Ouabain binding kinetics of the rat alpha two and alpha three isoforms of the sodium-potassium adenosine triphosphate.
The Na,K-ATPase has three alpha isoforms which differ in cardiac glycoside sensitivity and tissue distribution. The rodent alpha 1 isoform is relatively resistant to cardiac glycosides, while the alpha 2 and alpha 3 isoforms are quite sensitive. Because both the alpha 2 and alpha 3 isoforms are generally expressed in the same tissue, it has been difficult to differentiate and accurately determine the kinetics of ouabain binding to these isoforms. To more fully understand the interactions of the alpha 2 and alpha 3 isoforms with cardiac glycosides, the association and dissociation rates of ouabain binding were measured in transfected cell lines. cDNA's coding for the rat alpha 2 and alpha 3 isoforms were transfected into NIH 3T3 cells and characterized by Na,K-ATPase activity and [3H]ouabain binding. By individually expressing the alpha 2 and alpha 3 isoforms in ouabain-insensitive NIH 3T3 cells, the ouabain-binding characteristics of each isoform could be accurately determined. The association rate constants of the alpha 2 and alpha 3 isoforms were similar while the dissociation rate constant was 33 times slower for the alpha 3 isoform than the alpha 2 isoform. Calculation of the dissociation constant (Kd) from these rate constants yielded values of 115 and 1.6 nM for rat alpha 2 and alpha 3 isoforms, respectively. Scatchard analysis of the rat alpha 2 isoform produced a similar value for Kd of 37 +/- 9 nM. Inhibition of Na,K-ATPase activity indicates the rodent alpha 1 isoform has an IC50 1000-fold higher than the alpha 2 or alpha 3 isoform at 4.8 x 10(-5) M. The results are consistent with the hypothesis that the order of ouabain affinity between the rat alpha isoforms of the Na,K-ATPase is alpha 3 > alpha 2 >> alpha 1.
O'Brien
W J
WJ
Department of Molecular Genetics, Biochemistry, and Microbiology, University of Cincinnati College of Medicine, Ohio 45267-0524.
Lingrel
J B
JB
Wallick
E T
ET
D016475:3T3 Cells; D000818:Animals; D004305:Dose-Response Relationship, Drug; D007527:Isoenzymes / Q000378:metabolism*; D007700:Kinetics; D051379:Mice; D010042:Ouabain / Q000378:metabolism*; D051381:Rats; D011994:Recombinant Proteins / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000378:metabolism*
D007527:Isoenzymes; D011994:Recombinant Proteins; D010042:Ouabain; D000254:Sodium-Potassium-Exchanging ATPase