title: Biological implication of conformational flexibility in ouabain: observations with two ouabain phosphate isomers.
abstract: Ouabain is a highly polar and unusually potent sodium pump inhibitor that possesses uncommon conformational flexibility in its steroid A-ring moiety. The biological significance of ring flection in the cardiotonic steroids has not been described. Accordingly, we prepared ouabain 1,5,19- and 1,11,19-phosphates. The former stabilizes the steroid A-ring chair conformation and the latter locks the A-ring in the half-boat conformation and decreases flection of the ABC-ring moiety. Using a dog kidney cell line (MDCK) in a pH microphysiometer (Cytosensor), ouabain and its 1,5,19-phosphate at 10(-5) M reduced the rate of extracellular acidification by 15-20%. During inhibitor washout, the rate of recovery from the 1,5,19-phosphate analogue was approximately 3 times faster than ouabain. The 1,11,19-phosphate at 10(-4) M elicited a weak ( approximately 7%) response, and the effects reversed approximately 44-fold faster than ouabain. Studies with purified Na(+),K(+)-ATPase showed that ouabain and its 1,5,19-phosphate analogue were of similar efficacy (EC(50) = 1.1 and 5.2 x 10(-7) M, respectively) and >100-fold more potent than the 1,11,19-phosphate analogue. Studies of the binding kinetics showed that the 1,5,19-phosphate analogue bound 3-fold and dissociated 16-fold faster from the purified Na(+),K(+)-ATPase than ouabain. Both analogues were competitive inhibitors of 3H-ouabain binding. Taken together, these results suggest that the marked conformational flexibility of the A-ring in ouabain ordinarily slows the initial binding of this steroid to the sodium pump. However, once ouabain is bound, flection of the steroidal A- and BC-rings is critical for the maintenance of high-affinity binding. Our results indicate that the ouabain-binding site is comprised of structurally mobile elements and highlight the roles that synchronization between receptor and ligand dynamics play as determinants of biological activity in this system.
authors.affiliation: Department of Chemistry, Columbia University, New York, New York 10027, USA.
authors.forename: A
authors.initials: A
authors.lastname: Kawamura
authors.forename: L M
authors.initials: LM
authors.lastname: Abrell
authors.forename: F
authors.initials: F
authors.lastname: Maggiali
authors.forename: N
authors.initials: N
authors.lastname: Berova
authors.forename: K
authors.initials: K
authors.lastname: Nakanishi
authors.forename: J
authors.initials: J
authors.lastname: Labutti
authors.forename: S
authors.initials: S
authors.lastname: Magil
authors.forename: G T
authors.initials: GT
authors.lastname: Haupert
authors.forename: J M
authors.initials: JM
authors.lastname: Hamlyn
mesh_terms: D000818:Animals; D001667:Binding, Competitive; D015374:Biosensing Techniques; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002460:Cell Line; D004285:Dogs; D004791:Enzyme Inhibitors / Q000737:chemistry / Q000378:metabolism; D007536:Isomerism; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008969:Molecular Sequence Data; D019906:Nuclear Magnetic Resonance, Biomolecular; D010042:Ouabain / Q000031:analogs & derivatives* / Q000737:chemistry* / Q000378:metabolism; D010710:Phosphates / Q000737:chemistry* / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism; D014316:Tritium
chemical_list: D004791:Enzyme Inhibitors; D010710:Phosphates; D014316:Tritium; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase