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 depho sphorylation were observed by chasing phosphoenzyme formed from [P-32] 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 conversio n 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, wh ich 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, b ut 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 qualif y E1P to participate in Na,K-ATPase activity if this rapid state parti cipates in the normal reaction cycle. Phosphorylation in 0.02-0.1 m Na Cl produced mostly E2P. Upward jumps in [NaCl] converted E2P to E1P eq ually rapidly and transiently. Oligomycin and high [salt] cooperated i n stabilizing E1P. Jumps in [salt] greatly and transiently increased t he rate of conversion of one form of the phosphoenzyme to the other. T his extraordinary result required heterogeneous kinetics. A model is p roposed based on control of enzyme conformation by changes in separate unmixed phases of the lipid of the membrane.