title: Ca2(+)-dependent conformational change of the ATP-binding site of Ca2(+)-transporting ATPase of sarcoplasmic reticulum as revealed by an alteration of the target-site specificity of adenosine triphosphopyridoxal.
abstract: Adenosinetriphosphopyridoxal (AP3PL) specifically modifies Lys684 of Ca2(+)-ATPase of sarcoplasmic reticulum (SR-ATPase) in the presence of Ca2+, leading to its inactivation (Yamamoto, H. et al. (1988) J. Biochem. 103, 452-457). We have now investigated the effects of AP3PL on SR-ATPase in the absence of Ca2+. Similarly to its action in the presence of Ca2+, AP3PL inhibited the Ca2(+)-transporting activity in a dose-dependent manner in the absence of Ca2+ as well. ATP and ADP protected SR-ATPase against inactivation by this reagent. One mole of AP3PL was bound per mol of SR-ATPase with concomitant loss of the Ca2(+)-transporting activity. Binding of AP3PL to SR-ATPase was prevented by ATP. AP3PL-labeled SR membranes were digested with thermolysin and labeled thermolytic peptides were purified through C18 reversed-phase HPLC. Two major AP3PL-labeled peptides were obtained in approximately 1:1 ratio; one was an octapeptide corresponding to 679-ValGluProSerHisLys*SerLys-686, and the other, a nonapeptide corresponding to 487-PheSerArgAspSerLys*ArgMetSer-495 (Lys* indicates a labeled Lys residue) of SR-ATPase. Lys684 in the former turned out to be the same as the highly specific target of AP3PL in the presence of Ca2+ which was identified previously. The target site specificity of AP3PL thus changed significantly but not entirely on binding of Ca2+ to SR-ATPase. This indicates that the spatial arrangement around the gamma-phosphoryl group of the bound ATP is affected by Ca2+ ions bound at the transport site. It is also likely that Lys492 and Lys684 are situated close together in the ATP binding site of SR-ATPase.
authors.affiliation: Department of Pure and Applied Sciences, College of Arts and Sciences, University of Tokyo.
authors.forename: H
authors.initials: H
authors.lastname: Yamamoto
authors.forename: Y
authors.initials: Y
authors.lastname: Imamura
authors.forename: M
authors.initials: M
authors.lastname: Tagaya
authors.forename: T
authors.initials: T
authors.lastname: Fukui
authors.forename: M
authors.initials: M
authors.lastname: Kawakita
mesh_terms: D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000494:pharmacology; D000595:Amino Acid Sequence; D000818:Animals; D001665:Binding Sites; D002118:Calcium / Q000378:metabolism; D000252:Calcium-Transporting ATPases / Q000378:metabolism*; D006358:Hot Temperature; D008969:Molecular Sequence Data; D010446:Peptide Fragments / Q000302:isolation & purification; D010449:Peptide Mapping; D011485:Protein Binding; D011487:Protein Conformation; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013379:Substrate Specificity
chemical_list: D010446:Peptide Fragments; C049517:adenosine triphosphopyridoxal; D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D002118:Calcium