Radiation inactivation of (Na,K)-ATPase, an enzyme showing multiple radiation-sensitive domains.

Abstract

The radiation inactivation of membrane-bound (Na,K)-ATPase from pig kidney was studied by irradiating lyophilized enzyme preparations in vacuo. Various results were obtained depending upon the function assayed. The apparent target size of (Na,K)-ATPase activity was found to be 264 kDa an the high affinity binding sites for ATP, vanadate, and ouabain (bound in the presence of Na+, Mg2+, and ATP) had an apparent target size of 145 kDa. The binding of ouabain in the presence of Mg2+ alone seemed to depend upon the integrity of a domain with a target size of approximately 100 kDa. Na-ATPase and p-nitrophenyl phosphatase, assayed under a variety of conditions, gave inactivation kinetics that did not conform to classical target theory. All the results have been assembled into a model according to which 1) the membrane-bound enzyme is an (alpha beta)2-dimer; 2) there is radiation energy coupling between all four peptides in a molecule, and 3) each alpha-peptide consists of five discrete and independent radiation-sensitive domains with specific functions in the enzymatic reactions catalyzed by the sodium pump.