Biochemistry 1986, 25, 7 155-7 162
7155
Uncoupling of ATP Binding to Na+,K+-ATPase from Its Stimulation of Ouabain Binding: Studies of the Inhibition of Na+,K+-ATPase by a Monoclonal Antibody? William J. Ball, Jr. Department of Pharmacology and Cell Biophysics, University of Cincinnati College of Medicine, Cincinnati, Ohio 45267 Received May 6 , 1986; Revised Manuscript Received July 25, 1986
ABSTRACT: The effects of a monoclonal antibody, prepared against the purified lamb kidney Na+,K+-ATPase, on the enzyme's Na+,K+-dependent ATPase activity were analyzed. This antibody, designated M1 0-P5-C1 1, is directed against the catalytic subunit of the "native" holoenzyme. It inhibits greater than 90% of the ATPase activity and acts as a noncompetitive or mixed inhibitor with respect to the ATP, Na', and K+ dependence of enzyme activity. It inhibits the Na+- and Mg2+ATP-dependent phosphoenzyme intermediate formation. In contrast, it has no effect on K+-dependent p-nitrophenylphosphatase (pNPPase) activity, the interconversion of the phosphoenzyme intermediates, and ADP-sensitive or K+-dependent dephosphorylation. It does not alter A T P binding to the enzyme nor the covalent labeling of the enzyme at the presumed A T P site by fluorescein 5'-isothiocyanate (FITC), but it prevents the ATP-induced stimulation in the rate of cardiac glycoside [3H]ouabain binding to the Na+,K+-ATPase. M 10-P5-C11 binding appears to inhibit enzyme function by blocking the transfer of the y-phosphoryl of A T P to the phosphorylation site after A T P binding to the enzyme has occurred. In the presence of Mg2+ATP, it also prevents the ATP-induced transmembrane conformational change that enhances cardiac glycoside binding. This uncoupling of A T P binding from its stimulation of ouabain binding and enzyme phosphorylation demonstrates the existence of an enzyme-Mg2+ATP transitional intermediate preceding the formation of the Na+-dependent ADPsensitive phosphoenzyme intermediate. These results are also consistent with a model of the Na+,K+-ATPase active site being composed of two distinct but interacting regions, the ATP binding site and the phosphorylation site.
x e plasma membrane Na+,K+-ATPase regulates the active transport of Na+ and K+ across the cell membrane, and it is the pharmacological receptor for cardiac glycosides. It is composed of two subunits, the CY subunit (- 100000 daltons), which contains the ATP binding site, the phosphorylation site, and the cardiac glycoside receptor, and the p subunit (-50000 daltons), which is a glycoprotein with no clear functional role [see reviews by Glynn and Karlish (1975), Stekhoven and Bonting (1 98 l ) , and Jorgensen (1 982)]. The generally accepted reaction scheme for the turnover cycle is as follows (Fahn et al., 1966; Post et al., 1969; Mardh & Lindahl, 1977):
3. Nat
€1 t M B * ~ A T P
t ADP
Nat*Mg*E1-P
(1 )
Na+ Nat Ma2'* El- Pa Na
\\+ Na
M ~ ~ + * E ~ - P
-
(2)
Kt
M
