Use of a monoclonal antibody to quantify (Na+,K+)-ATPase activity and sites in normal and regenerating rat liver.

Quantitative measurements of (Na+,K+)-ATPase activity and numbers of (Na+,K+)-ATPase sites in membranes from quiescent and regenerating rat liver have been made using an anticatalytic monoclonal antibody (9-A5) that binds to alpha subunits of the sodium pump (Schenk, D. B., and Leffert, H. L. (1983) Proc. Natl. Acad. Sci. U.S.A. 80, 5281-5285). To validate the measurements, kinetic properties of 9-A5 binding to plasma membrane sodium pumps, specificity and requirements of the reactions, and mechanisms by which 9-A5 inhibits (Na+,K+)-ATPase were analyzed. 125I-9-A5 binding is saturable and reversible (k1 = 1.8 X 10(6) X M-1 X S-1; k2 = 2.7 X 10(-4) X S-1). At equilibrium, 9-A5 binds to a single class of sites revealed by Scatchard plots (KD[app] = 0.64 nM, Bmax = 29.3 pmol/mg of proteins; = 238,000 sites X cell-1). This binding requires monovalent cations (sodium, potassium, or lithium); is blocked by purified (Na+,K+)-ATPase; is inhibited noncompetitively by ATP (KI[app] = 0.5 mM); and is unaffected by ouabain. 9-A5 inhibits ATP-stimulated (Na+,K+)-ATPase noncompetitively by blocking sodium-dependent phosphorylation of alpha subunits of liver or kidney membrane (Na+,K+)-ATPase. Twelve h after 67% hepatectomy, maximal 125I-9-A5 binding to plasma membranes from regenerating liver falls 30 +/- 7% compared to sham-operated controls (p less than 0.01). In contrast, (Na+,K+)-ATPase activity in regenerating liver membranes rises 58 +/- 12% compared to controls (p less than 0.03). Similar experiments with particulate fractions from regenerating liver show insignificant decreases in maximal 125I-9-A5 binding (22 +/- 12%) but large increases in (Na+,K+)-ATPase activity (325 +/- 14%) compared to controls (p less than 0.001). No differences among groups are seen in KD values for 9-A5 binding or in the activities of plasma membrane 5'-nucleotidase (EC 3.1.3.5). Thus, stimulation of the sodium pump during the late prereplicative phase of liver regeneration is not accompanied by increases in the numbers of (Na+,K+)-ATPase sites. Instead, it appears that preexisting (Na+,K+)-ATPases are activated specifically before DNA replication starts.