Open AccessBinding of pyrene isothiocyanate to the E 1 ATP site makes the H 4 ‐H 5 cytoplasmic loop of Na + /K + ‐ATPase rigid
Author(s) -
Linnertz Holger,
Miksik Ivan,
Kvasnicka Peter,
Bertoli Enrico,
Mazzanti Laura,
Schoner Wilhelm,
Amler Evzen
Publication year - 1998
Publication title -
european journal of biochemistry
Language(s) - English
Resource type - Journals
eISSN - 1432-1033
pISSN - 0014-2956
DOI - 10.1046/j.1432-1327.1998.2510522.x
Subject(s) - fluorescein , atpase , binding site , isothiocyanate , chemistry , pyrene , stereochemistry , fluorescein isothiocyanate , fluorescence , fluorophore , enzyme , biochemistry , physics , organic chemistry , quantum mechanics
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 H 4 ‐H 5 loop of the α subunit and competition with the fluorescein 5′‐isothiocyanate for the E 1 ATP site. While fluorescein 5′‐isothiocyanate, the fluorescent ATP pseudo‐analog, was shown to be immobilized at the E 1 ATP site, there was no possibility to draw any conclusion about the flexibility of the E 1 ATP site due to its short lifetime. Employing 1pyreneisothiocyanate as a long‐lived fluorophore and a label for the E 1 ATP site, we found that the ATP‐binding site of Na + /K + ‐ATPase and, in fact, the whole large intracellularly exposed H 4 ‐H 5 loop of the catalytic α subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function.