A monoclonal antibody against horse kidney (Na+ + K+)-ATPase inhibits sodium pump and E2K to E1 conversion of (Na+ + K+)-ATPase from outside of the cell membrane

Abstract Monoclonal antibodies against horse kidney outer medulla (Na + + K + )-ATPase were prepared. One of these antibodies (M45–80), was identified as an IgM, recognized the α subunit of the enzyme. M45–80 had the following effects on horse kidney (Na + + K + )-ATPase; (1) it inhibited the enzyme activity by 50% in 140 mM Na + and by 80% in 8.3 mM Na + ; (2) it increased the Na + concentration necessary for half-maximal activation ( K 0.5 for Na + ) from 12.0 to 57.6 mM, but did not affect K 0.5 for K + ; (3) it slightly increased the K + -dependent p -nitrophenylphosphatase (K-pNPPase) activity; (4) it inhibited phosphorylation of the enzyme with ATP by 30%, but did not affect the step of dephosphorylation; and (5) it enhanced the ouabain binding rate. These data are compatible with a stabilizing effect on the E 2 form of (Na + + K + )-ATPase. M45–80 was concluded to binf to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45–80 inhibited by 50% the ouabain-sensitive 86 Rb + uptake in human intact erythrocytes from outside of the cells; (2) the inhibition of (Na + + K + )-ATPase activity in right-side-out vesicles of human erythrocytes was greater than that in inside-out vesicles; and (3) the fluorescence intensity due to FITC-labeled rabbit anti-mouse IgM that reacted with M45–80 bound to the right-side-out vesicles was much greater than that in the case of the inside-out vesicles.