Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The mechanism whereby events in and around the catalytic site/head of Ca2+-ATPase effect Ca2+ release to the lumen from the transmembrane helices remains elusive. We developed a method to determine deoccluded bound Ca2+ by taking advantage of its rapid occlusion upon formation of E1PCa(2) and of stabilization afforded by a high concentration of Ca2+. The assay is applicable to minute amounts of Ca2+-ATPase expressed in COS-1 cells. It was validated by measuring the Ca2+ binding properties of unphosphorylated Ca2+-ATPase. The method was then applied to the isomerization of the phosphorylated intermediate associated with the Ca2+ release process E1PCa(2) -> E2PCa(2) -> E2P + 2Ca(2+). In the wild type, Ca2+ release occurs concomitantly with EP isomerization fitting with rate-limiting isomerization (E1PCa(2) -> E2PCa(2)) followed by very rapid Ca2+ release. In contrast, with alanine mutants of Leu(119) and Tyr(122) on the cytoplasmic part of the second transmembrane helix (M2) and Ile(179) on the A domain, Ca2+ release in 10 mu M Ca2+ lags EP isomerization, indicating the presence of a transient E2P state with bound Ca2+. The results suggest that these residues function in Ca2+ affinity reduction in E2P, likely via a structural rearrangement at the cytoplasmic part of M2 and a resulting association with the A and P domains, therefore leading to Ca2+ release.

DOI： 10.1074/jbc.M115.693770

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The actuator (A) domain of sarco(endo) plasmic reticulum Ca2+-ATPase not only plays a catalytic role but also undergoes large rotational movements that influence the distant transport sites through connections with transmembrane helices M1 and M2. Here we explore the importance of long helix M2 and its junction with the A domain by disrupting the helix structure and elongating with insertions of five glycine residues. Insertions into the membrane region of M2 and the top junctional segment impair Ca2+ transport despite reasonable ATPase activity, indicating that they are uncoupled. These mutants fail to occlude Ca2+. Those at the top segment also exhibited accelerated phosphoenzyme isomerization E1P -> E2P. Insertions into the middle of M2 markedly accelerate E2P hydrolysis and cause strong resistance to inhibition by luminal Ca2+. Insertions along almost the entire M2 region inhibit the dephosphorylated enzyme transition E2 -> E1. The results pinpoint which parts of M2 control cytoplasm gating and which are critical for luminal gating at each stage in the transport cycle and suggest that proper gate function requires appropriate interactions, tension, and/or rigidity in the M2 region at appropriate times for coupling with A domain movements and catalysis.

DOI： 10.1074/jbc.M114.584086

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Language：Chinese   Publishing type：Research paper (scientific journal)   Publisher：CHINESE ACAD SCIENCES, INST BIOPHYSICS  

Sarco-endoplasmic reticulum calcium transporting ATPase expressed in adult fast-twitch skeletal muscle (SERCA1a) utilizes energy from ATP hydrolysis to transport Ca2+ from cytoplasm into sarcoplasmic reticulum against the Ca2+ concentration gradient. By this means, the Ca2+ concentration in cytoplasm may decrease and contracted muscle cells relax. SERCA1a is the structurally and functionally best studied representative of the P-type ion transporting ATPase. Studies of SERCA1a may provide with enlightening information for research about other SERCA isoforms and P type ATPase. To understand the functional roles of the linker between the transmembrane helix M7 and the transmembrane helix M8 (L78), mutational studies about some L78 amino acid residues were performed to evaluate the effect of their single substitutions on the SERCA1a reaction cycle. Mutant SERCA1a cDNAs were obtained by Quick Change site directed mutagenesis. SERCA1a wild type and mutant proteins were expressed in COS-1 cells separately and extracted by microsome preparation. Single mutant SERCA1a proteins, wild type SERCA1a proteins and control microsomes were checked with radioactive [gamma-P-32] ATP, (CaCl2)-Ca-45, and (32)Pi independently to determine their ATPase activities, Ca2+ transport rates, amount of total EP formed from ATP, and amount of E2P formed from Pi. Results showed that all single mutants except for G864A transported Ca2+ at lower rates than that of the SERCA1a wild type protein. Single mutations of G862 or P863 lead to severe decreases of the ATPase activity and the amount of EP formed from ATP or Pi. Measurements about ATP hydrolysis and EP formation of A893P gave data similar to those obtained from G(862) P-863 single mutant proteins, whereas single mutants of G864 and FMQ873-875 did not induce significant reduction of ATPase activity and EP amount. Experimental results above indicated that L78 is involved in the Ca2+ transport across the endoplasmic reticulum membrane, and moreover, appropriate turns at GPG862-864 and near A893 are essential for D351 phosphorylation at the cytoplasmic domain P. Thereby it is suggested that the accurate configuration and flexibility of L78 play important roles for the successive conformational changes accompanying the SERCA1a reaction cycle.

DOI： 10.3724/SP.J.1206.2013.00524

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.54.S159_3

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Sarcoplasmic reticulum Ca2+-ATPase couples the motions and rearrangements of three cytoplasmic domains (A, P, and N) with Ca2+ transport. We explored the role of electrostatic force in the domain dynamics in a rate-limiting phosphoenzyme (EP) transition by a systematic approach combining electrostatic screening with salts, computer analysis of electric fields in crystal structures, and mutations. Low KCl concentration activated and increasing salt above 0.1 M inhibited the EP transition. A plot of the logarithm of the transition rate versus the square of the mean activity coefficient of the protein gave a linear relationship allowing division of the activation energy into an electrostatic component and a non-electrostatic component in which the screenable electrostatic forces are shielded by salt. Results show that the structural change in the transition is sterically restricted, but that strong electrostatic forces, when K+ is specifically bound at the P domain, come into play to accelerate the reaction. Electric field analysis revealed long-range electrostatic interactions between the N and P domains around their hinge. Mutations of the residues directly involved and other charged residues at the hinge disrupted in parallel the electric field and the structural transition. Favorable electrostatics evidently provides a low energy path for the critical N domain motion toward the P domain, overcoming steric restriction. The systematic approach employed here is, in general, a powerful tool for understanding the structural mechanisms of enzymes.

DOI： 10.1074/jbc.M113.482711

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

During Ca2+ transport by sarcoplasmic reticulum Ca2+-ATPase, the conformation change of ADP-sensitive phosphoenzyme (E1PCa(2)) to ADP-insensitive phosphoenzyme (E2PCa(2)) is followed by rapid Ca2+ release into the lumen. Here, we find that in the absence of K+, Ca2+ release occurs considerably faster than E1PCa(2) to E2PCa(2) conformation change. Therefore, the lumenal Ca2+ release pathway is open to some extent in the K+-free E1PCa(2) structure. The Ca2+ affinity of this E1P is as high as that of the unphosphorylated ATPase (E1), indicating the Ca2+ binding sites are not disrupted. Thus, bound K+ stabilizes the E1PCa(2) structure with occluded Ca2+, keeping the Ca2+ pathway to the lumen closed. We found previously (Yamasaki, K., Wang, G., Daiho, T., Danko, S., and Suzuki, H. (2008) J. Biol. Chem. 283, 29144-29155) that the K+ bound in E2P reduces the Ca2+ affinity essential for achieving the high physiological Ca2+ gradient and to fully open the lumenal Ca2+ gate for rapid Ca2+ release (E2PCa(2)-> E2P + 2Ca(2+)). These findings show that bound K+ is critical for stabilizing both E1PCa(2) and E2P structures, thereby contributing to the structural changes that efficiently couple phosphoenzyme processing and Ca2+ handling.

