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

FEBS Letters

Urbanova, Petra¹  Amler, Evzen¹  Linnertz, Holger¹ 

[1] Institute of Physiology, Academy of Sciences of the Czech Republic, Videnska 1083, Cz-142 20 Prague 4, Czech Republic

关键词: Na+/K+-ATPase;    NBD-Cl;    ATP-binding site;    ATP analog;    Fluorescence anisotropy;    Fluorescence quenching;    Cr(H2O)4AdoPP[CH2]P;    β;    γ bidentate complex of chromium(III)-tetraaqua-adenylyl [β;    γ-methylene] diphosphonate;    Co(NH3)4ATP;    β;    γ bidentate complex of cobalt(III)-tetramino-adenosine-5′-triphosphate;    Co(NH3)4PO4;    tetramine cobalt(III)phosphate;    FITC;    fluorescein 5′isothiocyanate;    NBD-Cl;    7-chloro-4-nitrobenzo-2-oxa-1;    3-diazole;    7-chloro-4-nitro-benzofurazane;    E1ATP site;    nucleotide-binding site of Na+/K+-ATPase with high affinity for ATP;    E2ATP site;    nucleotide-binding site of Na+/K+-ATPase with low affinity for ATP;   

DOI  :  10.1016/S0014-5793(97)01460-9

学科分类：生物化学/生物物理

来源: John Wiley & Sons Ltd.

【 摘 要 】

7-Chloro-4-nitrobenzo-2-oxa-1,3-diazole (NBD-Cl) labeled Na⁺/K⁺-ATPase covalently with two different inactivation constants (K _i=2.5 μM; K _i′=10 μM). 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.

【 授权许可】

Unknown