2--3--o-(2-4-6-trinitrophenyl)adenosine-5--triphosphate and 5-iodoacetamidofluorescein

Topics: Adenosine Triphosphate; Animals; Dogs; Fluoresceins; Fluorescent Dyes; Kidney; Kinetics; Magnesium; Sodium-Potassium-Exchanging ATPase; Spectrometry, Fluorescence

This paper examines the transient kinetics of substrate binding to the Na+/K(+)-ATPase labelled with iodoacetamidofluorescein (IAF) using fluorescence quenching by trinitrophenyl-ATP (TNP-ATP). Earlier work (E.H. Hellen, P.R. Pratap, 1996, Fluorescence quenching of IAF-Na+/K(+)-ATPase via energy transfer to TNP-labelled nucleotide, Proceedings of the VIIIth International Conference on the Na+/K(+)-ATPase, in press) has shown that TNP-nucleotide binds to specific sites (from which unlabelled nucleotide can displace it) and nonspecific sites (from which unlabelled nucleotide cannot displace it). Under stopped-flow conditions, quenching of IAF-enzyme fluorescence was well described by a stretched exponential (F(t) = F infinity + delta F exp[-Bt alpha]). Physically, this function may be interpreted in terms of its inverse Laplace transform phi (k), which describes a distribution of rate-constants; alpha reflects the width of this distribution. As TNP-ATP concentration increased, alpha decreased, reflecting TNP-ATP binding to sites with higher energy barriers. alpha decreased by about the same amount with increasing [TNP-ATP] in the presence of saturating ATP, indicating that the distribution of rate-constants is largely associated with the nonspecific binding sites. However, alpha was significantly less than 1 for ATP-induced fluorescence recovery in the presence of TNP-ATP, indicating that rate-constants associated with specific binding site are also distributed. The distribution of rate-constants for binding to the specific site indicates a distribution in the energy of the transition state for substrate binding. These results suggest that the specific binding site (in either the empty or the full state) may exist in a series of conformations separated by small energy barriers. However, the energy barriers for binding associated with these conformations are significantly distributed.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Dimethylformamide; Dogs; Fluoresceins; Fluorescent Dyes; Fluorometry; Kidney; Kinetics; Protein Binding; Sodium-Potassium-Exchanging ATPase

The first indication of the size of a conformational change implicated in ion transport by sodium pump has been obtained by measuring the change in efficiency of fluorescence energy transfer between two specific locations on the alpha-subunit. The donor (5'-(iodoacetamido)fluorescein) attaches covalently to cysteine-457, and the acceptor (2'(or 3')-O-(trinitrophenyl)adenosine 5'-triphosphate) binds reversibly to the active site. The acceptor binds nearly 2 orders of magnitude tighter to the Na+ than to the K+ conformation of the enzyme and quenches donor fluorescence more efficiently in the Na+ than in the K+ conformation. The estimated distance between donor and acceptor, assuming random orientation of their emission and absorption dipoles, increases 2.9 +/- 0.6 A when the enzyme changes from the Na+ to the K+ conformation. Stopped-flow measurements of the change in fluorescence energy transfer efficiency with time when the doubly-labeled pump is mixed with Na+ or K+ demonstrate that the donor/acceptor pair reports the change between the E1 and E2 conformations of unphosphorylated enzyme. The observed first-order rate constant for the change in energy transfer efficiency depends sigmoidally on [K+] and inversely on [Na+], and both rate and amplitude data for the change in energy transfer efficiency can be fit with the same values of the rate and ion-dissociation constants as published data for the conformational change between E1 and E2 obtained by singly labeling the enzyme with fluorophores that report changes in protein microenvironment. The prerequisite for successfully measuring the distance change and equating the protein rearrangement with a step in the catalysis-transport cycle is that the donor by itself does not report the conformational change.

Topics: Adenosine Triphosphate; Animals; Binding Sites; Biological Transport, Active; Energy Transfer; Flow Injection Analysis; Fluoresceins; Fluorescence Polarization; Kidney; Models, Chemical; Potassium; Protein Conformation; Sodium; Sodium-Potassium-Exchanging ATPase; Spectrometry, Fluorescence; Swine; Titrimetry