Eosin, a fluorescent marker for the high-affinity ATP site of (K+ + H+)-ATPase

Eosin has been used as a fluorescent probe for studying conformational states in (K+ + H+)-ATPase. The eosin fluorescence level is increased by Mg2+ (K0.5 = 0.2 mM). This increase is counteracted by K+ (I0.5 = 1.3 mM) and choline (I0.5 = 17.2 mM) and by ATP. Binding studies with eosin indicate that the increase and decrease in fluorescence is due to changes in binding of eosin to the enzyme. The Mg2+-induced specific binding has a Kd of 0.7 microM and a maximal capacity of 3.5 nmol per mg enzyme, which is equivalent to 2.5 site per phosphorylation site. These experiments and the fact that eosin competitively inhibits (K+ + H+)-ATPase towards ATP, suggest that eosin binds to ATP binding sites.