Phosphoenzyme decomposition in dog cardiac sarcoplasmic reticulum Ca2+-ATPase

A five-syringe quench-flow apparatus was used in the transient-state kinetic study of intermediary phosphoenzyme (EP) decomposition in a Triton X-100 purified dog cardiac sarcoplasmic reticulum (SR) Ca2+-ATPase at 20 degrees C. Phosphorylation of the enzyme by ATP in the presence of 100 mM K+ for 116 ms gave 32% ADP-sensitive E1P, 52% ADP- and K+-reactive E2P, and 16% unreactive residual EPr. The EP underwent a monomeric, sequential E1P 17 s-1----E2P 10.5 s-1----E2 + Pi transformation and decomposition in the ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid quenched Ca2+-devoid medium. The calculated rate constant for the total EP (i.e., E1P + E2P) dephosphorylation was 7.8 s-1. The E1P had an affinity for ADP with an apparent Kd congruent to 100 microM. When the EP was formed in the absence of K+ for 116 ms, no appreciable amount of the ADP-sensitive E1P was detected. The EP comprised about 80% ADP- and K+-reactive E2P and 20% residual EPr, suggesting a rapid E1P----E2P transformation. Both the E2P's formed in the presence and absence of K+ decomposed with a rate constant of about 19.5 s-1 in the presence of 80 mM K+ and 2 mM ADP, showing an ADP enhancement of the E2P decomposition. The results demonstrate mechanistic differences in monomeric EP transformation and decomposition between the Triton X-100 purified cardiac SR Ca2+-ATPase and deoxycholate-purified skeletal enzyme [Wang, T. (1986) J. Biol. Chem. 261, 6307-6319].     

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.     

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+.     

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+.     

Functional consequences of mutations in the beta-strand sector of the Ca2(+)-ATPase of sarcoplasmic reticulum

Kinetic studies of the phosphoenzyme intermediates of site-specific mutants were used to examine the role of Gly233 in the reaction mechanism of the sarcoplasmic reticulum Ca2(+)-ATPase. When this glycine residue, which is highly conserved among cation-transporting ATPases, was replaced by valine, arginine, or glutamic acid, a complete loss of the ability to pump Ca2+ was observed. The mutant enzymes were able to form an ADP-sensitive phosphoenzyme intermediate (E1P) by reaction with ATP in the presence of Ca2+, but this intermediate decayed to the ADP-insensitive form (E2P) very slowly, relative to the wild-type enzyme. The mutant phosphoenzyme intermediate remained ADP-sensitive, even when phosphorylation from ATP was performed under conditions which permitted accumulation of the ADP-insensitive phosphoenzyme intermediate in the wild type. The mutants were also defective in their ability to form the ADP-insensitive phosphoenzyme intermediate by phosphorylation from inorganic phosphate. In addition, they displayed a higher affinity for Ca2+ and a lower cooperativity in Ca2+ binding than did the wild-type enzyme, as measured through the phosphorylation reaction with ATP. These findings can be rationalized either in terms of a parallel shift of E1 to E2 and E1P to E2P conformational equilibria toward the E1 and E1P forms, respectively, or in terms of destabilization of the phosphoryl-protein interaction in the E2P form. The roles of 7 other residues located in the vicinity of Gly233 were also examined by mutation. Although the side chains of these residues are potential Ca2+ ligands, their replacement did not affect the Ca2+ affinity of the enzyme, suggesting the lack of a role of this region of the peptide in formation of Ca2(+)-binding sites.     

Conformational changes in the vicinity of the N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine attached to the specific thiol of sarcoplasmic reticulum Ca2+-ATPase throughout the catalytic cycle

In the previous experiment (Suzuki, H., Obara, M., Kuwayama, H., and Kanazawa, T. (1987) J. Biol. Chem. 262, 15448-15456), the Ca2+-ATPase of sarcoplasmic reticulum vesicles was labeled with N-iodoacetyl-N'-(5-sulfo-1-naphthyl)ethylenediamine without a loss of the catalytic activity. The main labeled site was Cys674. A large monophasic fluorescence drop occurred upon ATP binding to the catalytic site of the Ca2+-activated enzyme in the presence of K+. The present results show that this fluorescence drop is biphasic in the absence of K+. The first and rapid phase of this drop accounts for most of the fluorescence drop. This phase reflects a conformational change in the enzyme.ATP complex. The second and slow phase, being much smaller than the first phase, coincides with phosphoenzyme (EP) isomerization from the ADP-sensitive form to the ADP-insensitive form. This phase disappears when accumulation of ADP-insensitive EP is inhibited by K+ or when EP isomerization is prevented by the N-ethylmaleimide treatment. These results show that this phase reflects a conformational change upon EP isomerization. When free Ca2+ is chelated after EP formation from ATP, the fluorescence intensity is restored to the initial level without Ca2+. This restoration coincides with EP decomposition. This suggests that the fluorescence restoration reflects a conformational change upon hydrolysis of ADP-insensitive EP. This probability is supported by the concurrent occurrence of the Pi-induced fluorescence drop and EP formation from Pi. The results demonstrate that the fluorescence drop upon ATP binding is predominant in the fluorescence change throughout the catalytic cycle.     

