2'(3')-O-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5'-triphosphate and its Cr(H2O)4 and Co(NH3)4 complex derivatives are new fluorescent tools for labelling ATP binding sites of Na+/K+-ATPase.
2'(3')-O-[N- [2- [3- [5-fluoresceinyl] thioureido] ethyl] carbamoyl] adenosine 5'-triphosphate (FEDA-ATP), a spectroscopic tool used for studying skeletal muscle myosin ATPase subfragment 1, was applied to Na+/K+-ATPase (EC 3.6.1.37). In contrast to the myosin subfragment, we found that FEDA-ATP is not a substrate for Na+/K+-ATPase. On the other hand, FEDA-ATP showed an affinity for both the low (E2, Kd=200 microM) and the high (E1, Kd=22 microM) affinity ATP-binding sites. When the microscopic affinities of FEDA-ATP were used for calculating the macroscopic affinity in the overall reaction according to Ki=(KdE1*KdE2)1/2, the experimentally measured inhibition constant of 66 microM was obtained. To evoke irreversible binding inhibitors, FEDA-ATP was transferred in its chromium(III) and cobalt(III) complex analogs, which are suitable tools for labelling the ATP binding sites of Na+/K+-ATPase in a specific way.
Linnertz
H
H
Institute of Biochemistry and Endocrinology, Justus-Liebig-University Giessen, Germany.
Lastres Becker
I
I
Krumscheid
R
R
Amler
E
E
Thoenges
D
D
Schoner
W
W
D000255:Adenosine Triphosphate / Q000031:analogs & derivatives* / Q000378:metabolism*; D000818:Animals; D001665:Binding Sites; D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; D006868:Hydrolysis; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*; D013379:Substrate Specificity; D013552:Swine
D004791:Enzyme Inhibitors; D005452:Fluoresceins; D005456:Fluorescent Dyes; C097960:caged FEDA-ATP; D000255:Adenosine Triphosphate; D000254:Sodium-Potassium-Exchanging ATPase