(Na++K+)-ATPase is a target receptor of digitalis (cardiac glycoside) drugs. It has been demonstrated that the H1-H2 domain of the alpha-subunit of the (Na++K+)-ATPase is one of the digitalis drug interaction sites of the enzyme, Despite the extensive studies of the inhibitory effect of digitalis on the (Na++K+)-ATPase, the functional property of the H1-H2 domain of the enzyme and its role in regulating enzyme activity is not completely understood. Here we report a surprise finding: instead of inhibiting the enzyme, binding of a specific monoclonal antibody SSA78 to the H1-H2 domain of the (Na++K+)-ATPase elevates the catalytic activity of the enzyme. In the presence of low concentration of ouabain, monoclonal antibody SSA78 significantly protects enzyme function against ouabain-induced inhibition. However, higher concentration of ouabain completely inactivates the (Na++K+)-ATPase even in the presence of SSA78. These results Suggest that the H1-H2 domain of the (Na++K+)-ATPase is capable of regulating enzyme function in two distinct ways for both ouabain-sensitive and -resistant forms of the enzyme: it increases the activity of the (Na++K+)-ATPase during its interaction with an activator; it also participates in the mechanism of digitalis OF ouabain-induced inhibition of the enzyme. Understanding the dual activity of the H1-H2 domain will help better understand the structure-function relationships of the (Na++K+)-ATPase and the biological processes mediated by the enzyme. (C) 2008 Elsevier Inc. All rights reserved.