DOI： 10.1074/jbc.M110.183343

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We have developed a stable analog for the ADP-insensitive phosphoenzyme intermediate with two occluded Ca2+ at the transport sites (E2PCa(2)) of sarcoplasmic reticulum Ca2+-ATPase. This is normally a transient intermediate state during phosphoenzyme isomerization from the ADP-sensitive to ADP-insensitive form and Ca2+ deocclusion/ release to the lumen; E1PCa(2) -> E2PCa(2) -> E2P + 2Ca(2+). Stabilization was achieved by elongation of the Glu(40)-Ser(48) loop linking the Actuator domain and M1 (1st transmembrane helix) with four glycine insertions at Gly(46)/Lys(47) and by binding of beryllium fluoride (BeFx) to the phosphorylation site of the Ca2+-bound ATPase (E1Ca(2)). The complex E2Ca(2)center dot BeF3- was also produced by lumenal Ca2+ binding to E2 center dot BeF3- (E2P ground state analog) of the elongated linker mutant. The complex was stable for at least 1 week at 25 degrees C. Only BeFx, but not AlFx or MgFx, produced the E2PCa(2) structural analog. Complex formation required binding of Mg2+, Mn2+, or Ca2+ at the catalytic Mg2+ site. Results reveal that the phosphorylation product E1PCa(2) and the E2P ground state (but not the transition states) become competent to produce the E2PCa(2) transient state during forward and reverse phosphoenzyme isomerization. Thus, isomerization and lumenal Ca2+ release processes are strictly coupled with the formation of the acylphosphate covalent bond at the catalytic site. Results also demonstrate the critical structural roles of the Glu(40)-Ser(48) linker and of Mg2+ at the catalytic site in these processes.

DOI： 10.1074/jbc.M110.144535

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Language：Japanese   Publisher：旭川医科大学  

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Other Link： http://search.jamas.or.jp/link/ui/2010188382

Language：Japanese   Publisher：PHARMACEUTICAL SOC JAPAN  

Sarco(endo)plasmic reticulum Ca2+-ATPase is a representative member of P-type cation transporting ATPases and catalyzes Ca2+ transport coupled with ATP hydrolysis. The ATPase possesses three cytoplasmic domains (N, P, and A) and ten transmembrane helices (M1-M10). Ca2+ binding at the transport sites in the transmembrane domain activates the ATPase and then the catalytic aspartate is auto-phosphorylated to form the phosphorylated intermediate (EP). Structural and functional studies have shown that, during the isomerization of EP in the Ca2+ transport cycle, large motions of the three cytoplasmic domains take place, which then rearranges the transmembrane helices thereby destroying the Ca2+ binding sites, opening the lumenal gate, and thus releasing the Ca2+ into lumen. Stable structural analogues for the Ca2+-occluded and -released states of phosphorylated intermediates and for the transition and product states of the phosphorylation and dephosphorylation reactions were developed for biochemical and atomic-level structural studies to reveal the coupled changes in the catalytic and transport sites. Mutation studies identified the residues and structural regions essential for the structural changes and Ca2+ transport function. Genetic dysfunction of Ca2+-ATPase causes various isoform-specific diseases. In this manuscript, recent understanding of the Ca-ATPase will be reviewed.

DOI： 10.1248/yakushi.130.179

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Roles of hydrogen bonding interaction between Ser(186) of the actuator (A) domain and Glu(439) of nucleotide binding (N) domain seen in the structures of ADP-insensitive phosphorylated intermediate (E2P) of sarco(endo)plasmic reticulum Ca2+-ATPase were explored by their double alanine substitution S186A/E439A, swap substitution S186E/E439S, and each of these single substitutions. All the mutants except the swap mutant S186E/E439S showed markedly reduced Ca2+-ATPase activity, and S186E/E439S restored completely the wild-type activity. In all the mutants except S186E/E439S, the isomerization of ADP-sensitive phosphorylated intermediate (E1P) to E2P was markedly retarded, and the E2P hydrolysis was largely accelerated, whereas S186E/E439S restored almost the wildtype rates. Results showed that the Ser(186)-Glu(439) hydrogen bond stabilizes the E2P ground state structure. The modulatory ATP binding at sub-mM similar to mM range largely accelerated the EP isomerization in all the alanine mutants and E439S. In S186E, this acceleration as well as the acceleration of the ATPase activity was almost completely abolished, whereas the swap mutation S186E/E439S restored the modulatory ATP acceleration with a much higher ATP affinity than the wild type. Results indicated that Ser(186) and Glu(439) are closely located to the modulatory ATP binding site for the EP isomerization, and that their hydrogen bond fixes their side chain configurations thereby adjusts properly the modulatory ATP affinity to respond to the cellular ATP level.

DOI： 10.1074/jbc.M109.034140

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca2+-ATPase E1PCa(2)center dot Mg, a complex of E1Ca(2)center dot BeFx, was successfully developed by addition of beryllium fluoride and Mg2+ to the Ca2+-bound state, E1Ca(2). In E1Ca(2)center dot BeFx, most probably E1Ca(2)center dot BeF3-, two Ca2+ are occluded at high affinity transport sites, its formation required Mg2+ binding at the catalytic site, and ADP decomposed it to E1Ca(2), as in E1PCa(2)center dot Mg. Organization of cytoplasmic domains in E1Ca(2)center dot BeFx was revealed to be intermediate between those in E1Ca(2)center dot AlF4- ADP (transition state of E1PCa(2) formation) and E2 center dot BeF3-(ADP-insensitive phosphorylated intermediate E2P center dot Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca(2)center dot BeFx in contrast to no superfluorescence of TNP-AMP bound to E1Ca(2)center dot AlFx. E1Ca(2)center dot BeFx with bound TNP-AMP slowly decayed to E1Ca(2), being distinct from the superfluorescent complex of TNP-AMP with E2 center dot BeF3-, which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca(2)center dot BeFx mimics E1PCa(2)center dot Mg, and between those of E1Ca(2)center dot AlF4-center dot ADP and E2 center dot BeF3-. E1Ca(2)center dot BeFx at low 50-100 mu M Ca2+ was converted slowly to E2 center dot BeF3- releasing Ca2+, mimicking E1PCa(2)center dot Mg -> E2P center dot Mg + 2Ca(2+)center dot Ca2+ replacement of Mg2+ at the catalytic site at approximately millimolar high Ca2+ decomposed E1Ca(2)center dot BeFx to E1Ca(2). Notably, E1Ca(2)center dot BeFx was perfectly stabilized for at least 12 days by 0.7 mM lumenal Ca2+ with 15 mM Mg2+. Also, stable E1Ca(2)center dot BeFx was produced from E2 center dot BeF3- at 0.7 mM lumenal Ca2+ by binding two Ca2+ to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P center dot Mg + 2Ca(2+) -> E1PCa(2)center dot Mg.

DOI： 10.1074/jbc.M109.029702

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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 Ca2+-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 Ca2+ release process in E2P. In the steady state, the amount of E2P decreased so that of E1PCa(2) increased with increasing lumenal Ca2+ concentration in the mutants with K-0.5 110-320 mu M at pH 7.3. These lumenal Ca2+ affinities in E2P agreed with those estimated from the forward and lumenal Ca2+-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 Ca2+ than that of the wild type. The kinetics further indicated that the rates of lumenal Ca2+ 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 Ca2+ affinity and opening the lumenal gate, thus for Ca2+ 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.

