The Kinetic properties of the [³H] ADP-ATP exchange reaction catalyzed by Na⁺, K⁺-dependent ATPase [EC 3. 6. 1. 3] were investigated, using Nal-treated microsomes from bovine brain, and the following results were obtained.
1. The rates of the Na⁺-dependent exchange reaction in the steady state were measured in a solution containing 45μM free Mg²⁺, 100mM NaCl, 80 μM ATP, and 160 μM ADP at pH 6.5 and 4-5°. The rate and amount of decrease in phosphorylated intermediate on adding ADP, i.e., the amount of ADP-sensitive EP, were measured while varying one of the reaction param-eters and fixing the others mentioned above. Plots of the exchange rate and the amount of ADP-sensitive EP against the logarithm of free Mg²⁺ concentration gave bell-shaped curves with maximum values at 50-60 μM free Mg²⁺. Plots of the exchange rate and the amount of ADP-sensitive EP against pH also gave bell-shaped curves with maximum values at pH 6.9-7. They both increased with increase in the concentration of NaCl to maximum values at 150-200 mM NaCl, and then decreased rapidly with increase in the NaCl concentration above 200 mM The dependences of the exchange rate and the amount of ADP-sensitive EP on the concentration of ADP followed the Michaelis-Menten equation, and the Michaelis constants, Km, for both were 43 μM. The dependence of the exchange rate on the ATP concentration also followed the Michaelis-Menten equation, and the Km value was 30 μM. The amount of ADP-sensitive EP increased with increase in the ATP concentration, and reached a maximum value at about 5 μM ATP.
2. The Na⁺-dependent [³H] ADP-ATP exchange reaction was started by adding [3H]ADP to EP at low Mg²⁺ concentration. The reaction consisted of a rapid initial phase and a slow steady phase. The amount of [³H] ATP formed during the rapid initial phase, i.e. the size of the ATP burst, was equal to that of ADP-sensitive EP, and was proportional to the rate in the steady state. At high Mg²⁺ concentration, the rate of Na⁺-dependent exchange in the steady state was almost zero, and EP did not show any ADP sensitivity. However, rapid formation of [³H]ATP was observed in the pre-steady state, and the size of the ATP burst increased with increase in the KCl concentration.
From these findings, we concluded that an enzyme-ATP complex (E₂ATP) formed at low Mg²⁺ concentration is in equilibrium with EP+ADP, that the rate-limiting step for the exchange reaction is the release of ATP from the enzyme-ATP complex, that the ADP-insensitive EP_??_produced at high Mg²⁺ concentration is in equilibrium with the enzyme-ATP complex, and that the equilibrium shifts towards the enzyme-ATP complex on adding KCl. Actually, the ratio of the size of the ATP burst to the amount of EP was equal to the reciprocal of the equilibrium constant of step_??_determined by a method previously reported by us.