1. Mechanistic Basis for Kinetic Differences between the Rat α1, α2, and α3 Isoforms of the Na,K-ATPase
- Author
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Rhoda Blostein, Laura Segall, and Stewart E. Daly
- Subjects
Gene isoform ,Cytoplasm ,Stereochemistry ,Chemistry ,Protein subunit ,Vanadate ,Cell Biology ,Na+/K+-ATPase ,Selectivity ,Molecular Biology ,Biochemistry ,IC50 ,Affinities - Abstract
Previous studies showed that the α1, α2, and α3 isoforms of the catalytic subunit of the Na,K-ATPase differ in their apparent affinities for 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 α2 and α3 compared with α1; relative to α1 and α2, α3 has a higher K′Na and lowerK′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 experiments described in the present study provide insight into the mechanistic basis for these differences. The results show that α2 differs from α1 primarily by a shift in the E 1 ⇌E 2 equilibrium in favor ofE 1 form(s) as evidenced by (i) a ∼20-fold increase in IC50 for vanadate, (ii) decreased catalytic turnover, and (iii) notable stability of Na,K-ATPase activity at acidic pH. In contrast, despite its lower K′ATPcompared with α1, the E 1 ⇌E 2 poise of α3 is not shifted towardE 1. Distinct intrinsic interactions with Na+ ions are underscored by the marked selectivity for Na+ over Li+ of α3 compared with either α1 or α2 and higher K′Na for cytoplasmic Na+, which persists over a 100-fold range in proton concentration, independent of the presence of K+. The kinetic analysis also suggests α3-specific differences in relative rates of partial reactions, which impact this isoform's distinct apparent affinities for both Na+ and K+.
- Published
- 2001