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 130mM NaClO4 caused an increase in the Kd values of both the high- and low-affinity ATP-binding sites, from values of 7.0 (± 0.6) μM and 143 (± 17) μM in 130mM NaCl solution to values of 42 (± 3) μM and 660 (± 100) μM in 130mM NaClO4 (pH 7.4, 24°C). The half-saturating concentration of the Na+-binding sites on the E1 conformation was found to decrease from 8–10mM in NaCl to 2.5–3.5mM 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 the E1P(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.
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