Kinetic heterogeneity of phosphoenzyme of Na,K-ATPase modeled by unmixed lipid phases. Competence of the phosphointermediate.

Interconversion of phosphoenzyme resistant to K+ and sensitive to ADP (E1P) and phosphoenzyme resistant to ADP and sensitive to K+ (E2P) was studied in bovine brain and dog and pig kidney. The kinetics of dephosphorylation were observed by chasing phosphoenzyme formed from [32P]ATP with unlabeled ATP with or without ADP or K+. Phosphorylation in 0.6-1.0 M NaCl produced mostly ADP-sensitive potassium-insensitive E1P. A potassium chase of this phosphoenzyme exposed its rate of conversion to potassium-sensitive ADP-insensitive E2P. At 20 degrees C the rate constant was approximately 1 s-1. Simultaneous dilution of [NaCl] in the chase to 100 mM increased the constant to approximately 60 s-1, which probably qualifies E1P as an intermediate in Na,K-ATPase activity. Anions inhibited conversion according to a Hofmeister series. Na+ had no specific effect. At 0 degrees C the rate constant was < 0.4 s-1, but downward jumps in [salt] produced an acceleration to > 1 s-1 for < 3 s followed by a return to the slow rate. The rapid rate would qualify E1P to participate in Na,K-ATPase activity if this rapid state participates in the normal reaction cycle. Phosphorylation in 0.02-0.1 M NaCl produced mostly E2P. Upward jumps in [NaCl] converted E2P to E1P equally rapidly and transiently. Oligomycin and high [salt] cooperated in stabilizing E1P. Jumps in [salt] greatly and transiently increased the rate of conversion of one form of the phosphoenzyme to the other. This extraordinary result required heterogeneous kinetics. A model is proposed based on control of enzyme conformation by changes in separate unmixed phases of the lipid of the membrane.