Binding of Adenosine Diphosphate to Reaction Intermediates in the Na+, K+Dependent ATPase from Porcine Kidney1

Abstract

Na⁺, K⁺-dependent ATPase [EC 3.6.1.3] was partially purified from porcine kidney by the method of Lane et al. with slight modifications. The properties of partial reactions of the enzyme were compared with those of the Nal-treated enzyme from bovine brain, which had been used in our previous studies. The following results were obtained: (1) no difference was observed in the elementary processes which constituted these two ATPase reactions, (2) but the affinity of the porcine kidney enzyme for K⁺ ions was about four times that of the porcine brain enzyme. On the basis of these results, the amount of ADP bound to the enzyme from porcine kidney was measured during ATP hydrolysis in the presence of 4 mM MgCl₂, 100 mM NaCl, and 100 mM Tris-HCl at pH 8.5, using sufficient amounts of creatine kinase (CK) [EC 2.7.3.2] and creatine phosphate (CP) to regenerate [¹⁴C]ATP from free [¹⁴UC]ADP. The amounts of ADP bound to the enzyme were determined by separating the nucleotides using TLC after stopping the reaction with TCA. The following results were obtained. We concluded that a phosphorylated intermediate with bound ADP, $EPADP$, exists during ATP hydrolysis, and that an enzyme-ADP complex, E^ADP, is one of the main reaction intermediates in high concentrations of KCl.