title: Interaction of melittin with the (Na+ + K+)ATPase: evidence for a melittin-induced conformational change.
abstract: The (Na+ + K+)ATPase is inhibited by the bee venom polypeptide, melittin. KCl and NaCl protect the enzyme from melittin inhibition. Analysis of the K+ and Na+ protection against melittin inhibition suggested a kinetic model which was consistent with slowly reversible melittin binding, and mutually exclusive binding of melittin with K+ and Na+. Accordingly, in the absence of salt, the KI for melittin inhibition = 1.2 microM, and the protection by KCl occurs with a KA,KCl = 0.6 mM. The protection by NaCl occurs with a KA,NaCl = 15 mM. Melittin inhibition of enzyme activity is due to direct interactions with the (Na+ + K+)ATPase, as demonstrated by photolabeling with [125I]azidosalicylyl melittin, which labeled the alpha subunit, but not the beta subunit of the (Na+ + K+)ATPase. Melittin and KCl reduced the extent of labeling. In non-covalent binding studies using [125I]azidosalicylyl melittin, the stoichiometry of binding was 1.6 melittin per (Na+ + K+)ATPase. Ligand-induced conformational changes of FITC-labeled (Na+ + K+)ATPase were examined in the presence and absence of melittin. K+ alone or melittin alone caused a fluorescence intensity quenching consistent with formation of an E2 form of the enzyme. The NaCl-induced (E2----E1) fluorescence intensity changes were maximal when the enzyme was treated with K+. NaCl-induced fluorescence changes did not occur when the enzyme was treated with melittin in the absence of K+. However, when K+ was present before the addition of melittin, NaCl-induced fluorescence intensity increases were observed, which were dependent upon the concentration of K+ in the preincubation mixture. The results of the labeling and conformational studies support the kinetic model and suggest a mechanism for inhibition of ion pumps by (poly)peptides.
authors.affiliation: Department of Physiology and Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0576.
authors.forename: J
authors.initials: J
authors.lastname: Cuppoletti
authors.forename: A J
authors.initials: AJ
authors.lastname: Abbott
mesh_terms: D000818:Animals; D004285:Dogs; D016650:Fluorescein-5-isothiocyanate; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007668:Kidney / Q000201:enzymology; D007700:Kinetics; D008555:Melitten / Q000378:metabolism / Q000494:pharmacology*; D011485:Protein Binding; D011487:Protein Conformation / Q000187:drug effects; D012756:Sheep; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism*; D013050:Spectrometry, Fluorescence; D013861:Thiocyanates
chemical_list: D005452:Fluoresceins; D005456:Fluorescent Dyes; D013861:Thiocyanates; D008555:Melitten; D000254:Sodium-Potassium-Exchanging ATPase; D016650:Fluorescein-5-isothiocyanate