| 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. 1. At 15°C and in the absence of CP all the phosphorylated intermediate (EP) was ADP insensitive. On the other hand, in the presence of CK and CP the amount of ADP bound to the Na+, K+-dependent ATPase in the absence of K+ ions was about one-third of the amount ofEP. 2. The amount of ADP bound to the enzyme decreased slightly with an increase in the KCl concentration to 0.5 mM, but remained almost constant in the KCl concentration range of 1-100 mM. On the other hand, the amount of EP decreased markedly with increasing KCl concentration. Thus, the amount of EP was much smaller than that of ADP bound to the enzyme in 1-100 mM KCl. The rate of the ATPase in the steady state, v0, was almost proportional to the amount of ADP bound to the enzyme in the range of 1-100 mM KC1. 3. However, at 1°C about half of the EP was ADP-sensitive even in the presence of a high concentration of MgCl2, and the amount of ADP-insensitive EP was equal to that of bound ADP in the NaCl concentration range of 10-100 mM. We concluded that a phosphorylated intermediate with bound ADP, EPADP, exists during ATP hydrolysis, and that an enzyme-ADP complex, EADP, is one of the main reaction intermediates in high concentrations of KCl.
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