The effect of sulphydryl-blocking reagents and of urea on the (Na + + K +)-activated enzyme system

Abstract

Incubation of the (Na+ + K+)-activated ATP-hydrolysing enzyme system with SH-blocking reagents decreases its activity. With SH-blocking reagents such as maleimide, N-ethylmaleimide and 1 fluoro-2,4-dinitrobenzene the activity with Mg2+ decreases more than the activity with Mg2+ + Na+ + K+, i.e. the (Mg2+ + Na+ + K+)/Mg2+ activity ratio is increased. ATP in the incubation medium protects against the decrease in activity with Mg2+ + Na+ + K+ but not against the decrease in activity with Mg2+; incubation with ATP therefore gives a further increase in activity ratio. The same effect is obtained by incubation with urea and ATP. Na+ in the incubation medium gives a further decrease in activity with Mg2+ + Na+ + K+, whereas K+ has less effect. With ATP, K+ decreases the activity more than Na+, and the concentrations of the cations which give half the maximum decrease are lower than without ATP. When G-strophanthin is present together with ATP, Na+ gives a further decrease in activity, whereas there is no change in the effect with K+. The experiments suggest (a) that ATP has an effect on the structure of the enzyme system and on the affinities of the enzyme system for Na+ and K+; and (b) that the activity with Mg2+ and with Mg2+ + Na+ + K+ is due to the same enzyme. When activated by Mg2+ the enzyme system is “locked” in a structural configuration in which ATP cannot induce the change in affinities which leads to the activation by Na+ + K+.