DOI： 10.1074/jbc.M804596200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

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 Ca2+-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 Ca2+ ions at the transport sites, and the Ca2+ deocclusion and release into lumen were blocked in the mutants. By contrast, the hydrolysis of the Ca2+-free form of E2P produced from Pi without Ca2+ 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 Ca2+-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 Ca2+-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 Ca2+-released E2P structure. Results showed that the EP isomerization/Ca2+-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 Ca2+ deocclusion and release.

DOI： 10.1074/jbc.M707665200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We examined possible defects of sarco(endo) plasmic reticulum Ca2+-ATPase 2b (SERCA2b) associated with its 51 mutations found in Darier disease (DD) pedigrees, i.e. most of the substitution and deletion mutations of residues reported so far. COS-1 cells were transfected with each of the mutant cDNAs, and the expression and function of the SERCA2b protein was analyzed with microsomes prepared from the cells and compared with those of the wild type. Fifteen mutants showed markedly reduced expression. Among the other 36, 29 mutants exhibited completely abolished or strongly inhibited Ca2+-ATPase activity, whereas the other seven possessed fairly high or normal ATPase activity. In four of the aforementioned seven mutants, Ca2+ transport activity was significantly reduced or almost completely lost, therefore uncoupled from ATP hydrolysis. The other three were exceptional cases as they were seemingly normal in protein expression and Ca2+ transport function, but were found to have abnormalities in the kinetic properties altered by the three mutations, which happened to be in the three DD pedigrees found by us previously (Sato, K., Yamasaki, K., Daiho, T., Miyauchi, Y., Takahashi, H., Ishida-Yamamoto, A., Nakamura, S., Iizuka, H., and Suzuki, H. (2004) J. Biol. Chem. 279, 35595 - 35603). Collectively, our results indicated that in most cases (48 of 51) DD mutations cause severe disruption of Ca2+ homeostasis by the defects in protein expression and/or transport function and hence DD, but even a slight disturbance of the homeostasis will result in the disease. Our results also provided further insight into the structure-function relationship of SERCAs and revealed critical regions and residues of the enzyme.

DOI： 10.1074/jbc.M601966200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER IRELAND LTD  

Background: ATP2C1 is a calcium/manganese-ATPase localized in the Golgi apparatus nd known as responsible gene for Hailey-Hailey disease. But its localization and roles in the epidermis are not fully elucidated.
Objective: To explore the localization and biological role of ATP2C1 in normal epidermis in terms of differentiation states.
Methods: We examined the immunohistochemical distribution of ATP2C1 in normal epidermis and measured the expression of ATP2C1 in cultured keratinocytes following forced detachment from culture dish or following treatment with high concentrations of calcium. Furthermore, we knockdown ATP2C1 expression in cultured keratinocytes by using RNA interference procedure to abrogate cation accumulation in cell organelles.
Results: ATP2C1 is specifically localized at the basal cell layer in normal epidermis. Neither detachment of keratinocyte from culture dish nor treatment with high concentrations of calcium suppressed ATP2C1 expression, while both procedures induced differentiation markers, K10 keratin and involucrin. In contrast, knockdown of ATP2C1 induced these differentiation markers of cultured keratinocytes. Furthermore, treatment of keratinocytes with a calcium ionophore, A23187, did not upregulate differentiation markers of keratinocytes, while a more manganese selective ionophore Br-A23187 up-regutated these differentiation markers.
Conclusion: Our results suggest that ATP2C1 plays an essential role for basal keratinocytes to keep in the undifferentiated state and that its reduction evokes differentiation and up-localization to suprabasat layers most likely via the manganese starvation in the Golgi apparatus of keratinocytes. (c) 2006 Japanese Society for Investigative Dermatology. Published by Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.jdermsci.2006.03.003

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Functional roles of seven hydrophobic residues on the interface between the actuator ( A) and phosphorylation (P) domains of sarcoplasmic reticulum Ca2+-ATPase were explored by alanine and serine substitutions. The residues examined were Ile(179)/Leu(180)/ Ile(232) on the A domain, Val(705)/Val(726) on the P domain, and Leu(119)/Tyr(122) on the loop linking the A domain and M2 ( the second transmembrane helix). These residues gather to form a hydrophobic cluster around Tyr(122) in the crystal structures of Ca2+-ATPase in Ca2+-unbound E2 (unphosphorylated) and E2P ( phosphorylated) states but are far apart in those of Ca2+- bound E1 ( unphosphorylated) and E1P ( phosphorylated) states. The substitution-effects were also compared with those of Ile(235) on the A domain/M3 linker and those of T(181)GE of the A domain, since they are in the immediate vicinity of the Tyr(122)-cluster. All these substitutions almost completely inhibited ATPase activity without inhibiting Ca2+-activated E1P formation from ATP. Substitutions of Ile(235) and T(181)GE blocked the E1P to E2P transition, whereas those in the Tyr(122)-cluster blocked the subsequent E2P hydrolysis. Substitutions of Ile(235) and Glu(183) also blocked EP hydrolysis. Results indicate that the Tyr(122)-cluster is formed during the E1P to E2P transition to configure the catalytic site and position Glu(183) properly for hydrolyzing the acylphosphate. Ile(235) on the A domain/M3 linker likely forms hydrophobic interactions with the A domain and thereby allowing the strain of this linker to be utilized for large motions of the A domain during these processes. The Tyr(122)-cluster, Ile(235), and T(181)GE thus seem to have different roles and are critical in the successive events in processing phosphorylated intermediates to transport Ca2+.

DOI： 10.1074/jbc.M503789200

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Language：English   Publisher：The Biophysical Society of Japan General Incorporated Association  

DOI： 10.2142/biophys.45.S87_4

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The possible functional abnormalities in three different Darier disease-causing Ca2+-ATPase (SERCA2b) mutants, Ile(274) --> Val at the lumenal end of M3, Leu(321) --> Phe on the cytoplasmic part of M4, and Met(719) --> Ile in P domain, were explored, because they exhibited nearly normal expression and localization in COS-1 cells and the high ATPase and coupled Ca2+ transport activities that were essentially identical (L321F) or slightly lower (I274V by similar to35% and M719I by similar to30%) as compared with those of the wild type. These mutations happened to be in Japanese patients found previously by us. Kinetic analyses revealed that each of the mutants possesses distinct types of abnormalities; M719I and L321F possess the 2-3-fold reduced affinity for cytoplasmic Ca2+, whereas I274V possesses the normal high affinity. L321F exhibited also the remarkably reduced sensitivity to the feedback inhibition of the transport cycle by accumulated lumenal Ca2+, as demonstrated with the effect of Ca2+ ionophore on ATPase activity and more specifically with the effects of Ca2+ (up to 50 mM) on the decay of phosphoenzyme intermediates. The results on I274V and M719I suggest that the physiological requirement for Ca2+ homeostasis in keratinocytes to avoid haploinsufficiency is very strict, probably much more than considered previously. The insensitivity to lumenal Ca2+ in L321F likely brings the lumenal Ca2+ to an abnormally elevated level. The three mutants with their distinctively altered kinetic properties will thus likely cause different types of perturbation of intracellular Ca2+ homeostasis, but nevertheless all types of perturbation result in Darier disease. It might be possible that the observed unique feature of L321F could possibly be associated with the specific symptoms in the pedigree with this mutation, neuropsychiatric disorder, and behavior problems. The results also provided further insight into the global nature of conformational changes of SERCAs for ATP-driven Ca2+ transport.

