Figure 9.

Effect of access-channel blockers on the PTX-induced action of the Na,K-ATPase in the absence of monovalent cations other than H⁺. Na,K-ATPase was phosphorylated in standard buffer by 0.5 mM Tris phosphate in the absence or presence of 15 μM Br₂-Titu³⁺ and/or 30 mM TPA⁺. After reaching a steady-state level of the fluorescence, corresponding to state P-E₂, 150 nM PTX was added. Only Br₂-Titu³⁺, the blocker of the cytoplasmic access channel, produced a striking difference by reducing the amplitude of the fluorescence decrease by about a factor of five. TPA⁺ had no significant effect. In all four cases, the rate constant of the exponential decay was 0.01 ± 0.0002 s⁻¹.