The time-dependent distribution of phosphorylated intermediates in native sarcoplasmic reticulum Ca2+-ATPase from skeletal muscle is not compatible with a linear kinetic model

Quenched-flow mixing was used to characterize the kinetic behavior of the intermediate reactions of the skeletal muscle sarcoplasmic reticulum (SR) Ca-ATPase (SERCA1) at 2 and 21 degrees C. At 2 degrees C, phosphorylation of SR Ca-ATPase with 100 microM ATP labeled one-half of the catalytic sites with a biphasic time dependence [Mahaney, J. E., Froehlich, J. P., and Thomas, D. D. (1995) Biochemistry 34, 4864-4879]. Chasing the phosphoenzyme (EP) with 1.66 mM ADP 10 ms after the start of phosphorylation revealed mostly ADP-insensitive E2P (95% of EP(total)), consistent with its rapid formation from ADP-sensitive E1P. The consecutive relationship of the phosphorylated intermediates predicts a decrease in the proportion of E1P ([E1P]/[EP(total)]) with increasing phosphorylation time. Instead, after 10 ms the proportion of E1P increased and that of E2P decreased until they reached a constant 1:1 stoichiometry ([E1P]:[E2P] approximately 1). At 21 degrees C, phosphorylation displayed a transient overshoot associated with an inorganic phosphate (P(i)) burst, reflecting increased turnover of E2P at the higher temperature. The P(i) burst exceeded the decay of the EP overshoot, suggesting that rephosphorylation of the enzyme occurs before the recycling step (E2 --> E1). This behavior and the reversed order of accumulation of phosphorylated intermediates at 2 degrees C are not compatible with the conventional linear consecutive reaction mechanism: E1 + ATP --> E1.ATP --> E1P + ADP --> E2P --> E2.P(i) --> E1 + P(i). Solubilization of the Ca-ATPase into monomers using the nonionic detergent C(12)E(8) gave a pattern of phosphorylation in which E1P and E2P behave like consecutive intermediates. Kinetic modeling of the C(12)E(8)-solubilized SR Ca-ATPase showed that it behaves according to the conventional Ca-ATPase reaction mechanism, consistent with monomeric catalytic function. We conclude that the nonconforming features of native SERCA1 arise from oligomeric protein conformational interactions that constrain the subunits to a staggered or out-of-phase mode of operation.     

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+.     

Calcium-dependent calcium occlusion in the sarcoplasmic reticulum Ca2+-ATPase. Its enhancement by phosphorylation of the enzyme

45Ca2+-40Ca2+ exchangeability of 45Ca bound to the calcium transport sites of unphosphorylated sarcoplasmic reticulum Ca2+-ATPase at equilibrium has been found to be heterogeneous: Half of the bound calcium is [Ca2+]-dependent in a slowly exchangeable (k less than 0.3 s-1), "occluded" state in the Ca2+-ATPase, and the other calcium is [Ca2+]-independent in a rapidly exchangeable (k approximately 0.3 s-1), "unoccluded" state (Nakamura, J. (1986) Biochim. Biophys. Acta 870, 495-501). In this paper, the two different forms of exchangeable calcium were studied after phosphorylation of the enzyme by ATP without added Mg2+ at pH 7.0 and 0 degree C. By the phosphorylation, the degree of the occlusion became higher (k less than 0.03 s-1). The unoccluded calcium was, however, not significantly affected. The more highly occluded calcium exchanged at the same rate as the decay rate of the phosphoenzyme (EP) in the steady state at a ratio of about 1:1. The occluded calcium was relieved by dephosphorylation of EP by ADP. These results suggest that 1 mol of ADP-sensitive EP more highly occluded 1 mol of calcium, already occluded before phosphorylation. After transformation of ADP-sensitive EP to its ADP-insensitive form by the addition of 20 mM Mg2+ at pH 8.8, the unoccluded calcium was rapidly (k = 0.1-0.3 s-1) released from the transformed EP. However, the occluded calcium was maintained in an occluded state in which the calcium was slowly (k approximately 0.01 s-1) released from the EP without exchange. The results suggest that calcium occlusion in the ADP-sensitive EP is not relieved by the loss of ADP sensitivity of the EP itself.     

