Kinetic characterization of α₁ and α_2/3Na⁺,K⁺-ATPase isoforms in cerebral cortex and hippocampus from rats

Kinetic properties of α₁ and α_2/3 isoforms of Na⁺, K⁺-ATPase (NKA) were evaluated in cerebral cortex (CX) and hippocampus (HP) homogenates of Wistar rats. In CX ATP curves, α₁NKA (K_0.5a = 0.04 ± 0.002 mM; V_Ma = 1.11 ± 0.02 U/mg; K_0.5b = 2.56 ± 1.08 mM; V_Mb = 9.72 ± 3.56 U/mg) and α_2/3NKA (K_0.5a = 0.01 ± 0.001 mM; V_Ma = 1.17 ± 0.30 U/mg; K_0.5b = 2.71 ± 1.87 mM; V_Mb = 1.88 ± 0.56 U/mg) isoforms showed biphasic curves. In HP, α₁NKA showed K_0.5a = 0.05 ± 0.03 mM; V_Ma = 2.77 ± 0.28 U/mg; K_0.5b = 2.06 ± 0.60 mM; V_Mb = 4.91 ± 1.48 U/mg, while α_2/3NKA showed monophasic curve (K_0.5 = 1.02 ± 0.01 mM; V_M = 5.22 ± 1.88 U/mg). Under Na⁺ stimulation, α₁NKA in CX reached higher V_M and affinity than α_2/3NKA. Whereas HP V_M was similar, but K_0.5 was higher in α₁NKA. α_2/3NKA exhibited higher affinity for K⁺ and smaller V_M than α₁NKA in CX, but in HP both isoforms presented similar K_0.5. In Mg²⁺ assays, α₁NKA showed higher affinities and activity in CX. In HP, the V_M are similar across isoforms, but K_0.5 was higher in α₁NKA than α_2/3NKA. In increasing NH₄⁺ concentrations, α₁NKA showed higher affinities than α_2/3NKA in CX and HP, but α_2/3NKA demonstrated higher V_M. These results provide the first comparative kinetic characterization of α₁ and α_2/3NKA isoforms in rat brain, highlighting regional and isoform-specific differences that may reflect distinct physiological demands.