Properties of a Phosphorylated Protein as a Reaction Intermediate of Na+-K+ Sensitive ATPase*

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Abstract
1. The dependence of Na+-K+ sensitive ATPase [EC 3.6.1.3] activity on temperature was investigated between 0° and 40°C. The Arrhenius plot of the total activity of a system containing Mg++, Na+ and K+ ions consisted of two linear portions which intersected at about 25°C. The experimental activation energies calculated from the plot were 29.5 kcal per mole in the low temperature range (0°–25°C) and 18.4 kcal per mole in the high temperature range (25°–40°C). The Arrhenius plot of the system containing Mg++ and Na+ ions was linear with an activation energy of 16.3 kcal per mole over the entire temperature range (0°–40°C). 2. The dependence of ATPase activity at the steady state on the ATP concentration was measured at 10°C. At high ATP concentrations, the activity of the system containing Mg++, Na+ and K+ ions (Mg-Na-K ATPase activity) was much higher than that of the system containing Mg++ and Na+ ions (Mg-Na ATPase activity). But the former decreased rapidly with reduction in the ATP concentration, becoming much lower than the latter, and reached the level of the activity of the system containing Mg++ alone (Mg ATPase activity) at extremely low concentrations of ATP. 3. The Na+-dependent ATPase activity at the steady state (the difference between Mg-Na ATPase activity and Mg ATPase activity) and the amount of Na+-dependent P32-incorporation from ATP32 (γ-P32) into the enzyme were measured simultaneously in the presence of various concentrations of ATP (0.33–10 μM) at 0°C. The amount of P32-in-corporation was 1.23 moles per 107g. of protein at 10 μM ATP and diminished with decrease in the concentration of ATP. The rate of ATPase activity was always lower than the initial rate of P32-incorporation. The difference between these two rates became greater as the ATP concentration became lower. Furthermore, the apparent Michaelis constant of the ATPase activity (1.8 μM) was very different from that of P32-incorporation (0.31 μM). From these results, it was concluded that the P32-incorporation observed at 0°C cannot be due to an intermediate of the Na+-dependent ATPase reaction at this temperature. 4. The rate of the ATPase reaction at the steady state (u) and the amount of P32-incorporation ([E∼P]) were measured simultaneously in the presence of various concentrations of KC1 (0–28mM) and a fixed concentration of NaCl (140 mM) at 37°C. The following two relations were observed: Here, V and A are constants and s indicates the molar concentration of phosphorylation site. Next, the values of u and [E∼P] were measured simultaneously in the presence of various concentrations of NaCl (0–140 mM) and a fixed concentration of KC1 (1 mM) at 37°C. The value of v was found to be in proportion to [E∼P] over a wide range of NaCl concentrations. These facts can be explained quantitatively if phosphory-lated enzyme (E∼P) is an intermediate of the ATPase reaction at 37°C and the ATPase reaction occurs according to the following mechanism: 5. By comparing the properties of Na+-K+ sensitive ATPase with those of myosin B-ATPase, the following new reaction mechanism is suggested: the Na+-K+ sensitive ATPase is a double headed enzyme, which can hydrolyze ATP by two routes, simple hydrolysis through the Michaelis complex and hydrolysis via phosphorylated enzyme. The former is the main pathway of ATP-hydrolysis in the presence of Na+ and K+ at relatively low temperatures or in the presence of Na+ alone over the entire range of temperatures, while the latter is the main pathway in the presence of Na+ and K+ at relatively high temperatures.