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.     

Phosphorylated intermediates of Na,K-ATPase proteoliposomes controlled by bilayer cholesterol. Interaction with cardiac steroid

Fragmental Na,K-ATPase from the electric eel forms three phosphorylated intermediates (EP) with MgATP and Na+: ADP-sensitive K+-insensitive EP (E1P), ADP- and K+-sensitive EP (E*P), and K+-sensitive ADP-insensitive EP (E2P). The EP composition varied with the Na+ concentration. In the reconstituted Na,K-ATPase proteoliposomes (PL), the EP composition of the inside-out form was controlled not only by the intravesicular (extracellular) Na+ concentration, but also by the temperature and the cholesterol content of the lipid bilayer. When the lipid bilayer of PL contained less than 30 mol % cholesterol, the E*P content did not change significantly while the E2P content increased with an elevation in temperature (3-20 degrees C). In contrast, when the lipid bilayer contains more than 35 mol % cholesterol, the E*P content increased while the E2P content stayed less than 10% under the same temperature change. These observations suggest that a high cholesterol content in the lipid bilayer interferes with the E*P to E2P conversion. This cholesterol effect was reversed by ionophores (monensin, nigericin, and A23187). Therefore, E1P-rich EP, E*P-rich EP, or E2P-rich EP could be obtained in the PL under a constant Na+ concentration by using various concentrations of cholesterol and ionophores. The reaction between the proteoliposomal EPs and digitoxigenin (lipid-soluble cardiac steroid) occurred in a single turnover, thereby avoiding unphysiologically high Na+ concentrations. The increase in the ADP- and K+-insensitive EP, which indicated formation of the digitoxigenin-Na,K-ATPase complex, was equivalent to the decrease in the E*P under six different sets of conditions, without any significant change in the E1P and E2P contents. This result indicated that E*P is the active intermediate of the Na,K-ATPase for cardiac steroid binding. Although the E2P has been thought to be the active form for binding, it cannot bind with the cardiac steroid in the presence of Na+ and in the absence of free Mg2+.     

Changes in Ca2+ affinity related to conformational transitions in the phosphorylated state of soluble monomeric Ca2+-ATPase from sarcoplasmic reticulum

Changes in Ca2+ binding after phosphorylation of membranous or detergent-solubilized preparations of sarcoplasmic reticulum Ca2+-ATPase with ATP were followed spectrophotometrically by the use of murexide. Distinct Ca2+ release from the two high-affinity translocation sites was observed, particularly at alkaline pH and at low Ca2+/Mg2+ concentration ratios. Phosphorylation also induced additional binding of Ca2+ at a third site in competition with Mg2+. Ca2+ release was increased after solubilization of Ca2+-ATPase in predominantly monomeric form with the nonionic detergent octaethyleneglycol monododecyl ether. At 0 degree C, chemical-quench studies with [32P]ATP indicated that release of Ca2+ is correlated with the level of ADP-insensitive phosphoenzyme (2 mol of Ca2+ released per mol of E2P formed), both for membranous and detergent solubilized Ca2+-ATPase. Ca2+ release was also found to be accompanied by changes in intrinsic fluorescence. Analysis of the data at 20 degrees C, pH 8.0, showed that binding of Ca2+ to transport sites on E2P occurs with a half-saturation constant of 0.7 mM and a Hill coefficient of 1.8. This is consistent with a drastic decrease in Ca2+ affinity following conversion of ADP-sensitive E1P to ADP-insensitive E2P. The similarity between membranous and detergent-solubilized Ca2+-ATPase supports the view that not more than a single Ca2+-ATPase polypeptide chain is required to complete the conformational transitions which are the basis for active transport of Ca2+.     

