Effects of Mg(2+) on activation of the (Na(+) + K(+)-dependent ATPase by Na(+1).

The effects of MgCl₂ on Na activation of three different enzymatic reactions catalyzed by a rat brain (Na + K)-dependent ATPase (adenosine 5′-triphosphatase) were studied. For the Na⁺-dependent ATPase reaction measured with 6 μm ATP, the K_0.5 for Na increased from 0.4 to 1.7 mm as the MgCl₂ concentration was raised from 50 to 2000 μm; the half-maximal effect occurred at a free Mg²⁺ concentration near 0.8 mm. By contrast, with 3 mm ATP and 3 mm MgCl₂ the K_0.5 for Na was again 0.4 mm, but further addition of 2 mm MgCl₂ then had little effect on the K_0.5 for Na. For the Na-dependent phosphorylation of the enzyme, measured with 6 μm ATP, the K_0.5 for Na increased similarly, from 0.2 to 0.8 mM, as the MgCl₂ concentration was raised from 50 to 2000 μm, but for the (Na + K)-dependent ATPase reaction the K_0.5 for Na was 13 mm and increased by only one-third as the MgCl₂ concentration was raised. The K_0.5 for K was also little affected by changes in MgCl₂ concentration. Finally, with 3 mm ATP and 3 mm MgCl₂ the K_0.5 for Na in the (Na + K)-dependent ATPase reaction decreased to 5 mm. These observations are considered in terms of an enzyme having high-affinity and low-affinity substrate sites, with occupancy of the low-affinity sites modifying Na activation differently, depending both on the specific reaction catalyzed and on whether occupancy is by free Mg²⁺ or by Mg-ATP.