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.

This research is about the production of monoclonal antibodies against an enzyme in horse kidneys known as (Na+ + K+)-ATPase, to observe their effects on the enzyme’s activity. One antibody, identified as M45-80, was found to inhibit the enzyme’s activity and it is suggested that it binds to the outer surface of cell membranes.

Preparation of Monoclonal Antibodies and Identification of M45-80

• The researchers prepared monoclonal antibodies against an enzyme from the kidney outer medulla of horses, known as (Na+ + K+)-ATPase, which plays a crucial role in maintaining the sodium-potassium balance in cells.
• Among these antibodies, one named M45-80 was identified as an IgM type, recognizing the alpha subunit of the enzyme. The alpha subunit holds the sites necessary for ATP binding, ions transport, and ouabain reception.

Effects of M45-80 on (Na+ + K+)-ATPase

• M45-80 was found to inhibit the enzyme’s activity: by 50% in high sodium concentration (140 mM) and by 80% in lower sodium concentration (8.3 mM).
• It increased the sodium concentration necessary for half-maximal activation of the enzyme, without affecting the equivalent condition for potassium. Which shows sodium and potassium have different affinities with (Na+ + K+)-ATPase.
• M45-80 slightly increased the activity of a related enzyme, K-pNPPase.
• It inhibited phosphorylation of the enzyme by 30%, but didn’t affect the step of dephosphorylation. Phosphorylation and dephosphorylation are important processes that modify the activity of the enzyme.
• The antibody also increased the binding rate of ouabain, a plant-derived poison that’s known to inhibit (Na+ + K+)-ATPase.

Evidence M45-80 Binds to the Extracellular Surface of the Plasma Membrane

• The researchers provide evidence that M45-80 binds to the cell’s outer surface.
• It inhibited 50% of the ouabain-sensitive uptake of 86Rb+ (a substitute for potassium) in intact human red blood cells, indicating that the antibody interferes with the mechanism of ion transport across the cell membrane from the outside.
• The inhibition of enzyme activity was greater in right-side-out vesicles of human red blood cells compared to inside-out vesicles, suggesting an external site of action for M45-80.
• Fluorescence intensity due to a reaction with FITC-labeled rabbit anti-mouse IgM (which reacts with M45-80) was significantly greater in right-side-out vesicles, further suggesting that M45-80 binds to the exterior of cell membranes.

Conclusion

• These results suggest that M45-80 has a stabilizing effect on one state of the enzyme, the E2 form. This can change the enzyme’s function and the overall balance between sodium and potassium ions within the cell.
• This study could be used as a base for further research on the effects of antibodies on enzyme activity and might be valuable for medical fields dealing with body ion homeostasis.