Two-step internalization of Ca2+ from a single E approximately P.Ca2 species by the Ca2+-ATPase

Phosphorylation by ATP of E.*Ca2 (sarcoplasmic reticulum vesicles (SRV) with bound 45Ca2+) during 5-10 ms leads to the occlusion of 2 *Ca2+/EPtot [quench by ethylene glycol bis(beta-aminoethyl ether)-N,N,N',N'-tetraacetic acid (EGTA) alone] in both "empty" (10 microM free Ca2+in) or "loaded" SRV (20-40 mM free Ca2+in). The rate of Ca2+ "internalization" from the occluded E approximately P.*Ca2 was measured by using an ADP + EGTA quench; a *Ca2+ ion that is not removed by this quench is defined as internalized. In the presence of 20-40 mM unlabeled Ca2+ inside SRV, 1 *Ca2+/EPtot is internalized from 45Ca-labeled E approximately P.*Ca2 with a first-order rate constant of kl = 34 s-1. Empty SRV take up 2 *Ca2+/EPtot with the same initial rate, but the overall rate constant is kobsd = 17 s-1. The apparent rate constant (kb = 17 s-1) for internalization of the second *Ca2+ is inhibited by [Ca]in, with K0.5 approximately 1.3 mM and a Hill coefficient of n = 1.1. These data show that the two Ca2+ ions are internalized sequentially, presumably from separate sequential sites in the channel. [32P]EP.Ca2 obtained by rapid mixing of E.Ca2 with [gamma-32P]ATP and EGTA disappears in a biphasic time course with a lag corresponding to approximately 34 s-1, followed by EP* decay with a rate constant of approximately 17 s-1. This shows that both Ca2+ ions must be internalized before the enzyme changes its specificity for catalysis of phosphoryl transfer to water instead of to ADP. Increasing the concentration of ATP from 0.25 to 3 mM accelerates the rate of 45Ca2+ internalization from 34 to 69 s-1 for the first Ca2+ and from 17 to 34 s-1 for the second Ca2+. High [ATP] also accelerates both phases of [32P]EP.Ca2 disappearance by the same factor. The data are consistent with a single form of ADP-sensitive E approximately P.Ca2 that sequentially internalizes two ions. The intravesicular volume was estimated to be 2.0 microL/mg, so that one turnover of the enzyme gives 4 mM internal [Ca2+].     

Critical role of Glu40-Ser48 loop linking actuator domain and first transmembrane helix of Ca2+-ATPase in Ca2+ deocclusion and release from ADP-insensitive phosphoenzyme

The functional importance of the length of the A/M1 linker (Glu(40)-Ser(48)) connecting the actuator domain and the first transmembrane helix of sarcoplasmic reticulum Ca(2+)-ATPase was explored by its elongation with glycine insertion at Pro(42)/Ala(43) and Gly(46)/Lys(47). Two or more glycine insertions at each site completely abolished ATPase activity. The isomerization of phosphoenzyme (EP) intermediate from the ADP-sensitive form (E1P) to the ADP-insensitive form (E2P) was markedly accelerated, but the decay of EP was completely blocked in these mutants. The E2P accumulated was therefore demonstrated to be E2PCa(2) possessing two occluded Ca(2+) ions at the transport sites, and the Ca(2+) deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca(2+)-free form of E2P produced from P(i) without Ca(2+) was as rapid in the mutants as in the wild type. Analysis of resistance against trypsin and proteinase K revealed that the structure of E2PCa(2) accumulated is an intermediate state between E1PCa(2) and the Ca(2+)-released E2P state. Namely in E2PCa(2), the actuator domain is already largely rotated from its position in E1PCa(2) and associated with the phosphorylation domain as in the Ca(2+)-released E2P state; however, in E2PCa(2), the hydrophobic interactions among these domains and Leu(119)/Tyr(122) on the top of second transmembrane helix are not yet formed properly. This is consistent with our previous finding that these interactions at Tyr(122) are critical for formation of the Ca(2+)-released E2P structure. Results showed that the EP isomerization/Ca(2+)-release process consists of the following two steps: E1PCa(2) --> E2PCa(2) --> E2P + 2Ca(2+); and the intermediate state E2PCa(2) was identified for the first time. Results further indicated that the A/M1 linker with its appropriately short length, probably because of the strain imposed in E2PCa(2), is critical for the correct positioning and interactions of the actuator and phosphorylation domains to cause structural changes for the Ca(2+) deocclusion and release.     

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.     

Role of divalent cation bound to phosphoenzyme intermediate of sarcoplasmic reticulum ATPase

