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Kinetic Investigations of the Mechanism of the Rate‐Determining Step of the Na + ,K + ‐ATPase Pump Cycle
Author(s) -
CLARKE RONALD J.,
HUMPHREY PAUL A.,
LÜPFERT CHRISTIAN,
APELL HANSJÜRGEN,
CORNELIUS FLEMMING
Publication year - 2003
Publication title -
annals of the new york academy of sciences
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.712
H-Index - 248
eISSN - 1749-6632
pISSN - 0077-8923
DOI - 10.1111/j.1749-6632.2003.tb07154.x
Subject(s) - chemistry , conformational change , reaction rate constant , tris , chloride , enzyme , dissociation (chemistry) , histidine , choline chloride , kinetics , crystallography , analytical chemistry (journal) , biophysics , stereochemistry , chromatography , biochemistry , organic chemistry , physics , quantum mechanics , biology
A bstract : The kinetics of the E 2 → E 1 conformational change of unphosphorylated Na + ,K + ‐ATPase from rabbit kidney were investigated via the stopped‐flow technique using the fluorescent label RH421 (pH 7.4, 24°C). The enzyme was preequilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to initially stabilize the E 2 conformation. On mixing enzyme with NaCl alone, tris‐ATP alone, or NaCl and tris‐ATP simultaneously, a fluorescence decrease was observed. The reciprocal relaxation time, 1/τ, of the fluorescent transient was found to increase with increasing NaCl concentration and reached a saturating value in the presence of 1 mM tris‐ATP of 54 (±3) s −1 . The experimental behavior could be described by a binding of Na + to the enzyme in the E 2 state with a dissociation constant of 31 (±7) mM, which induces a subsequent rate‐limiting conformational change to the E 1 state. Similar behavior, but with a decreased saturating value of 1/τ, was found when NaCl was replaced by choline chloride. Experiments performed with enzyme from shark rectal gland showed similar effects, but with a lower amplitude of the fluorescence change and a higher saturating value of 1/τ for both the NaCl and choline chloride titrations. The results suggest that Na + ions or salt in general play a regulatory role, similar to ATP, in enhancing the rate of the rate‐limiting E 2 → E 1 conformational transition by interaction with the E 2 state.