EFFECT OF LIGANDS ON ROTATIONAL MOBILITY OF NA-ATPASE(,K+)

The phosphorescence anisotropy of eosin-5'-isothiocyanate-labeled duck salt gland Na+,K+-ATPase was measured. The initial anisotropy was 0.2 35 +/- 0.015 at room temperature and did not depend on the conformatio n of the enzyme (sodium or potassium). The phosphorescence anisotropy decay curve was well fitted with two exponentials plus a residual term , the fast component being presumed to describe the mobility of functi onal units of the enzyme (protomers) and the slow component that of ol igomers. In the presence of ligands modifying the conformational state of Na+,K+-ATPase (sodium, potassium, ATP), the fast component of the experimental curve was not changed, whereas the slow component changed substantially. Comparison of the rotational mobility of the labeled e nzyme in the presence of ligands inducing different stages of the hydr olytic cycle suggests that interprotomer interactions are changed duri ng ATP hydrolysis: the share of large associates is enhanced when pota ssium interacts with the enzyme, while the mobility of oligomers is st rongly increased after ATP binding. After addition of 2% nonionic dete rgent C(12)E(9), the initial phosphorescence anisotropy is decreased t o 0.1 and the residual term disappears while the curve remains biexpon ential. The difference between the rotational correlation time for the enzyme measured in the presence of sodium or potassium is decreased. The difference in the protomer/oligomer ratio between potassium or sod ium containing medium becomes insignificant. This means that in the pr esence of detergent high molecular weight aggregates of the enzyme are solubilized, the mobility of both protomeric and oligomeric forms of the ATPase is increased, and the difference in mobility between sodium and potassium conformers disappears.