| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
Biophysical Journal 61: 553-568 (1992)
© 1992 the Biophysical Society
Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Ohio 45267-0575.
ABSTRACT
The oligomeric nature of the purified lamb kidney Na+,K(+)-ATPase was investigated by measuring the fluorescence energy transfer between catalytic (alpha) subunits following sequential labeling with fluorescein 5'-isothiocyanate (FITC) and erythrosin 5'-isothiocyanate (ErITC). Although these two probes had different spectral responses upon reaction with the enzyme, our studies suggest that a sizeable proportion of their binding occurs at the same ATP protectable, active site domain of alpha. Fluorescence energy transfer (FET) from donor (FITC) to acceptor (ErITC) revealed an apparent 56 A distance between the putative ATP binding sites of alpha subunits, which is consistent with (alpha beta)2 dimers rather than randomly spaced alpha beta heteromonomers. In this work, methods were introduced to eliminate the contribution of nonspecific probe labeling to FET values and to determine the most probable orientation factor (K2) for these rigidly bound fluorophores. FET measurements between anthroylouabain/ErITC, 5'-iodoacetamide fluorescein (5'IAF)/ErITC, and TNP-ATP/FITC, donor/acceptor pairs were also made. Interestingly, none of these distances were affected by ligand-dependent changes in enzyme conformation. These results and those from electron microscopy imaging (Ting-Beall et al. 1990. FEBS Lett. 265:121) suggest a model in which ATP binding sites of (alpha beta)2 dimers are 56 A apart, and reside 30 A from the intracellular surface of the membrane contiguous with the phosphorylation domain.
This article has been cited by other articles:
 |
|
 |
|
  H. Linnertz, P. Urbanova, T. Obsil, P. Herman, E. Amler, and W. Schoner Molecular Distance Measurements Reveal an (alpha beta )2 Dimeric Structure of Na+/K+-ATPase. HIGH AFFINITY ATP BINDING SITE AND K+-ACTIVATED PHOSPHATASE RESIDE ON DIFFERENT alpha -SUBUNITS J. Biol. Chem., October 30, 1998; 273(44): 28813 - 28821. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. G. Ward and J. D. Cavieres Binding of 2`(3`)-O-(2,4,6-Trinitrophenyl)ADP to Soluble alphabeta Protomers of Na,K-ATPase Modified with Fluorescein Isothiocyanate J. Biol. Chem., May 24, 1996; 271(21): 12317 - 12321. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
M. Ganjeizadeh, N. Zolotarjova, W.-H. Huang, and A. Askari Interactions of Phosphorylation and Dimerizing Domains of the [IMAGE]-Subunits of Na[IMAGE]/K[IMAGE]-ATPase J. Biol. Chem., June 30, 1995; 270(26): 15707 - 15710. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
D. W. Martin and J. R. Sachs Ligands Presumed to Label High Affinity and Low Affinity ATP Binding Sites Do Not Interact in an (alpha beta )2 Diprotomer in Duck Nasal Gland Na+,K+-ATPase, nor Do the Sites Coexist in Native Enzyme J. Biol. Chem., August 4, 2000; 275(32): 24512 - 24517. [Abstract] [Full Text] [PDF]
|
|
|
|
|
|
C. Donnet, E. Arystarkhova, and K. J. Sweadner Thermal Denaturation of the Na,K-ATPase Provides Evidence for alpha -alpha Oligomeric Interaction and gamma Subunit Association with the C-terminal Domain J. Biol. Chem., March 2, 2001; 276(10): 7357 - 7365. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 D. W. Martin, J. Marecek, S. Scarlata, and J. R. Sachs alpha beta protomers of Na+,K+-ATPase from microsomes of duck salt gland are mostly monomeric: Formation of higher oligomers does not modify molecular activity PNAS, March 28, 2000; 97(7): 3195 - 3200. [Abstract] [Full Text] [PDF]
|
|
| HOME | HELP | FEEDBACK | SUBSCRIPTIONS | ARCHIVE | SEARCH | TABLE OF CONTENTS |
