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Abstract

The kinetics of the E₂ → E₁ conformational change of unphosphorylated Na⁺⁺-ATPase from rabbit kidney and shark rectal gland were investigated via the stopped-flow technique using the fluorescent label RH421 (pH 7.4, 24 °C). The enzyme was pre-equilibrated in a solution containing 25 mM histidine and 0.1 mM EDTA to stabilize initially the E₂ conformation. When rabbit kidney enzyme was mixed 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 in the case of rabbit kidney enzyme. The experimental behavior could be described by a binding of Na⁺ to the enzyme in the E₂ state with a dissociation constant of 31 ± 7 mM, which induces a subsequent rate-limiting conformational change to the E₁ state. Similar behavior, but with a decreased saturating value of 1/τ, was found when NaCl was replaced by choline chloride. Analogous experiments performed with enzyme from shark rectal gland showed similar effects, but with a significantly 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 that of ATP, in enhancing the rate of the rate-limiting E₂ → E₁ conformational transition by interaction with the E₂ state.