title: Characterization of calcium, nucleotide, phosphate, and vanadate bound states by derivatization of sarcoplasmic reticulum ATPase with ThioGlo1.
abstract: Sarcoplasmic reticulum vesicles were incubated with the maleimide-directed probe ThioGlo1, resulting in ATPase inactivation. Reacted ThioGlo1, revealed by its enhanced fluorescence, was found to be associated with the cytosolic but not with the membrane-bound region of the ATPase. The dependence of inactivation on ThioGlo1 concentration suggests derivatization of approximately four residues per ATPase, of which Cys(364), Cys(498), and Cys(636) were identified in prominently fluorescent peptide fragments. These cysteines reside within the phosphorylation and nucleotide-binding region of the ATPase. Accordingly, protection is observed in the presence of ATP, 2'(3')-O-(2,4,6-trinitrophenyl)adenosine 5'-diphosphate (TNP-AMP), or an fluoroisothiocyanate label of Lys(515). Furthermore, protection is observed in the presence of vanadate (or decavanadate), but not in the presence of phosphate. Labeling occurs equally well in the presence or in the absence of Ca(2+) and thapsigargin, excluding a role of the E1-to-E2 transition in the protective effect of vanadate. It is concluded that protection by vanadate is due to formation of a pentacoordinated orthovanadate complex at the phosphorylation site, corresponding to a stable transition state analog of the phosphorylation reaction, with intermediate characteristics of the EP1 and EP2 states. The lack of protection by phosphate is attributed to instability of its complex with the enzyme (EP2). These findings are discussed with respect to different structural images obtained from diffraction studies of ATPase in the presence or in the absence of Ca(2+) and/or decavanadate (Ogawa et al., 1998, Biophys. J. 75:41-52).
authors.affiliation: Department of Biochemistry and Molecular Biology, University of Maryland School of Medicine Baltimore, Maryland 21201, USA.
authors.forename: S
authors.initials: S
authors.lastname: Hua
authors.forename: D
authors.initials: D
authors.lastname: Fabris
authors.forename: G
authors.initials: G
authors.lastname: Inesi
mesh_terms: D000249:Adenosine Monophosphate / Q000031:analogs & derivatives / Q000378:metabolism; D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism*; D000252:Calcium-Transporting ATPases / Q000037:antagonists & inhibitors / Q000737:chemistry* / Q000378:metabolism*; D003545:Cysteine / Q000378:metabolism; D005456:Fluorescent Dyes / Q000737:chemistry / Q000378:metabolism; D006863:Hydrogen-Ion Concentration; D008301:Maleimides / Q000378:metabolism; D008958:Models, Molecular; D009711:Nucleotides / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D010710:Phosphates / Q000378:metabolism*; D010766:Phosphorylation; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013696:Temperature; D019284:Thapsigargin / Q000494:pharmacology; D013997:Time Factors; D014638:Vanadates / Q000378:metabolism*
chemical_list: D005456:Fluorescent Dyes; D008301:Maleimides; D009711:Nucleotides; D010446:Peptide Fragments; D010710:Phosphates; D014638:Vanadates; D000249:Adenosine Monophosphate; D019284:Thapsigargin; C036744:2',3'-O (2,4,6-trinitrocyclohexadienylidine)adenosine 5'-monophosphate; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D003545:Cysteine; D002118:Calcium