Time-resolved phosphorescence measurement of conformational transitions in cation transport Na+ K+-ATPase

摘要

Abstract: LU$/, K$+$PLU$/)-activated adenosine triphosphatase in a crude microsomal membrane preparation isolated from duck salt glands was covalently labelled with the phosphorescent probe, eosin-5'-isothiocyanate, and the labelled protein transformed into either of its putative conformeric states, designed E$-1$/ and E$-2$/ by addition of Na$+$PLU$/ and K$+$PLU$/ respectively. Samples in the different conformeric states were excited into the triplet state by a short laser pulse from a frequency double Nd:YAG laser. The resulting time-resolved phosphorescence depolarization was then measured by a 'T' format phosphorimeter, and the anisotropy, which is in part molecular conformation-dependent, was calculated. The initial experimental results showed that shifting the enzyme into an E$-2$/ conformer results in an increase in rotational correlation time, while conversely shifting the enzyme into its E$-1$/ conformer reduces its rotational correlation time. The labelled microsomal preparation which was isolated free of monovalent cations yielded a rotational correlation time intermediate between the values obtained from E$-1$/ and E$-2$/ conformers. The ratio of the rotational correlation times of the enzyme in E$-2$/ and E$-1$/ conformers was found to be about 2 at 0$DGR@C and steadily decreased as the temperature was increased. This suggests that the ATPase in its E$-2$/ conformation rotates at a slower rate than that of its E$-1$/ conformer. The relative contribution of the conformational transition and radius of rotation of the protein about its axis perpendicular to the plane of the membrane to the observed phosphorescence anisotropy has yet to be determined.!22