Mechanistic basis for kinetic differences between the rat alpha 1, alpha 2, and alpha 3 isoforms of the Na,K-ATPase

Previous studies showed that the alpha1, alpha2, and alpha3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities f or the ligands ATP, Na+, and K+. For the rat isoforms transfected into HeLa cells, K ' (ATP) for ATP binding at its low affinity site is lower for a2 and a3 compared with al; relative to al and a2, a3 has a higher K ' (Na) an d lower K ' (K) (Jewell, E. A., and Lingrel, J. B (1991) J. Biol. Chem. 266 , 16925-16930; Munzer, J. S., Daly, S. E., Jewell-Motz, E. A., Lingrel, J. B, and Blostein, R. (1994) J. Biol. Chem. 269, 16668-16676). The experiment s described in the present study provide insight into the mechanistic basis for these differences. The results show that a2 differs from al primarily by a shift in the E-1 reversible arrow E-2 equilibrium in favor of E-1 form (s) as evidenced by (i) a similar to 20-fold increase in IC50 for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATP ase activity at acidic pH. In contrast, despite its lower K ' (ATP) compare d with alpha (1) the E-1 <----> E-2 poise of A is not shifted toward E-1. D istinct intrinsic interactions with Na+ ions are underscored by the marked selectivity for Na+ over Li+ of alpha3 compared with either al or a2 and hi gher K ' (Na) for cytoplasmic Na+, which persists over a 100-fold range in proton concentration, independent of the presence of K+. The kinetic analys is also suggests alpha3-specific differences in relative rates of partial r eactions, which impact this isoform's distinct apparent affinities for both Na+ and K+.