Binding of pyrene isothiocyanate to the E1ATP site makes the H4-H5 cytoplasmic loop of Na+/K+-ATPase rigid.

1-Pyreneisothiocyanate was shown to be an inhibitor of Na⁺/K⁺-ATPase. Reverse-phase HPLC and activity studies indicated binding of 1-pyreneisothiocyanate at the H₄-H₅ loop of the α subunit and competition with the fluorescein 5′-isothiocyanate for the E₁ATP site. While fluorescein 5′-isothiocyanate, the fluorescent ATP pseudo-analog, was shown to be immobilized at the E₁ATP site, there was no possibility to draw any conclusion about the flexibility of the E₁ATP site due to its short lifetime. Employing 1pyreneisothiocyanate as a long-lived fluorophore and a label for the E₁ATP site, we found that the ATP-binding site of Na⁺/K⁺-ATPase and, in fact, the whole large intracellularly exposed H₄-H₅ loop of the catalytic α subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function. [ABSTRACT FROM AUTHOR]