Kinetics of Luminal Proton Binding to the SR Ca-ATPase

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• 作者：Andreas Fibich ; Hans-Jü ; rgen Apell  ; ;   ; ^{h-j.apell@uni-konstanz.de}
• 刊名：Biophysical Journal
• 出版年：2011
• 出版时间：19 October 2011
• 年：2011
• 卷：101
• 期：8
• 页码：1896-1904
• 全文大小：420 K

文摘

An open membrane preparation containing SR Ca-ATPase was prepared from sarcoplasmic-reticulum vesicles to study the ion binding kinetics in the P-E₂ conformation. Because Ca²⁺ 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-E₂ conformation showed that three Ca-ATPase-specific processes could be identified, fast H⁺ binding (¦Ó?<?100?¦Ìs) and pH-insensitive conformational relaxations after the release of the Ca²⁺ ion (¦Ó ?60?ms), and a slow process (¦Ó ?.4?s) whose origin could not be unambiguously revealed. The Ca²⁺-binding affinity in the P-E₂ conformation was reduced with increasing pH, a behavior that can be explained by a reversible transition of the empty P-E₂ 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.