ausblenden:
Schlagwörter:
Titration Curve; Conformational State; Adenosine Triphosphatase; Fluorescent State; High Affinity Binding Site
Zusammenfassung:
The transfer of Na+ and K+ across membranes by the Na+/K+-pump is coupled to the transition between two conformational states of the enzyme denoted E1 and E2 (1,12). In the E1 state the enzyme binds preferentially Na+, while in the E2 state it binds preferentially K+. Fluorescent probes like fluorescein-isothiocyanate (FITC) covalently bound to the protein (8) or potential-sensitive styryl dyes such as RH 421 incorporated into the membrane (2,9,13) change their fluorescent properties in the presence of Na+ or K+, and thus offer the possibility of studying the binding of these ions and conformational transitions of the enzyme spectroscopically. FITC-labeled Na+/K+-ATPase is strongly fluorescent in the presence of high Na+ concentrations, but exhibits a much lower fluorescence if K+ or one of its congeners is bound. Accordingly the two fluorescence emission intensity states have been assigned to the conformational states E1 and E2 (6,8,11). Na+ binding alone can also be studied by means of the fluorescence changes of RH 421, which has been used to study phosphorylation of the native enzyme by ATP in the presence of Na+ and Mg++ under conditions where no K+ is present (2,9,13). Titrations of the enzyme with Na+ in the presence of these dyes showed that the two different fluorescent labels report binding of Na+ to two different sites (4). The implications of these findings to the assignment of fluorescent states to conformations of the enzyme will be discussed here in more detail.
