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 alpha 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 1-pyreneisothiocyanate 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 alpha subunit is rigid and rotationally immobilized. This has important consequences for the molecular mechanism of the transport function.
Linnertz
H
H
Institute of Biochemistry and Endocrinology, Justus-Liebig-University, Giessen, Germany.
Miksik
I
I
Kvasnicka
P
P
Bertoli
E
E
Mazzanti
L
L
Schoner
W
W
Amler
E
E
D000255:Adenosine Triphosphate / Q000378:metabolism*; D001665:Binding Sites; D003593:Cytoplasm / Q000378:metabolism*; D004789:Enzyme Activation; D006207:Half-Life; D017879:Isothiocyanates / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000737:chemistry / Q000378:metabolism*; D013997:Time Factors
C110887:1-pyreneisothiocyanate; D017879:Isothiocyanates; D010446:Peptide Fragments; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase