title: Kinetic heterogeneity of phosphoenzyme of Na,K-ATPase modeled by unmixed lipid phases. Competence of the phosphointermediate.
abstract: 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.
authors.affiliation: Institute of Biophysics, University of Aarhus, Denmark.
authors.forename: I
authors.initials: I
authors.lastname: Klodos
authors.forename: R L
authors.initials: RL
authors.lastname: Post
authors.forename: B
authors.initials: B
authors.lastname: Forbush
mesh_terms: D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D001921:Brain / Q000201:enzymology; D002417:Cattle; D002462:Cell Membrane / Q000378:metabolism; D004285:Dogs; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008954:Models, Biological; D009840:Oligomycins / Q000494:pharmacology; D009994:Osmolar Concentration; D010710:Phosphates / Q000378:metabolism; D010750:Phosphoproteins / Q000737:chemistry / Q000378:metabolism*; D010761:Phosphorus Radioisotopes; D011188:Potassium / Q000494:pharmacology; D012965:Sodium Chloride / Q000494:pharmacology; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013816:Thermodynamics
chemical_list: D009840:Oligomycins; D010710:Phosphates; D010750:Phosphoproteins; D010761:Phosphorus Radioisotopes; D012965:Sodium Chloride; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D011188:Potassium