DOI： 10.1074/jbc.M404887200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

The structural natures of stable analogues for the ADP-insensitive phosphoenzyme (E2P) of Ca2+-ATPase formed in sarcoplasmic reticulum vesicles, i.e. the enzymes with bound beryllium fluoride (BeF.E2), bound aluminum fluoride (AlF.E2), and bound magnesium fluoride (MgF.E2), were explored and compared with those of actual E2P formed from Pi without Ca2+. Changes in trinitrophenyl-AMP fluorescence revealed that the catalytic site is strongly hydrophobic in BeF.E2 as in E2P but hydrophilic in MgF.E2 and AlF.E2; yet, the three cytoplasmic domains are compactly organized in these states. Thapsigargin, which was shown in the crystal structure to fix the transmembrane helices and, thus, the postulated Ca2+ release pathway to lumen in a closed state, largely reduced the tryptophan fluorescence in BeF.E2 as in E2P, but only very slightly ( hence, the release pathway is likely closed without thapsigargin) in MgF.E2 and AlF.E2 as in dephosphorylated enzyme. Consistently, the completely suppressed Ca2+-ATPase activity in BeF-treated vesicles was rapidly restored in the presence of ionophore A23187 but not in its absence by incubation with Ca2+ ( over several millimolar concentrations) at pH 6, and, therefore, lumenal Ca2+ is accessible to reactivate the enzyme. In contrast, no or only very slow restoration was observed with vesicles treated with MgF and AlF even with A23187. BeF.E2 thus has the features very similar to those characteristic of the E2P ground state, although AlF.E2 and MgF.E2 most likely mimic the transition or product state for the E2P hydrolysis, during which the hydrophobic nature around the phosphorylation site is lost and the Ca2+ release pathway is closed. The change in hydrophobic nature is probably associated with the change in phosphate geometry from the covalently bound tetrahedral ground state (BeF3-) to trigonal bipyramidal transition state (AlF3 or AlF4-) and further to tetrahedral product state (MgF42-), and such change likely rearranges transmembrane helices to prevent access and leakage of lumenal Ca2+.

DOI： 10.1074/jbc.M313363200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

We explored, by mutational substitutions and kinetic analysis, possible roles of the four residues involved in the hydrogen-bonding or ionic interactions found in the Ca2+-bound structure of sarcoplasmic reticulum Ca2+-ATPase, Tyr(122)-Arg(324), and Glu(123)-Arg(334) at the top part of second transmembrane helix (M2) connected to the A domain and fourth transmembrane helix (M4) in the P domain. The observed substitution effects indicated that Glu(123), Arg(334), and Tyr(122) contributed to the rapid transition between the Ca2+-unbound and bound states of the unphosphorylated enzyme. Results further showed the more profound inhibitory effects of the substitutions in the M4/P domain (Arg(324) and Arg(334)) upon the isomeric transition of phosphorylated intermediate (EP) (loss of ADP sensitivity) and those in M2/A domain (Tyr(122) and Glu(123)) upon the subsequent processing and hydrolysis of EP. The observed distinct effects suggest that the interactions seen in the Ca2+-bound structure are not functionally important but indicate that Arg(334) with its positive charge and Tyr(122) with its aromatic ring are critically important for the above distinct steps. On the basis of the available structural information, the results strongly suggest that Arg(334) moves downward and forms new interactions with M2 (likely Asn(111)); it thus contributes to the inclination of the M4/P domain toward the M2/A domain, which is crucial for the appropriate gathering between the P domain and the largely rotated A domain to cause the loss of ADP sensitivity. On the other hand, Tyr(122) most likely functions in the subsequent Ca2+-releasing step to produce hydrophobic interactions at the A-P domain interface formed upon their gathering and thus to produce the Ca2+-released form of EP. During the Ca2+-transport cycle, the four residues seem to change interaction partners and thus contribute to the coordinated movements of the cytoplasmic and transmembrane domains.

DOI： 10.1074/jbc.M309398200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Possible roles of the Glu(40)-Ser(48) loop connecting A domain and the first transmembrane helix (M1) in sarcoplasmic reticulum Ca(2+)-ATPase (SERCA1a) were explored by mutagenesis. Deletions of any single residues in this loop caused almost complete loss of Ca(2+)-ATPase activity, while their substitutions had no or only slight effects. Single deletions or substitutions in the adjacent N- and C-terminal regions of the loop (His(32)-Asn(39) and Leu(49)-Ile(54)) had no or only slight effects except two specific substitutions of Asn(39) found in SERCA2b in Darier's disease pedigrees. All the single deletion mutants for the Glu(40)-Ser(48) loop and the specific Asn(39) mutants formed phosphoenzyme intermediate (EP) from ATP, but their isomeric transition from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was almost completely or strongly inhibited. Hydrolysis of E2P formed from P(i) was also dramatically slowed in these deletion mutants. On the other hand, the rates of the Ca(2+)-induced enzyme activation and subsequent E1P formation from ATP were not altered by the deletions and substitutions. The results indicate that the Glu(40)-Ser(48) loop, with its appropriate length (but not with specific residues) and with its appropriate junction to A domain, is a critical element for the E1P to E2P transition and formation of the proper structure of E2P, therefore, most likely for the large rotational movement of A domain and resulting in its association with P and N domains. Results further suggest that the loop functions to coordinate this movement of A domain and the unique motion of M1 during the E1P to E2P transition.

DOI： 10.1074/jbc.M305200200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Possible roles of the Lys(189)-Lys(205) outermost loop on the A domain of sarcoplasmic reticulum Ca2+-ATPase were explored by mutagenesis. Both nonconservative and conservative substitutions of Val(200) caused very strong inhibition of Ca2+-ATPase activity, whereas substitutions of other residues on this loop reduced activity only moderately. All of the Val(200) mutants formed phosphoenzyme intermediate (EP) from ATP. Isomerization from ADP-sensitive EP (E1P) to ADP-insensitive EP (E2P) was not inhibited in the mutants, and a substantially larger amount of E2P, actually accumulated in the mutants than in wild-type sarcoplasmic reticulum Ca2+-ATPase at steady state. In contrast, decay of EP formed from ATP in the presence of Ca2+ was strongly inhibited in the mutants. Hydrolysis of E2P formed from P-i in the absence of Ca2+ was also strongly inhibited but was faster than the decay of EP formed from ATP, indicating that the main kinetic limitation of the decay comes after loss of ADP sensitivity but before E2P hydrolysis. On the basis of the well accepted mechanism of the Ca2+-ATPase, the limitation is likely associated with the Ca2+-releasing step from E2P(.)Ca(2). On the other hand, the rate of activation of dephosphorylated enzyme on high affinity Ca2+ binding was not altered by the substitutions. In light of the crystal structures, the present results strongly suggest that Val(200) confers appropriate interactions of the Lys(189)-Lys(205) loop with the P domain in the Ca2+-released form of E2P. Results further suggest that these interactions, however, do not contribute much to domain organization in the dephosphorylated enzyme and thus would be mostly lost on E2P hydrolysis.

