Item

Abstract

The effects of lyotropic anions, particularly perchlorate, on the kinetics of partial reactions of the Na⁺,K⁺-ATPase from pig kidney were investigated by two different kinetic techniques: stopped flow in combination with the fluorescent label RH421 and a stationary electrical relaxation technique. It was found that 130 mM NaClO₄ caused an increase in the K_d values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (± 0.6) μM and 143 (± 17) μM in 130 mM NaCl solution to values of 42 (± 3) μM and 660 (± 100) μM in 130 mM NaClO₄ (pH 7.4, 24°C). The half-saturating concentration of the Na⁺-binding sites on the E₁ conformation was found to decrease from 8–10 mM in NaCl to 2.5–3.5 mM in NaClO₄ solution. The rate of equilibration of the reaction, E₁P(Na⁺)₃ ↔ E₂P + 3Na⁺, decreased from 393 (±51)s_-1 in NaCl solution to 114 (± 15) s−1 in NaClO₄.
This decrease is attributed predominantly to an inhibition of theE₁ P(Na⁺)₃ → E₂P(Na⁺) ₃ transition. The effects can be explained in terms of electrostatic interactions due to perchlorate binding within the membrane and/or protein matrix of the Na⁺,K⁺-ATPase membrane fragments and alteration of the local electric field strength experienced by the protein. The kinetic results obtained support the conclusion that the conformational transition E₁P(Na⁺)₃ → E₂P(Na⁺)₃ is a major charge translocating step of the pump cycle.51)