-limiting. This is suggested by the biphasic profile (V-shape) of phosphoenzyme accumulation at different ritodrine concentrations, This was also confirmed by chase experiments of radioactive phosphoenzyme decomposition at steady state. These data reveal a complex pattern of inhibition involving two sites for interaction with low and high ritodrine concentrations. It is envisaged that ritodrine does not exert any direct effect on the smooth muscle sarcoplasmic reticulum Ca2+-ATPase when used in the treatment of preterm birth.

Relationship between protein rotational dynamics and phosphoenzyme decomposition in the sarcoplasmic reticulum Ca-ATPase.

We have investigated the role of large-scale protein rotational mobility in the reaction mechanism of the Ca-ATPase in sarcoplasmic reticulum using conditions that have previously been found to inhibit selectively phosphoenzyme decomposition, i.e. 1) partial delipidation (by detergent extraction or phospholipase treatment) and 2) the addition of nonaqueous solvents (dimethyl sulfoxide, glycerol, and sucrose). Using saturation-transfer electron paramagnetic resonance to probe the microsecond rotational motion of the spin-labeled Ca-ATPase, we find that both calcium-dependent ATPase activity and protein rotational mobility decrease in parallel, suggesting that protein mobility is important to the enzymatic step(s) involving phosphoenzyme decomposition. Using conventional EPR to measure the nanosecond rotational dynamics of spin-labeled lipid hydrocarbon chains, we find that neither the removal of lipid nor the addition of nonaqueous solvents significantly affects the lipid dynamics. We propose that the physical mode of inactivation under these conditions is the reduction in protein mobility through enforced protein-protein interactions, the result of which is a reduction in a motion essential for Ca-ATPase activity.

Phosphoenzyme conformational states and nucleotide-binding site hydrophobicity following thiol modification of the Ca2+-ATPase of sarcoplasmic reticulum from skeletal muscle.

Enhanced fluorescence of the ATP analogue 2',3'-O-(2,4,6-trinitrocyclohexyldienylidine)adenosine 5'-triphosphate (TNP-ATP), bound to the Ca2+-ATPase of skeletal muscle sarcoplasmic reticulum, is closely related to phosphoenzyme levels (Bishop, J. E., Johnson, J. D., and Berman, M. C. (1984) J. Biol. Chem. 259, 15163-15171) and has an emission maximum consistent with decreased polarity of the TNP-ATP-binding site. The phosphoenzyme conformation responsible for increased nucleotide-binding site hydrophobicity has been studied by redistribution of phosphoenzyme intermediates following specific thiol group modification. N-Ethylmaleimide, in the presence of 50 microM Ca2+, 1 mM adenyl-5'-yl imidodiphosphate, pH 7.0, at 25 degrees C for 30 min, selectively modified the SH group essential for phosphoenzyme decomposition, which resulted in decreased ATPase activity, Ca2+ uptake, and a decrease in ATP-induced TNP-ATP fluorescence. Phosphorylated (Ca2+, Mg2+)-ATPase levels from [gamma-32P] ATP remained relatively unaffected (3.1 nmol/mg), but the ADP-insensitive fraction decreased from 56 to 15%. Phosphoenzyme levels from 32Pi were also decreased to the same extent as turnover, with equivalent loss of Pi-induced TNP-ATP fluorescence. The E1 to E2 transition, as monitored by the change in intrinsic tryptophan fluorescence, was unaffected. Modification of thiol groups of unknown function did not modify turnover-induced TNP-ATP fluorescence. It is concluded that the ADP-insensitive phosphoenzyme, E2-P, is responsible for enhanced TNP-ATP fluorescence. This suggests that the conformational transition, 2Ca2+outE1 approximately P----2Ca2+inE2-P, is associated with altered properties of the noncatalytic, or regulatory, nucleotide-binding site.

Relationship of the regulatory nucleotide site to the catalytic site of the sarcoplasmic reticulum Ca2+-ATPase.

The purpose of this study was to probe the regulatory nucleotide site of the Ca2+-ATPase of sarcoplasmic reticulum and to study its relationship with the catalytic nucleotide site. Our approach was to use the nucleotide analogue 2'(3')-O-(2,4,6-trinitrocyclohexadienylidene)adenosine 5'-phosphate (TNP-AMP), which is known to bind the Ca2+-ATPase with high affinity and to undergo a manyfold increase in fluorescence upon enzyme phosphorylation with ATP in the presence of Ca2+. TNP-AMP was shown to bind the regulatory site in that it competitively inhibited (Ki = 0.6 microM) the secondary activation of turnover induced by millimolar ATP, thus providing a high affinity probe for the site. Observation of the high phosphoenzyme-dependent fluorescence upon monomerization of the enzyme without an increase in phosphoenzyme levels showed the regulatory site to be on the same subunit as the catalytic site and excluded an uncovering of "silent" nucleotide sites resulting from dissociation of enzyme subunits. Identical stoichiometric levels of [3H]TNP-AMP binding (4 nmol/mg of protein) to either the free enzyme or the enzyme phosphorylated with 250 microM ATP excluded models of two nucleotide sites per subunit. Finally, transient kinetic experiments in which TNP-AMP was found to block the ADP-induced burst of phosphoenzyme decomposition showed that TNP-AMP was bound to the phosphorylated catalytic site. We conclude that the regulatory nucleotide site is not a separate and distinct site on the Ca2+-ATPase but, rather, results from the nucleotide catalytic site following formation of the phosphorylated enzyme intermediate.