DOI： 10.1074/jbc.M208861200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Conditions were developed in the absence of Ca2+ for purification, delipidation, and long term stabilization of octaethylene glycol monododecyl ether (C12E8)-solubilized sarcoplasmic reticulum Ca2+-ATPase with tightly bound Mg2+ and F-, an analog for the phosphoenzyme intermediate without bound Ca2+. The Ca2+-ATPase activity to monitor denaturation was assessed after treatment with 20 mm Ca2+ to release tightly bound Mg2+/F-. The purification and delipidation was successfully achieved with Reactive Red-agarose affinity chromatography. The solubilized Mg2+/F--bound Ca2+-ATPase was very rapidly denatured at pH 8, but was perfectly stabilized at pH 6 against denaturation for over 20 days at 4degreesC even without exogenously added phospholipid and at a high C12E8/enzyme weight ratio (10:1). The activity was not restored unless the enzyme was treated with 20 mm Ca2+, showing that tightly bound Mg2+/F- was not released during the long term incubation. The perfect stability was attained with or without 0.1 mm dithiothreitol, but inactivation occurred with a half-life of 10 days in the presence of 1 mm dithiothreitol, possibly due to reduction of a specific disulfide bond(s). The remarkable stability is likely conferred by intimate gathering of cytoplasmic domains of Ca2+-ATPase molecule induced by tight binding of Mg2+/F-. The present study thus reveals an essential property of the Mg2+/F-/Ca2+-ATPase complex, which will likely provide clues to understanding structure of the Ca2+-released form of phosphoenzyme intermediate at an atomic level.

DOI： 10.1074/jbc.M200625200

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

In order to characterize the domain organization of sarcoplasmic reticulum Ca(2+)-ATPase in different physiological states, limited proteolysis using three proteases (proteinase K (prtK), V8 and trypsin) was conducted systematically and quantitatively. The differences between E(2) and E(2)P were examined in our previous study and E(2)P was characterized by the complete resistance to all three proteases (except for trypsin attack at the very top of the molecule (T1 site)). The same strategies were employed in this study for E(1)ATP, E(1)PADP and E(1)P states. Because of the transient nature of these states, they were either stabilized by non-hydrolyzable analogues or made predominant by adjusting buffer conditions. Aluminum fluoride (without ADP) was found to stabilize E(1)P. All these states were characterized by strong (E(1)ATP) to complete (E(1)PADP and E(1)P) resistance to prtK and to V8 but only weak resistance to trypsin at the T2 site. Because prtK and V8 primarily attack the loops connecting the A domain to the transmembrane helices whereas the trypsin T2 site (Arg(198)) is located on the outermost loop in the A domain, these results lead us to propose that the A domain undergoes a large amount of rotation between Ell? and E2P. Combined with previous results, we demonstrated that four states can be clearly distinguished by the susceptibility to three proteases, which will be very useful for establishing the conditions for structural studies. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Disulfide-containing peptides in pepsin digest of sarcoplasmic reticulum vesicles were identified by using a fluorogenic thiol-specific reagent 4-fluoro-7-sulfamoylbenzofurazan and a reductant tributylphosphine. Sequencing of the purified peptides revealed the presence of a Cys(876)-Cys(888) disulfide bond on the luminal loop connecting the 7th and 8th transmembrane helices (loop 7-8) of the Ca2+-ATPase (SERCA1a). We substituted either or both of these cysteine residues with alanine and made three mutants (C876A, C888A, C876A/C888A), in which the disulfide bond is disrupted. The mutants and the wild type were expressed in COS-1 cells, and functional analysis was performed with the microsomes isolated from the cells. Electrophoresis performed under reducing and non-reducing conditions confirmed the presence of Cys(876)-Cys(888) disulfide bond in the expressed wild type. All the three mutants possessed high Ca2+-ATPase activity. In contrast, no Ca2+ transport activity was detected with these mutants. These mutants formed almost the same amount of phosphoenzyme intermediate as the wild type from ATP and from Pi. Detailed kinetic analysis showed that the three mutants hydrolyze ATP in the mechanism well accepted for the Ca2+-ATPase; activation of the catalytic site upon high affinity Ca2+ binding, formation of ADP-sensitive phosphoenzyme, subsequent rate-limiting transition to ADP-insensitive phosphoenzyme, and hydrolysis of the latter phosphoenzyme. It is likely that the pathway for delivery of Ca2+ from the binding sites into the lumen of vesicles is disrupted by disruption of the Cys(876)-Cys(888) disulfide bond, and therefore that the loop 7-8 having the disulfide bond is important for formation of the proper structure of the Ca2+ pathway.

DOI： 10.1074/jbc.M101229200

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (international conference proceedings)   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Sarcoplasmic reticulum vesicles were modified with diethyl pyrocarbonate (DEPC), a histidine-modifying reagent, Phosphoenzyme formation from P-i in the Ca2+-ATPase (reversal of hydrolysis of the phosphoenzyme intermediate) was almost completely inhibited by this modification, Tight binding of F- and Mg2+ and high affinity binding of vanadate in the presence of Mg2+, both of which produce transition state analogs for phosphoenzyme formation from the magnesium-enzyme-phosphate complex, were also inhibited, Formation of the phosphoenzyme from acetyl phosphate in the forward reaction was only weakly inhibited, but hydrolysis of the phosphoenzyme was strongly inhibited, The enzyme was protected by tight binding of F- and Mg2+ or by high affinity binding of vanadate in the presence of Mg2+ against the DEPC-induced inhibition of phosphoenzyme formation from P-i. The enzyme was also pro tected by tight binding of F- and Mg2+ against the DEPC-induced inhibition of phosphoenzyme hydrolysis, Peptide mapping of the tryptic digests, detection of peptides containing DEPC-modified histidine by UV absorption at 240 nm, amino acid analysis, sequencing, and mass spectrometry showed that His-5 was a single major residue protected by the above transition state analogs against the modification with DEPC, These results indicate that modification of His-Fi with DEPC is responsible for the DEPC induced inhibition of phosphoenzyme formation from P-i and of phosphoenzyme hydrolysis and suggest that His-5 is located in or very close to the catalytic site in the transition state for phosphoenzyme formation from the magnesium-enzyme-phosphate complex and is likely involved in the catalytic process of this reaction step.

