title: Kinetics of luminal proton binding to the SR Ca-ATPase.
abstract: An open membrane preparation containing SR Ca-ATPase was prepared from sarcoplasmic-reticulum vesicles to study the ion binding kinetics in the P-E(2) conformation. Because Ca(2+) and H(+) binding are electrogenic reactions, fluorescent styryl dyes could be used to determine changes in the binding site occupation in equilibrium titration experiments and time-resolved relaxation processes triggered by a pH jump. By photo release from caged proton the pH of the electrolyte could be decreased in a step of 0.1 pH units by a single ultraviolet-laser flash. Analysis of the pH-jump induced relaxation process in the P-E(2) conformation showed that three Ca-ATPase-specific processes could be identified, fast H(+) binding (τ < 100 μs) and pH-insensitive conformational relaxations after the release of the Ca(2+) ion (τ ∼160 ms), and a slow process (τ ∼3.4 s) whose origin could not be unambiguously revealed. The Ca(2+)-binding affinity in the P-E(2) conformation was reduced with increasing pH, a behavior that can be explained by a reversible transition of the empty P-E(2) state to an inactivated state of the ion pump. All findings are interpreted in the framework of the Post-Albers pump cycle introduced previously, supplemented by an additional transition to an inhibited state of the ion pump.
authors: Department of Biology, University of Konstanz, Konstanz, Germany.
authors: Andreas
authors: A
authors: Fibich
authors: Hans-Jürgen
authors: HJ
authors: Apell
mesh_terms: D000818:Animals; D001665:Binding Sites; D000252:Calcium-Transporting ATPases / Q000737:chemistry / Q000378:metabolism*; D007700:Kinetics; D011485:Protein Binding; D011487:Protein Conformation; D011522:Protons; D011817:Rabbits; D012519:Sarcoplasmic Reticulum / Q000201:enzymology*; D013696:Temperature
chemical_list: D011522:Protons; D000252:Calcium-Transporting ATPases