Stopped-flow kinetic investigations of conformational changes of pig kidney Na+,K+-ATPase.

作者: David J. Kane , Klaus Fendler , Ernst Grell , Ernst Bamberg , Kazuya Taniguchi

DOI: 10.1021/BI970598W

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摘要: The kinetics of Na+-dependent partial reactions the Na+,K+-ATPase were investigated via stopped-flow technique using fluorescent labels RH421 and BIPM. After enzyme is mixed with MgATP, both give almost identical kinetic responses. Under chosen experimental conditions two exponential time functions are necessary to fit data. dominant fast phase, 1/τ1 ≈ 180 s-1 (saturating [ATP] [Na+], pH 7.4 24 °C), attributed phosphorylation a subsequent conformational change (E1ATP(Na+)3 → E2P(Na+)3 + ADP). rate reaction measured by acid quenched-flow was 190 at 100 μM ATP, suggesting that controls signal very (≥600 s-1). optimal 7.5. Na+ concentration dependence showed half-saturation 8−10 mM positive cooperativity involved in occupation t...

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