DOI： 10.1074/jbc.272.49.30627

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Sarcoplasmic reticulum vesicles were treated with 1,2-cyclohexanedione (CHD) in sodium berate (pH 8.0), The Ca2+-ATPase activity was completely inhibited, Inhibition of Mg . ATP and Mg . ADP binding to the high affinity ATP binding site as well as inhibition of phosphorylation with ATP occurred simultaneously with the inhibition of the Ca2+-ATPase activity. Phosphorylation with acetyl phosphate was not inhibited, The Ca2+-ATPase was strongly protected by Mg . ATP, Mg . ADP, and Mg . AMP against this inhibition, Binding of acetyl phosphate or P-i to the enzyme gave no protection, Phosphorylation with acetyl phosphate also had no protective effect, Peptide mapping of the tryptic digests, detection of peptides containing CHD-modified arginyl residues with Girard's reagent T, and sequencing revealed that Arg-489, Arg-505, and Arg-678 were modified with CHD. Arg-489 and Arg-678 were almost completely protected by Mp . ATP against this modification, but partially protected by prelabeling with fluorescein 5-isothiocyanate, which occupies the adenosine binding region in the ATP binding site, In contrast, Arg-505 was slightly protected by Mg . ATP and almost completely protected by prelabeling with fluorescein 5-isothiocyanate, Taken together, these findings suggest that Arg-489 and Arg-678 are located in or near the region occupied by the triphosphate moiety of ATP, either or both of these residues being in or close to the region occupied by the alpha-phosphoryl group in the high affinity ATP binding site and involved in the CHD-induced inhibition of this enzyme and that Arg-505 is very close to (but slightly out of) the adenosine binding region in the ATP binding site. The acetyl phosphatase activity and phosphorylation with P-i were also inhibited by the CHD treatment, but the inhibitions were considerably slower than those described above, This suggests that the arginyl residues involved in these inhibitions are distinct from that involved in the inhibition of the Ca2+-ATPase activity.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Sarcoplasmic reticulum vesicles were labeled with 40 mu M 3'-O-(5-fluoro-2,4-dinitrophenyl)-ATP (FDNP-ATP) at 25 degrees C and pH 7.0 for 4 h. The Ca-2+-ATPase was inhibited strongly. The enzyme was almost completely protected either by 20 mM Mg.ATP or by 50 mM acetyl phosphate against this inhibition. P-i gave no protection. There was a linear relationship between the extent of this inhibition and the Mg.ATP-sensitive part of the content of bound FDNP-ATP. Extrapolation showed that the enzyme is completely inhibited by Mg.ATP sensitive binding of 3.6 nmol of FDNP-ATP/mg of the vesicle protein. This value is in good agreement with the content of the phosphorylation site (3.3 nmol/mg of the vesicle protein) in the vesicles used. These findings indicate that binding of 1 mol of FDNP-ATP per mol of the active sites leads to a complete inhibition of the enzyme. The acetylphosphatase activity and phosphorylation with ATP were also strongly inhibited by this labeling, whereas phosphorylation with P-i was not inhibited. The labeled vesicles were solubilized in SDS, and the Ca2+-ATPase was purified by size exclusion high performance liquid chromatography. Mapping the labeled peptides in the tryptic digest by reversed-phase high performance liquid chromatography and sequencing showed that Lys-492 was exclusively labeled with FDNP-ATP These results show that Lys-492 is located in or near the ATP binding site and apart from the phosphorylation site and P-i binding site. Molecular modeling of FDNP-ATP suggests that this Lys-492 residue is situated on the 3'-OH side of the ribose moiety of bound ATP and is close to the alpha-phosphoryl group.

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Language：English   Publisher：(公社)日本生化学会  

高,および低親和性部位へのvanadateの結合がCa-ATPaseのリン酸化中間体(E2P)形成にどのような影響を及ぼすかを調べた.SRにCa非存在下でリン酸を加えてE2Pを形成させた後vanadateを添加すると,E2P形成が抑制され,定常状態でのE2Pレベルは時間とともに減少した.vanadate添加後のE2P減少は,反応初期の急速な減少とそれに続く一次反応に従うゆるやかな減少からなる二相性の時間経過を示した.遅い相でのE2P減少の時間経過を反応時間0秒に外挿して得た速い相でのE2P減少量はvanadate濃度を増すにつれ増大し(Kd=17 μM),50 μMで最大となり,その値はE2P量とほぼ同じであった.速い相でのvanadateによるE2Pの減少はリン酸によって拮抗的に阻害され,1/2阻害に必要なリン酸濃度(7.4 mM)は,同一条件で測定したE2P形成反応のKmとほぼ等しかった.vanadate添加後に見られる遅いE2P減少の時間経過は,高親和性部位へのvanadate結合のそれとよく一致した.低親和性部位への結合が無視できる5 μM vanadateを添加すると,時間とともにCa-ATPaseのリン酸化量は低下し,10分後では5%以下となった.高親和性部位へのvanadateの結合によるE2Pレベルの減少はリン酸濃度を増すにつれて阻害された.上記の測定から求めたリン酸の解離定数は約0.4 mMと推定され,E2P形成のKm (=7.4 mM)と著しく異なっていた

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Language：English   Publisher：(公社)日本生化学会  

SR Ca2+-ATPaseに対するvanadateの結合を,遠心分離とDowex 2×8カラムを用いて測定した.筋小胞体に対するvanadate結合のvanadate濃度依存性は二相性を示し,親和性の異なる2種類のvanadate結合部位に分けることができた.またdeoxycholateを用いて部分精製したCa2+-ATPaseでも,同様に2種類のvanadate結合が存在した.vanadate結合の解離定数の値は,それぞれ0.1 μM以下と36 μMとなった.vanadate結合部位の数は高親和性,低親和性ともに2.6〜3.6 nmol/mgであった.この値は,Ca2+-ATPaseモル当たり0.5モルずつのvanadate結合部位が存在していることを示唆している.低親和性vanadate結合部位に対するvanadateの結合は,リン酸によって拮抗的に阻害された.Ca2+-ATPaseのリン酸からのリン酸化中間体の形成は,高親和性部位へのvanadate結合によって非拮抗的に阻害された.SRをC12E9で可溶化するとCa2+-ATPaseに対するvanadate結合のvanadate濃度依存性は一相性となり,結合部位の数は7.2 nmol/mg,解離定数は13 μMであった.このことは,可溶化したCa2+-ATPaseでは1種類のvanadate結合部位がCa2+-ATPase 1モル当たり1モル存在することを示唆している.可溶化Ca2+-ATPaseに対するvanadateの結合は,リン酸によって拮抗的に阻害された.可溶化Ca2+-ATPaseをリポソーム中に再構成すると,vanadate結合のvanadate濃度依存性は再び二相性を示した.以上の結果より著者等は,Ca2+-ATPaseにはvanadate結合部位が1種類のみ存在しているが,SR膜中ではCa2+-ATPaseが多量体として存在しているため,分子間相互作用により見掛け上2種類のvanadate結合部位が存在するように見えていると考えられている

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Language：English   Publisher：(公社)日本生化学会  

筋小胞体膜上でのCa2+-ATPaseの分子間相互作用について調べるため,N,N′-(1,4-phenylene)-bismaleimide (PBM)による分子間架橋の条件を検討した.ウサギ骨格筋より調製した筋小胞体を23°C, pH 7.0で種々の時間1 mM PBM処理を行ったのち,SDS-PAGEにより反応産物の分析を行った.その結果,Ca2+-ATPase単量体の量は,分子間架橋により速やかに減少し20分間の処理で半分になった.これと同時に二量体以上の多量体が生成したが,特定の多量体の蓄積は観察されなかった.1 mMのadenyl-5′-imidodiphosphate (AMP-PNP)の存在下でSRをPBM処理すると,分子間架橋の速度は低下し,約60分で単量体の量は半減した.さらにCa2+-ATPase二量体の量は時間とともに増大し,1時間の処理で全Ca2+-ATPaseの40%が二量体として蓄積された.このようなAMP-PNPの,PBMによる分子間架橋への影響は,1モルのCa2+-ATPaseに1モルのFITCが結合すると完全に失われた.0.1 mM vanadate存在下およびCa2+非存在下でSRをPBM処理すると,Ca2+-ATPaseの分子間架橋は強くおさえられた.しかしCa2+とAMP-PNPを同時に加えると,vanadateによる分子間架橋の阻害はまったく見られなかった.AMP-PNP存在下でPBM処理をしたSRをトリプシン消化すると,Ca2+-ATPase二量体(220 kDa)は170 kDaの消化断片を経て130 kDaへ低分子化された.130 kDaは,リン酸化部位を持っていた.さらにトリプシン処理を続けてもCa2+-ATPase二量体からはA1断片(30 kDa)が遊離せずA2断片(20 kDa)のみ遊離することから,PBMによる分子間架橋がA1断片間で起こっていると結論した

