Kinetics of Na-ATPase activity by the Na,K pump. Interactions of the phosphorylated intermediates with Na+, Tris+, and K+.

Abstract

To determine the biochemical events of Na+ transport, the interactions of Na+, Tris+ and K+ with the phosphorylated intermediates of Na,K-ATPase from ox brain were studied. The enzyme was phosphorylated by incubation at 0.degree. C with 1 mM Mg2+, 25 .mu.M [32P]ATP and 20-600 mM Na+ with or without Tris+, and the dephosphorylation kinetics of [32P]EP were studied after addition of (1) 1 mM ATP, (2) 2.5 mM ADP, (3) 1 mM ATP plus 20 mM K+ and (4) 2.5 mM ADP plus Na+ up to 600 mM. In dephosphorylation types 2-4, the curves were bi- or multiphasic. ADP-sensitive EP and K+-sensitive EP were determined by extrapolation of the slow phase of the curves to the ordinate and their sum was always larger than Etotal. These results required a minimal model consisting of 3 consecutive EP pools, A, B and C, where A was ADP sensitive and both B and C were K+ sensitive. At high [Na+], B was converted rapidly to A (type 4 experiment). The 7 rate coefficients were dependent on [Na+], [Tris+] and [K+], and to explain this a comprehensive model for cation interaction with EP was developed. The model has the following features: A, B and C are equilibrium mixtures of EP forms; EP in A has 2-3 Na ions bound at high-affinity (internal) sites, pool B has 3 and pool C 2-3 low-affinity (external) sites. The putative high-affinity outside Na+ site may be on E2P in pool C. The A .fwdarw. B conversion is blocked by K+ (and Tris+). Pool A can be an intermediate only in the Na-ATPase reaction and not in the normal operation of the Na,K pump.