Application of Time-Resolved Phosphorescence Anisotropy Measurement for the Study of Rotational Mobility of Eosin-Labelled Na⁺/K⁺-ATPase and Ca²⁺ ATPase

Abstract

To study the rotational mobility of E₁-E₂-type ATPases under different conditions the membrane preparations of Na⁺/K⁺-ATPase from duck salt glands and Ca²⁺-ATPase from rabbit skeletal muscle sarcoplasmic reticulum were specifically labelled at single lysine residue in or near putative ATP-binding site by eosin-5′-isothiocyanate (EITC) (1,4). Samples were excited into the triplet state by a short laser pulse from a frequency-doubled Nd:YAG laser. The resulting time-resolved phosphorescence depolarization was then measured by a “T”-format phosphorimeter and anisotropy was calculated and analyzed using a non-linear exponential function curve fitting method (Marquardt procedure) (5). Incorporation of EITC into Ca²⁺-ATPase protein provides for a complete inhibition of enzyme hydrolytic activity when level of dye incorporation reaches 1 mole per mole Ca²⁺-ATPase; incorporation of the same amount of EITC into Na⁺/K⁺-ATPase preparations provides only 30% inhibition of enzyme activity (data not shown).