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 ( 32 P i 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.

Role of lipids in sarcoplasmic reticulum: A higher lipid content is required to sustain phosphoenzyme decomposition than phosphoenzyme formation

Enzyme preparations with variable phospholipid contents were obtained by removing lipids from sarcoplasmic reticulum with deoxycholate. Preparations containing from 90 to 37 phospholipids per enzyme showed normal values of both Ca 2+-ATPase activity and steady-state phosphoenzyme levels. Fractions containing 37 to 23 phospholipids per enzyme had a reduced ATPase activity but normal phosphoenzyme levels, showing that in this range of lipid content the ATPase reaction is inhibited in a reaction step subsequent to phosphoenzyme formation but prior to phosphoenzyme decomposition. Delipidation below 23 lipids per enzyme caused a marked reduction of the amount of phosphoenzyme formed, so that although both reactions require lipids, fewer lipids are required for phosphoenzyme formation than for decomposition. The effect of lipid removal could be completely reversed by readdition of lipids to fractions containing more than 11 lipids per enzyme. It is proposed that phosphoenzyme formation requires full occupancy of a boundary domain of 23 lipids per enzyme, and that the selective inhibition of phosphoenzyme decomposition at higher lipid contents is caused by a decrease in the rotational mobility of the enzyme.

Kinetic effects of calcium and ADP on the phosphorylated intermediate of sarcoplasmic reticulum ATPase.

The decomposition of 32P phosphorylated enzyme intermediate formed by incubation of sarcoplasmic reticulum ATPase with [gamma-32P]ATP was studied following dilution of the reaction medium with a large excess of nonradioactive ATP. The phosphoenzyme decomposition includes two kinetic components. The fraction of intermediate undergoing slower decomposition is minimal in the presence of low (microM) Ca2+ and maximal in the presence of high (mM) Ca2+. A large fraction of phosphoenzyme undergoes slow decomposition when the Ca2+ concentration is high inside the vesicles, even if the Ca2+ concentration in the medium outside the vesicles is low. Parallel measurements of ATPase steady state velocity in the same experimental conditions indicate that the apparent rate constant for the slow component of phosphoenzyme decomposition is inadequate to account for the steady state ATPase velocity observed under the same conditions and cannot be the rate-limiting step in a single, obligatory pathway of the catalytic cycle. On the contrary, the steady state enzyme velocity at various Ca2+ concentrations is accounted for by the simultaneous contribution of both phosphoenzyme fractions undergoing fast and slow decomposition. Contrary to its slow rate of decomposition in the forward direction of the cycle, the phosphoenzyme pool formed in the presence of high Ca2+ reacts rapidly with ADP to form ATP in the reverse direction of the cycle. Detailed analysis of these experimental observations is consistent with a branched pathway following phosphoryl transfer from ATP to the enzyme, whereby the phosphoenzyme undergoes an isomeric transition followed by ADP dissociation, or ADP dissociation followed by the isomeric transition. The former path is much faster and is prevalent when the intravesicular Ca2+ concentration is low. When the intravesicular Ca2+ concentration rises, a pool of phosphoenzyme is formed by reverse equilibration through the alternate path. In the absence of ADP this intermediate decays slowly in the forward direction, and in the presence of ADP it decays rapidly in the reverse direction of the cycle.

ADP stimulates hydrolysis of the “ADP-insensitive” phosphoenzyme in Na+, K+-ATPase and Ca2+-ATPase.

ADP-sensitive (E1P) and K+-sensitive (E2P) phosphoenzymes are sequentially formed intermediates in the reaction pathways catalyzed by the Na+,K+- and Ca2+-ATPases. The kinetics of dephosphorylation of these intermediates were examined by means of rapid quenching with acid at 21 degrees C. Under conditions favoring the formation of E2P (25 mM Na+ and O K+), addition of 5 mM ADP + 10 mM EDTA to the Na+,K+-ATPase phosphoenzyme produced a biphasic pattern of dephosphorylation. Both phases of phosphoenzyme decomposition were accompanied by approximately stoichiometric amounts of inorganic phosphate (Pi) release. The rate of decay of the rapid phase was 10 times faster than the rate of phosphoenzyme turnover under phosphorylating conditions indicating acceleration of E2P hydrolysis by ADP. Similar patterns of ADP-stimulated phosphoenzyme decay and Pi release were observed in the Ca2+-ATPase from sarcoplasmic reticulum phosphorylated at low (0.1 mM) Mg2+ in the absence of KCl. These results demonstrate that ADP can enhance the rate of E2P hydrolysis in the cases of the Na+,K+-ATPase and Ca2+-ATPase. As a consequence measurement of "ADP-sensitive EP" may overestimate E1P.

