title: Formation of the stable structural analog of ADP-sensitive phosphoenzyme of Ca2+-ATPase with occluded Ca2+ by beryllium fluoride: structural changes during phosphorylation and isomerization.
abstract: As a stable analog for ADP-sensitive phosphorylated intermediate of sarcoplasmic reticulum Ca(2+)-ATPase E1PCa(2).Mg, a complex of E1Ca(2).BeF(x), was successfully developed by addition of beryllium fluoride and Mg(2+) to the Ca(2+)-bound state, E1Ca(2). In E1Ca(2).BeF(x), most probably E1Ca(2).BeF(3)(-), two Ca(2+) are occluded at high affinity transport sites, its formation required Mg(2+) binding at the catalytic site, and ADP decomposed it to E1Ca(2), as in E1PCa(2).Mg. Organization of cytoplasmic domains in E1Ca(2).BeF(x) was revealed to be intermediate between those in E1Ca(2).AlF(4)(-) ADP (transition state of E1PCa(2) formation) and E2.BeF(3)(-).(ADP-insensitive phosphorylated intermediate E2P.Mg). Trinitrophenyl-AMP (TNP-AMP) formed a very fluorescent (superfluorescent) complex with E1Ca(2).BeF(x) in contrast to no superfluorescence of TNP-AMP bound to E1Ca(2).AlF(x). E1Ca(2).BeF(x) with bound TNP-AMP slowly decayed to E1Ca(2), being distinct from the superfluorescent complex of TNP-AMP with E2.BeF(3)(-), which was stable. Tryptophan fluorescence revealed that the transmembrane structure of E1Ca(2).BeF(x) mimics E1PCa(2).Mg, and between those of E1Ca(2).AlF(4)(-).ADP and E2.BeF(3)(-). E1Ca(2).BeF(x) at low 50-100 microm Ca(2+) was converted slowly to E2.BeF(3)(-) releasing Ca(2+), mimicking E1PCa(2).Mg --> E2P.Mg + 2Ca(2+). Ca(2+) replacement of Mg(2+) at the catalytic site at approximately millimolar high Ca(2+) decomposed E1Ca(2).BeF(x) to E1Ca(2). Notably, E1Ca(2).BeF(x) was perfectly stabilized for at least 12 days by 0.7 mm lumenal Ca(2+) with 15 mm Mg(2+). Also, stable E1Ca(2).BeF(x) was produced from E2.BeF(3)(-) at 0.7 mm lumenal Ca(2+) by binding two Ca(2+) to lumenally oriented low affinity transport sites, as mimicking the reverse conversion E2P. Mg + 2Ca(2+) --> E1PCa(2).Mg.
authors: Department of Biochemistry, Asahikawa Medical College, Asahikawa 078-8510, Japan.
authors: Stefania
authors: S
authors: Danko
authors: Takashi
authors: T
authors: Daiho
authors: Kazuo
authors: K
authors: Yamasaki
authors: Xiaoyu
authors: X
authors: Liu
authors: Hiroshi
authors: H
authors: Suzuki
mesh_terms: D017607:Aluminum Compounds / Q000737:chemistry; D000818:Animals; D001608:Beryllium / Q000737:chemistry*; D002118:Calcium / Q000737:chemistry* / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000737:chemistry* / Q000378:metabolism*; D005459:Fluorides / Q000737:chemistry*; D008954:Models, Biological; D010766:Phosphorylation; D011485:Protein Binding; D017433:Protein Structure, Secondary; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism
chemical_list: D017607:Aluminum Compounds; C050992:tetrafluoroaluminate; C035982:beryllium fluoride; D053498:Sarcoplasmic Reticulum Calcium-Transporting ATPases; D000252:Calcium-Transporting ATPases; D001608:Beryllium; D005459:Fluorides; D002118:Calcium