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ROCKEFELLER UNIV PRESS  

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Language：English   Publishing type：Research paper, summary (international conference)   Publisher：ROCKEFELLER UNIV PRESS  

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Authorship：Lead author   Language：English  

Multiple and Distinct Effects of Mutations of Tyr122, Glu123, Arg324, and Arg334 Involved in Interactions between the Top Part of Second and Fourth Transmembrane Helices in Sarcoplasmic Reticulum Ca2+-ATPase: CHANGES IN CYTOPLASMIC DOMAIN ORGANIZATION DURING ISOMETRIC TRANSITION OF PHOSPHOENZYME INTERMEDIATE AND SUBSEQUENT Ca2+ RELEASE

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Sarcoplasmic reticulum Ca2+-ATPase was digested with proteinase K, V8 protease and trypsin in the absence of Ca2+. Unphosphorylated enzyme was rapidly degraded. In contrast, ADP-insensitive phosphoenzyme formed with Pi and phosphorylated state analogues produced by the binding of F- or orthovanadate, were almost completely resistant to the proteolysis except for tryptic cleavage at the T1 site (Arg(505)). The results indicate that the phosphoenzyme and its analogues have a very compact form in the cytoplasmic region, being consistent with large domain motions (gathering of three cytoplasmic domains). Results further show that the structure of the enzyme with bound decavanadate is very similar to ADP-insensitive phosphoenzyme. Thapsigargin did not affect the changes in digestion time course induced by the formation of the phosphorylated state analogues. (C) 2001 Federation of European Biochemical Societies. Published by Elsevier Science B.V. All rights reserved.

DOI： 10.1016/S0014-5793(01)02111-1

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Language：English   Publisher：ELSEVIER SCIENCE BV  

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Language：English   Publisher：AMER SOC BIOCHEMISTRY MOLECULAR BIOLOGY INC  

Amino acid residues in the NH2-terminal region (Glu(2) - Ala(14)) of adult fast twitch skeletal muscle sarcoplasmic reticulum Ca2+-ATPase (SERCA1a) were deleted or substituted, and the mutants were expressed in COS-1 cells. Deletion of any single residue in the Ala(3)-Ser(6) region or deletion of two or more consecutive residues in the Ala(3)-Thr(9) region caused strongly reduced expression. Substitution mutants A4K, A4D, and H5K also showed very low expression levels. Deletion of any single residue in the Ala(3)-Ser(6) region caused only a small decrease in the specific Ca2+ transport rate/mg of SERCA1a protein. In contrast, other mutants showing low expression levels had greatly reduced specific Ca2+ transport rates. In vitro expression experiments indicated that translation, transcription, and integration into the microsomal membranes were not impaired in the mutants that showed very low expression levels in COS-1 cells. Pulse-chase experiments using [S-35]methionine/cysteine labeling of transfected COS-1 cells demonstrated that degradation of the mutants showing low expression levels was substantially faster than that of the wild type. Lactacystin, a specific inhibitor of proteasome, inhibited the degradation accelerated by single-residue deletion of Ala(3). These results suggest that the NH2-terminal region (Ala(3)-Thr(9)) of SERCA1a is sensitive to the endoplasmic reticulum-mediated quality control and is thus critical for either correct folding of the SERCA1a protein or stabilization of the correctly folded SERCA1a protein or both.

DOI： 10.1074/jbc.274.34.23910

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Language：English   Publisher：ELSEVIER SCIENCE BV  

Arg(198) of sarcoplasmic reticulum Ca2+-ATPase was substituted with lysine, glutamine, glutamic acid, alanine, and isoleucine by site-directed mutagenesis. Kinetic analysis was performed with microsomal membranes isolated from COS-I cells which were transfected,vith the mutated cDNAs. The rate of dephosphorylation of the ADP-insensitive phosphoenzyme was determined by first phosphorylating the Ca2+-ATPase with P-32(i) and then diluting the sample with non-radioactive P-i. This rate mas reduced substantially in the mutant R198Q, more strongly in the mutants R198A and R198I, and most strongly in the mutant R198E, but to a much lesser extent in R198K. The reduction in the rate of dephosphorylation was consistent with the observed decrease in the turnover rate of the Ca2+-ATPase accompanied by the steady-state accumulation of the ADP-insensitive phosphoenzyme formed from ATP. These results indicate that the positive charge and high hydrophilicity. of Arg(198) are critical for rapid hydrolysis of the ADP-insensitive phosphoenzyme. (C) 1999 Federation of European Biochemical Societies.

DOI： 10.1016/S0014-5793(99)00027-7

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DOI： 10.1016/S0014-5793(99)00027-7

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Language：Japanese  

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DOI： 10.1074/jbc.271.46.28933

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Language：English   Publishing type：Book review, literature introduction, etc.   Publisher：Kluwer Academic Publishers-Plenum Publishers  

Changes in the fluoresence of N-acetyl-N′-(5-sulfo-1-naphthyl)ethylenediamine (EDANS), being attached to Cys-674 of sarcoplasmic reticulum Ca2+-ATPase without affecting the catalytic activity, as well as changes in the intrinsic tryptophan fluorescence were followed throughout the catalytic cycle by the steady-state measurements and the stopped-flow spectrofluorometry. EDANS-fluorescence changes reflect conformational changes near the ATP binding site in the cytoplasmic domain, while tryptophan-fluorescence changes most probably reflect conformational changes in or near the transmembrane domain in which the Ca2+ binding sites are located. Formation of the phosphoenzyme intermediates (EP) was also followed by the continuous flow-rapid quenching method. The kinetic analysis of EDANS-fluorescence changes and EP formation revealed that, when ATP is added to the calcium-activated enzyme, conformational changes in the ATP binding site occur in three successive reaction steps
conformational change in the calcium enzyme substrate complex, formation of ADP-sensitive EP, and transition of ADP-sensitive EP to ADP-insensitive EP. In contrast, the ATP-induced tryptophan-fluorescence changes occur only in the latter two steps. Thus, we conclude that conformational changes in the ATP binding site in the cytoplasmic domain are transmitted to the Ca2+-binding sites in the transmembrane domain in these latter two steps. © 1995 Plenum Publishing Corporation.

DOI： 10.1007/BF01788364

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Language：English   Publisher：OXFORD UNIV PRESS  

The sarcoplasmic reticulum (SR) of rabbit skeletal muscle was irradiated with ultraviolet light (UV) in the presence of vanadate plus 2 mM EGTA, 10 mM MgCl2, 20% DMSO, and 50 mM PIPES (pH 6.5) at room temperature. In the presence of 100 mu M vanadate, the Ca2+ uptake activity of SR rapidly decreased and was almost lost in 20 min. The activity was inhibited as a function of vanadate concentration with an apparent K-i of about 20 mu M. On the other hand, Ca2+-dependent ATP hydrolytic activity as well as phosphoenzyme (EP) formation activity decreased very slowly, and more than 50% of these activities remained 20 min after initiation of the vanadate-UV treatment. Half inhibition of these activities required about 100 mu M vanadate. The loss of the relationship between Ca2+-uptake and ATPase reaction was found to be mainly caused by an increase in the Ca2+ permeability of the SR membrane, which was raised by increasing the vanadate concentration or UV irradiation time in a manner similar to that observed for the Ca2+ uptake. No rise in Ca2+ permeability occurred in liposomes reconstituted from SE lipid when they were irradiated with UV in the presence of 100 mu M vanadate. When the vanadate-UV-treated SR was allowed to react with fluoral-P (4-amino-3-penten-2-one), an indicator of aldehyde, and the membrane proteins were separated by HPLC in the presence of SDS, the fluorescent probe was found to be closely associated with the Ca2+-ATPase fraction. Furthermore, the amount of fluoral-P incorporation into the Ca2+-ATPase increased with UV irradiation time in the presence of vanadate and reached the maximum level at 100 min. About 7-8 nmol of fluoral-P was bound to 1 mg of Ca2+-ATPase at the steady level. These results suggest that when SR is incubated with vanadate under UV irradiation, specific residues in Ca2+ ATPase are oxidized to form aldehyde, which causes an increase in the Ca2+ permeability of the SR membrane.

