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.
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 alpha 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 (K0.5 for Na+) from 12.0 to 57.6 mM, but did not affect K0.5 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 E2 form of (Na+ + K+)-ATPase. M45-80 was concluded to bind to the extracellular surface of the plasmamembrane, based on the following evidence: (1) M45-80 inhibited by 50% the ouabain-sensitive 86Rb+ 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.
Satoh
K
K
Department of Toxicology, Tokyo Metropolitan Research Laboratory of Public Health, Japan.
Nakao
T
T
Nagai
F
F
Kano
I
I
Nakagawa
A
A
Ushiyama
K
K
Urayama
O
O
Hara
Y
Y
Nakao
M
M
D009597:4-Nitrophenylphosphatase / Q000378:metabolism; D000818:Animals; D000937:Antigen-Antibody Reactions; D001693:Biological Transport, Active; D004910:Erythrocyte Membrane / Q000378:metabolism; D006736:Horses; D006801:Humans; D005779:Immunodiffusion; D066298:In Vitro Techniques; D007668:Kidney / Q000201:enzymology; D010042:Ouabain / Q000494:pharmacology; D012413:Rubidium / Q000378:metabolism; D012964:Sodium / Q000378:metabolism; D000254:Sodium-Potassium-Exchanging ATPase / Q000037:antagonists & inhibitors / Q000276:immunology*
D010042:Ouabain; D012964:Sodium; D009597:4-Nitrophenylphosphatase; D000254:Sodium-Potassium-Exchanging ATPase; D012413:Rubidium