Effect of divalent cations bound to the phosphoenzyme intermediate of the ATPase of sarcoplasmic reticulum was investigated at 0 degree C and pH 7.0 using the purified ATPase preparations. Our previous study (Shigekawa, M., Wakabayashi, S., and Nakamura, H. (1983) J. Biol. Chem. 258, 14157-14161) indicated that 1 mol of the ADP-sensitive phosphoenzyme (E1P) formed from CaATP has 3 mol of high affinity binding sites for Ca2+, of which two are transport sites for calcium while the remainder is the acceptor site for calcium derived from the substrate, CaATP ("substrate site"). When incubated with a chelator of divalent cation, E1P formed from CaATP released all of its bound calcium to form a divalent cation-free phosphoenzyme. Evidence was presented that calcium dissociation from the substrate site was faster than that from the transport sites and primarily responsible for the ADP sensitivity loss of E1P induced by the chelator. Divalent cation-free phosphoenzyme was kinetically stable but when treated with divalent cations, it behaved similarly to the ADP-insensitive phosphoenzyme (E2P) which is the normal reaction intermediate of ATP hydrolysis. 45Ca bound at the substrate site on E1P formed from 45CaATP exchanged readily with nonradioactive ionized Ca2+ in the reaction medium whereas 45Ca at the transport sites on E1P was displaced only at a very slow rate which was almost the same as that for the phosphoenzyme hydrolysis. It was suggested that calcium at the transport sites on E1P formed from CaATP is released only after the rate-limiting conformational transition of the phosphoenzyme from E1P to E2P and that removal of calcium by a chelator from the substrate site facilitates this conformational transition, thereby allowing calcium bound at the transport sites to be released readily from the phosphoenzyme.     

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.     

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.     

Use of helper enzymes for ADP removal in infrared spectroscopic experiments: application to Ca2+-ATPase

Adenylate kinase (AdK) and apyrase were employed as helper enzymes to remove ADP in infrared spectroscopic experiments that study the sarcoplasmic reticulum Ca(2+)-ATPase. The infrared absorbance changes of their enzymatic reactions were characterized and used to monitor enzyme activity. AdK transforms ADP to ATP and AMP, whereas apyrase consumes ATP and ADP to generate AMP and inorganic phosphate. The benefits of using them as helper enzymes are severalfold: i), both remove ADP generated after ATP hydrolysis by ATPase, which enables repeat of ATP-release experiments several times with the same sample without interference by ADP; ii), AdK helps maintain the presence of ATP for a longer time by regenerating 50% of the initial ATP; iii), apyrase generates free P(i), which can help stabilize the ADP-insensitive phosphoenzyme (E2P); and iv), apyrase can be used to monitor ADP dissociation from transient enzyme intermediates with relatively high affinity to ADP, as shown here for ADP dissociation from the ADP-sensitive phosphoenzyme intermediate (Ca(2)E1P). The respective infrared spectra indicate that ADP dissociation relaxes the closed conformation immediately after phosphorylation partially back toward the open conformation of Ca(2)E1 but does not trigger the transition to E2P. The helper enzyme approach can be extended to study other nucleotide-dependent proteins.     

Roles of Tyr122-hydrophobic cluster and K+ binding in Ca2+ -releasing process of ADP-insensitive phosphoenzyme of sarcoplasmic reticulum Ca2+ -ATPase

Tyr(122)-hydrophobic cluster (Y122-HC) is an interaction network formed by the top part of the second transmembrane helix and the cytoplasmic actuator and phosphorylation domains of sarcoplasmic reticulum Ca(2+)-ATPase. We have previously found that Y122-HC plays critical roles in the processing of ADP-insensitive phosphoenzyme (E2P) after its formation by the isomerization from ADP-sensitive phosphoenzyme (E1PCa(2)) (Wang, G., Yamasaki, K., Daiho, T., and Suzuki, H. (2005) J. Biol. Chem. 280, 26508-26516). Here, we further explored kinetic properties of the alanine-substitution mutants of Y122-HC to examine roles of Y122-HC for Ca(2+) release process in E2P. In the steady state, the amount of E2P decreased so that of E1PCa(2) increased with increasing lumenal Ca(2+) concentration in the mutants with K(0.5) 110-320 microm at pH 7.3. These lumenal Ca(2+) affinities in E2P agreed with those estimated from the forward and lumenal Ca(2+)-induced reverse kinetics of the E1PCa(2)-E2P isomerization. K(0.5) of the wild type in the kinetics was estimated to be 1.5 mM. Thus, E2P of the mutants possesses significantly higher affinities for lumenal Ca(2+) than that of the wild type. The kinetics further indicated that the rates of lumenal Ca(2+) access and binding to the transport sites of E2P were substantially slowed by the mutations. Therefore, the proper formation of Y122-HC and resulting compactly organized structure are critical for both decreasing Ca(2+) affinity and opening the lumenal gate, thus for Ca(2+) release from E2PCa(2). Interestingly, when K(+) was omitted from the medium of the wild type, the properties of the wild type became similar to those of Y122-HC mutants. K(+) binding likely functions via producing the compactly organized structure, in this sense, similarly to Y122-HC.