The ADP- and Mg 2+-reactive calcium complex of the phosphoenzyme in skeletal sarcoplasmic reticulum Ca 2+-ATPase

The effects of ATP on Ca 2+ binding in the absence of added Mg 2+ to the purified sarcoplasmic reticulum Ca 2+-ATPase were studied at pH 7.0 and 0°C. ATP increased the number of Ca 2+-binding sites of the enzyme from 2 to 3 mol per mol of phosphorylatable enzyme. The association constant for the ATP-induced Ca 2+ binding was 4·10 5 M −1, which was not significantly different from that obtained in the absence of ATP. Ado P[CH 2] PP has little effect on the Ca 2+-binding process. The amount of phosphoenzyme formed was equivalent to the level of ATP-induced Ca 2+ binding. ADP decreased the level of ATP-induced Ca 2+ binding and phosphoenzyme by the same amount. These results suggest that ATP-induced Ca 2+ binding exists in the form of an ADP-reactive phosphoenzyme·Ca complex. In addition, the Ca 2+ bound to the enzyme in the presence of ATP was released on the addition of 1 mM MgCl 2; after the release of Ca 2+, the phosphoenzyme decayed. These observations suggest that Mg 2+, added after the ATP-induced Ca 2+-binding process, may replace the Ca 2+ on the phosphoenzyme and initiate phosphoenzyme decomposition.

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.

Phosphoenzyme decomposition in sarcoplasmic reticulum isolated from cat caudofemoralis, tibialis and soleus

Decomposition of phosphoenzyme (E ∼ P) in sarcoplasmic reticulum isolated from caudofemoralis, tibialis and soleus of cat hind leg skeletal muscles was studied under various conditions of monovalent cations. In the presence of Li +, Na + and K + chosen for E ∼ P formation and decomposition after quenching of E ∼ P with EGTA, E ∼ P in the caudofemoralis and tibialis sarcoplasmic reticulum decomposed faster than that in the soleus sarcoplasmic reticulum. Quenching the E ∼ P formation with EGTA and ADP revealed that 30–40% of the total E ∼ P formed in all types of sarcoplasmic reticulum was ‘ADP sensitive’. Decomposition of the remaining E ∼ P in caudofemoralis and tibialis sarcoplasmic reticulum was enhanced by ADP, which resulted in a multiphasic decomposition pattern. A larger portion of the remaining E ∼ P in the soleus sarcoplasmic reticulum, on the other hand, decomposed in a monophasic manner and was not significantly influenced by ADP. The data on E ∼ P decomposition clearly differentiate between the fast and slow muscle types.

Role of phospholipids in the calcium-dependent ATPase of the sarcoplasmic reticulum. Enzymatic and ESR studies with phospholipid-replaced membranes.

Three types of partially purified ATPase enzymes having different phospholipid contents and compositions have been prepared: (a) an enzyme whose phospholipid moiety has been replaced predominantly by dioleoyl lecithin (DOL-enzyme), with about the same phospholipid content as the original sarcoplasmic reticulum, (b) dipalmitoyl lecithin-replaced enzyme whose phospholipid content is 30% of that of DOL-enzyme (DPL-enzyme), and (c) a partially delipidated enzyme with about the same phospholipid content as DPL-enzyme but with the original sarcoplasmic reticulum phospholipid composition (del-enzyme). The temperature dependence of Ca2+-activated ATPase activity of these preparations showed clearcut differences; with DOL-enzyme there was no appreciable break in the Arrhenius plot in the 3-40 degrees range; DPL-enzyme showed a break at 29 degrees, and del-enzyme and sarcoplasmic reticulum one at 18 degrees. Transition temperatures obtained from ESR studies with the use of spin-labeled stearic acid incorporated into the membranes agreed with those derived from ATPase assays. Thermo-dynamic analysis of the ATP hydrolysis rates shows that DPL-enzyme has considerably larger values of activation enthalpy and activation entropy below the transition temperature (29 degrees) than those of the other preparations, while all enzyme preparations show similar free energies of activation. The ESR data show that below their transition temperatures DPL-enzyme, and to a lesser degree del-enzyme, have a strongly restricted motion of their phospholipid molecules as compared with either DOL-enzyme or sarcoplasmic reticulum. Studies on the formation and decomposition of phosphoenzyme have been carried out with the three types of ATPase preparations. At 0 degrees, the rate of inorganic phosphate liberation is 8 times lower in DPL-enzyme than in del-enzyme with little difference in the steady state level of phosphoenzyme. In DOL-enzyme, the level of phosphoenzyme and the rate of inorganic phosphate liberation are 1.8 and 3.5 times higher than the corresponding values obtained with del-enzyme. Addition of ADP to the phosphorylated intermediate of DPL-enzyme induces a fast reversal of the phosphorylation reaction. These results indicate that the physical state of the phospholipid molecules associated with the enzyme affects the decomposition of phosphoenzyme, with little effect on the phosphorylation reaction and its reversal.