Biochimica et Biophysica Acta, 307 (1973) 386-398 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands BBA 76303 OUABAIN-RECEPTOR INTERACTIONS IN (Na++ K+)-ATPase PREPARA TIONS FROM DIFFERENT TISSUES AND SPECIES DETERMINATION OF KINETIC CONSTANTS AND DISSOCIATIOb CONSTANTS ERLAND ERDMANN and WILHELM SCHONER lnstitut fiir Biochemie und Endokrinologie, Universitdt Giessen, Frankfurterstr. 112, D-63-Giesset (Germany) (Received December 4th, 1972) SUMMARY 1. [3H]Ouabain-receptor association in (Na++K+)-ATPase preparation, from beef (kidney, brain, and heart), from dog (heart), and guinea pig (kidneyl in the presence of MgE++P~ is a bimolecular reaction, while the dissociation i., strictly a first-order process. For the ouabain-receptor subunit of the enzyme frorr beef kidney, rate constants at 37 °C for the association are 1.85.104 M -1 .s -1 ant for the dissociation 0.94.10-4.s -1. A dissociation constant of 0.47.10-8 M wa~, calculated from these rate constants and is determined from a Scatchard plot undel equilibrium conditions. The dissociation constant determined in the presence ol Na +, Mg 2+, and ATP was 1.1 • l0 -8 M. Scatchard plots indicate one single type ol receptor. The ouabain-receptor dissociation constants varied with the tissue sourc~ of (Na++ K+)-ATPase. The considerable variation of the dissociation constant ir guinea pig kidney (1.6.10 -7 M) was preferentially caused by changes of the dissocia- tion rate constants. 2. From the dissociation constant at 37 °C a AG ° of -11 kcal.mole -~ L, calculated for the ouabain-receptor subunit of the enzymes from beef kidney brain. and heart and from dog heart. For the beef kidney enzyme AH ° is -5 kcal-mole -1 and AS ° 21 cal .mole -1 .deg -1. For the guinea pig subunit thermodynamic calcula- tions reveal a AG ° of -9 kcal.mole -1, a AH ° of - 11 kcal.mole -2, and a AS ° of - 4 cal. mole- J •deg- L. 3. Dissociation constants of the ouabain-receptor complex are 10- to 100- fold lower than the ouabain concentrations necessary for half-maximal inhibition of the (Na++K+)-ATPase. The stoichiometry of [3H]ouabain-binding sites: phosphorylated intermediate varied between 4 (guinea pig kidney) and 1 (beef enzymes). It is assumed that the ouabain receptor and the ATP hydrolysing subunit are not tightly linked. INTRODUCTION (Na++K+)-activated ATPase (ATP phosphohydrolase, EC 3.6.1.3), which is closely related to active Na + transport, is specifically inhibited by cardiac glyco-