Interaction of fluorescein isothiocyanate with the (H+ + K+)-ATPase |
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Authors: | R.J. Jackson J. Mendlein G. Sachs |
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Affiliation: | 1. Laboratory of Membrane Biology, University of Alabama in Birmingham, The Medical Center, Birmingham, AL 35294 U.S.A.;2. CURE, Wadsworth Veterans Administration Hospital, Los Angeles, CA 90073 U.S.A. |
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Abstract: | Fluorescein isothiocyanate was used to covalently label the gastric (H+ + K+)-ATPase. FITC treatment of the enzyme inhibited the ATPase activity while largely sparing partial reactions such as the associated activity. ATP protected against inhibition suggesting the ligand binds at or near an ATP binding site. At 100% inhibition the stoichiometry of binding was 1.5 nmol FITC per mg Lowry protein a value corresponding to maximal phosphoenzyme formation. Binding occurred largely to a peptide of 6.2 isoelectric point, although minor labelling of a peptide of 5.6 was also noted. Fluorescence was quenched by K+, Rb+ and Tl+ in a dose-dependent manner, and the values of 0.28, 0.83 and 0.025 mM correspond rather well to the values required for dephosphorylation at a luminal site. Vanadate, a known inhibitor of the gastric ATPase produced a slow Mg2+-dependent fluorescent quench. Ca2+ reversed the K+-dependent loss of fluorescence and inhibited it when added prior to K+. This may relate to the slow phosphorylation in the presence of ATP found when Ca2+ was substituted for Mg2+ and the absence of K+-dependent dephosphorylation. The results with FITC-modified gastric ATPase provide evidence for a conformational change with K+ binding to the enzyme. |
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Keywords: | Phosphorylation Fluorescein isothiocyanate Fluorescence (Hog gastric mucosa) FITC fluorescein isothiocyanate CDTA |
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