DOI： 10.1093/jb/117.2.324

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Language：English   Publisher：JAPANESE BIOCHEMICAL SOC  

In the preceding paper, we suggested that 1 mol Ca2+-ATPase of sarcoplasmic reticulum (SR) contains 0.5 mol of high-affinity vanadate binding sites as well as 0.5 mol of low-affinity vanadate binding sites [Yamasaki, K. & Yamamoto, T. (1991) J. Biochem. 110, 915-921]. In the present study, we examined the effects of vanadate binding to the high- and low-affinity sites upon phosphorylation of the enzyme by inorganic phosphate (P(i)). When vanadate was added to the reaction medium in which the Ca2+-ATPase had been phosphorylated by P(i) in the absence of Ca2+, the steady-state level of phosphoenzyme (E2P) decreased due to inhibition of its formation. The decrease of E2P after addition of vanadate exhibited biphasic kinetics consisting of an initial fast decay process followed by a slower first-order decay process. The size of the fast E2P decay, which was estimated by extrapolating the slow phase decay to time 0, varied depending on the vanadate concentration with a dissociation constant of 17 muM, and reached maximum at 50 muM vanadate. The maximum value of the fast E2P decay was almost equal to the initial E2P level. The initial fast decay of E2P was competitively prevented by P(i) with a dissociation constant of 7.4 mM, which was very close to K(m) for the E2P formation under similar conditions. These observations suggested that vanadate inhibits E2P formation by competition with P(i) at a phosphorylation site on the Ca2+-ATPase. The slow first-order decay of E2P corresponded well to the vanadate binding to the high-affinity site of the Ca2+-ATPase. When SR was incubated with 5 muM vanadate prior to the phosphorylation, the amount of E2P decreased to below 5% of the initial level in 10 min. Since vanadate binding to the low-affinity site was negligibly small at this concentration, the inhibition of E2P formation was considered to be caused by vanadate binding to the high-affinity site. P(i) also interrupted the slow decay of E2P which was induced by vanadate binding to the high-affinity site. The apparent dissociation constant of P(i) binding under this condition was estimated to be about 0.4 mM which is much lower than the K(m) value for the E2P formation. Based on these results, we discuss the involvement of dimeric interaction of the Ca2+-ATPase in the E2P formation.

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Language：English   Publisher：OXFORD UNIV PRESS  

The binding of vanadate to isolated sarcoplasmic reticulum (SR) membranes was measured colorimetrically by equilibrium sedimentation and ion exchange column filtration. The concentration dependence of vanadate binding exhibited a biphasic curve with two phases of equal amplitude. A similar biphasic curve of the vanadate dependence was observed with the purified Ca2+-ATPase prepared by deoxycholate extraction. Sites of vanadate binding could be classified into two distinct species based on apparent affinity; the high-affinity binding sites have a dissociation constant below 0.1-mu-M, and the low-affinity sites one of 36-mu-M. The maximum amount of vanadate bound to each of the high- or low-affinity sites was estimated to be 2.6-3.6 nmol/mg SR protein, which corresponds to approximately 0.5 mol of vanadate bound per mol of Ca2+-ATPase. These results indicate that 1 mol of Ca2+-ATPase contains 0.5 mol of high-affinity vanadate-binding sites as well as 0.5 mol of low-affinity vanadate-binding sites. Vanadate binding to the low-affinity sites was competitively inhibited by inorganic phosphate, while vanadate binding to the high-affinity sites resulted in a non-competitive inhibition of the phosphoenzyme formation from inorganic phosphate. When SR membrane were solubilized with polyoxyethylene-9-laurylether (C12E9), the vanadate binding exhibited a monophasic concentration dependency curve with a dissociation constant of 13-mu-M. The number of vanadate-binding sites was estimated to be 7.2 nmol/mg SR protein which represents about 1 mol of site per mol of Ca2+-ATPase. Vanadate binding to the solubilized Ca2+-ATPase was competitively inhibited by inorganic phosphate. When the detergent was removed to reconstitute SR membrane, vanadate binding again exhibited a biphasic concentration dependency curve. These results indicate that interactions between the ATPase molecules in intact SR membranes may involve the cooperative binding of vanadate to the enzyme.

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Language：English   Publisher：JAPAN BIOCHEMICAL SOC  

Web of Science

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Language：English   Publisher：The Japanese Biochemical Society  

The functional significance of the molecular interaction of Ca<SUP>2+</SUP>-ATPase in the sarcoplasmic reticulum (SR) membrane was examined using intermolecular cross-linking of Ca<SUP>2+</SUP>-ATPase with <I>N, N</I>'-(1, 4-phenylene) bismaleimide (PBM). When SR vesicles were allowed to react with 1mM PBM at pH 7 and 23°C for various intervals and subjected to SDS-PAGE, the amount of the major band of monomeric ATPase decreased with a half life of about 20min. Higher orders of oligomers were concurrently formed without accumulation of any particular species of oligomer. When SR vesicles were allowed to react with 1 mM PBM in the presence of 1 mM adenyl-5'-imidodiphosphate (AMP-PNP), the rate of oligomerization was markedly reduced and the amount of dimeric Ca<SUP>2+</SUP>-ATPase increased with time. After 1h, more than 40% of the Ca<SUP>2+</SUP>-ATPase had accumulated in the dimeric form. When 1 mol of fluorescein isothiocyanate (FITC) was bound per mol of ATPase, the effects of AMP-PNP on the cross-linking with PBM were completely abolished. When SR vesicles were treated with PBM in the presence of 0.1 mM vanadate in Ca<SUP>2+</SUP> free medium, the oligomerization of the Ca<SUP>2+</SUP>-ATPase by PBM was strongly inhibited. The vanadate effect on the cross-link formation was completely removed by the presence of Ca<SUP>2+</SUP> and AMP-PNP in the reaction medium. When SR vesicles were pretreated with PBM in the presence of AMP-PNP and digested with trypsin for a short time, the dimeric ATPase was degraded to a peptide with an apparent molecular mass of about 170kDa. Further digestion resulted in degradation to a 130 kDa peptide. As they were phosphorylated by [γ-<SUP>32</SUP>P] ATP in the presence of Ca<SUP>2+</SUP>, these peptides contained the subfragment A. In addition, the SDS-PAGE pattern of the dimeric ATPase after the prolonged digestion lacked subfragment A<SUB>1</SUB>, suggesting that ATPase molecules were cross-linked by PBM through an SH group in the A<SUB>1</SUB> region of this enzyme.

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Event date： 2022.12

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