oligomycins and anthroylouabain

The beta-subunit associated with the catalytic (alpha) subunit of the mammalian Na+, K(+) -ATPase is a transmembrane glycoprotein with three extracellularly located N-glycosylation sites. Although beta appears to be essential for a functional enzyme, the role of beta and its sugars remains unknown. In these studies, steady-state and dynamic fluorescence measurements of the fluorophore lucifer yellow (LY) covalently linked to the carbohydrate chains of beta have demonstrated that the bound probes are highly solvent exposed but restricted in their diffusional motions. Furthermore, the probes' environments on beta were not altered by Na+ or K+ or ouabain-induced enzyme conformational changes, but both divalent cation and oligomycin addition evoked modest changes in LY fluorescence. Frequency domain measurements reflecting the Förster fluorescence energy transfer (FET) occurring between anthroylouabain (AO) bound to the cardiac glycoside receptor site on alpha and the carbohydrate-linked LY demonstrated their close proximity (18 A). Additional FET determinations made between LY as donor and erythrosin-5-isothiocyanate, covalently bound at the enzyme's putative ATP binding site domain, indicated that a distance of about 85 A separates these two regions and that this distance is reduced upon divalent cation binding and increased upon the Na+E1-->K+E2 conformational transition. These data suggest a model for the localization of the terminal moieties of the oligosaccharides that places them, on average, about 18 A from the AO binding site and this distance or less from the extracellular membrane surface.

Topics: Adenosine Triphosphate; Animals; Anthracenes; Binding Sites; Biophysical Phenomena; Biophysics; Carbohydrates; Cardiac Glycosides; Cations; Energy Transfer; Fluorescent Dyes; In Vitro Techniques; Isoquinolines; Kinetics; Models, Biological; Motion; Oligomycins; Ouabain; Protein Conformation; Sheep; Sodium-Potassium-Exchanging ATPase; Solvents

Topics: Animals; Anthracenes; Brain; Cattle; Fluorescent Dyes; Isoenzymes; Kinetics; Mathematics; Models, Theoretical; Oligomycins; Ouabain; Phosphorylation; Protein Binding; Sodium-Potassium-Exchanging ATPase

We present experiments designed to test whether or not the ADP-sensitive (E1P) and the K-sensitive (E2P) phosphoenzyme forms of (Na,K)-ATPase are intermediates of ATP hydrolysis in the presence of Na and K. We have used the apparent rate constant of anthroylouabain binding, k' on, and its inhibition by oligomycin to monitor the steady-state levels of E2P and E1P, respectively. We have measured k' on in purified dog kidney (Na,K)-ATPase at 37 degrees C as a function of Na, K, and MgATP or Mg plus inorganic phosphate in the presence and absence of oligomycin. In the presence of 35 mM Na and no K, k' on was saturated with [ATP] greater than 25 microM, and oligomycin (10 microM) inhibited less than 10%. Inhibition by oligomycin increased when [Na] was increased. At 1.9 M Na oligomycin inhibited k' on and equilibrium binding of anthroylouabain with Ki = 136 nM. Oligomycin had no effect on k' on in the presence of Mg + inorganic phosphate. In the presence of 35 mM Na and 4.5 mM K, k' on was much slower, inhibition by oligomycin increased, and ATP stimulated both k' on and inhibition by oligomycin with K0.5 greater than 0.1 mM. From the k' on measurements in the presence and absence of K, we estimated that the steady-state level of E2P was between 4 and 15% of the enzyme with 25 microM to 6.5 mM ATP, 35 mM Na, and 4.5 mM K at 37 degrees C. For these conditions we estimated the rate constant of E2P hydrolysis to be 46,000 (+/- 1,000) min-1. These results are contrary to the predictions derived from a model in which acid-stable phosphoenzymes are not intermediates of ATP hydrolysis in the presence of Na and K. They are fully consistent with a model in which E1P and E2P are intermediates of ATP hydrolysis coupled to transport of Na and K.

Topics: Adenosine Triphosphate; Animals; Anthracenes; Dogs; Fluorescent Dyes; Kidney; Kinetics; Mathematics; Models, Chemical; Oligomycins; Ouabain; Potassium; Sodium-Potassium-Exchanging ATPase