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Abstract

Upon hydrolysis of ATP the integral membrane protein Na,K-ATPase actively transports the cations Na⁺ and K⁺ across its membrane. According to earlier kinetic studies, this pumping activity against the existing concentration gradient has been characterized in terms of two major conformational states of the enzyme that have been denoted E1 (Na⁺-bound state) and E2 (K⁺-bound state) [1,2]. Since it is difficult to separate the ATP binding process, which is assumed to lead to the formation of E1 in the presence of Na⁺, from the subsequent hydrolytic reaction steps considerable interest has been devoted to the investigation of molecules mimiking the binding of the substrate. A molecule exhibiting such features as well as fluorescence properties that are sensitive to changes in its local environment is eosin Y (Fig. 1), introduced by M. Esmann and J.C. Skou [3]. Up to now binding of the negatively charged eosin Y has been interpreted in terms of an interaction with the enzyme induced primarily by Na⁺. This interaction can be characterized by a time-resolvable fluorescence intensity increase employing the stopped flow technique [4,5]. This intensity increase in the millisecond time range has been attributed to a conformational change leading to the state E1.