Quenching of 7-chloro-4-nitrobenzo-2-oxa-1,3-diazole-modified Na+/K+-ATPase reveals a higher accessibility of the low-affinity ATP-binding site.
7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) labeled Na+/K+-ATPase covalently with two different inactivation constants (Ki = 2.5 microM; Ki' = 10 microM). It apparently modified the two different ATP-binding sites of the enzyme since it decreased the activity of the E2ATP site, i.e. the K+-activated para-nitrophenylphosphatase activity, in an enzyme whose high-affinity E1ATP site had been blocked by fluorescein 5'isothiocyanate (FITC). It also reduced the activity of the E1ATP site, i.e. the Na+-activated protein phosphorylation, in an enzyme whose low-affinity E2ATP site had been blocked by Co(NH3)4PO4. Fluorescence quenching experiments with KI, CsCl and MnCl2 of the NBD-Cl-labeled Na+/K+-ATPase revealed two differently accessible types of fluorophores depending on the ATP site: The E2ATP site apparently differs from the E1ATP site in that it is more open because the fluorophore labeling in the E2ATP site was sterically better accessible for quenchers.
Linnertz
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Institute of Physiology, Academy of Sciences of the Czech Republic, Prague.
Urbanova
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Amler
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D009327:4-Chloro-7-nitrobenzofurazan; D000255:Adenosine Triphosphate / Q000737:chemistry / Q000378:metabolism*; D001665:Binding Sites; D005456:Fluorescent Dyes; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*
D005456:Fluorescent Dyes; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase; D009327:4-Chloro-7-nitrobenzofurazan