Effects of some organic solvents on the reactivity of sodium plus potassium ion-transport ATPase

Abstract

1. 1. Organic solvents including aliphatic alcohols, acetone, diethyl ether and chloroform inhibited (Na + + K +)-ATPase (EC 3.6.1.3) of the plasma membranes of guinea pig kidney. Inhibition was uncompetitive with respect to ATP if the ATP concentration was larger than 5 μM. Below this concentration of ATP, K + inhibits rather than stimulates the Na +-dependent splitting of ATP by the native enzyme. The tested organic solvents converted this K + inhibition into K + stimulation. In the presence of these solvents, the apparent affinity of the enzyme increased for Na + and ATP and decreased for K + as estimated by the kinetics of ATP hydrolysis. Apparent homotropic allosteric interaction between the Na + and K + sites increased. 2. 2. Organic solvents interfered with different steps of the transient phosphorylation of the enzyme. As estimated by pulse labeling, organic solvents enhanced interaction of ATP with the enzyme prior to phosphorylation both in the absence or in the presence of K +. The solvents decreased the rate of dephosphorylation of the phospho-enzyme either in the presence of K + (native enzyme) or in the presence of ADP ( N-ethylmaleimide-treated enzyme) but they did not affect the ratio of the ADP-sensitive form of the native phospho-enzyme to the K +-sensitive form. Rephosphorylation by ATP of the dephospho-enzyme, formed by adding Rb +, was accelerated by organic solvents. 3. 3. Interaction of the enzyme with these organic solvents appeared to be primarily hydrophobic, as the half-maximal inhibitory concentrations of these solvents correlated with their octanol-water partition coefficients and with the length of the hydrophobic side chain in the series of homologous aliphatic alcohols. 4. 4. It is considered that these modifications of (Na + + K +)-ATPase were due partly to conformational changes of the enzyme, and partly to changes in the water structure around the active center which is proposed to be in a hydrophobic environment.