Specific effects of spermine on ouabain-sensitive and potassium-dependent phosphatase activity of kidney plasma membranes Specificity of the potassium sites

Abstract

Specific inhibition of ouabain-sensitive and K +-dependent p-nitrophenyl phosphatase activity of rabbit kidney plasma membranes by spermine ( N, N′bis(3-aminopropyl)-1,4-butanediamine) was characterized kinetically. 1. 1. Inhibition by spermine was competitive with K +. The K i for spermine was 31 μM in the presence of 1 mM Mg 2+. 2. 2. Excess Mg 2+ inhibited the ouabain-sensitive phosphatase activity in competition with K +. The K i for Mg 2+ was 2.6 mM. 3. 3. Increasing Mg 2+ concentrations reduced the spermine inhibition. This could be observed at Mg 2+ concentrations higher than that of K +. 4. 4. In the absence of inhibition by Mg 2+, spermine was noncompetitive with Mg 2+ which was essential for the ouabain-sensitive phosphatase activity. This could be observed at Mg 2+ concentrations lower than that of K +. 5. 5. Although Ca 2+ was a strong inhibitor of the ouabain-sensitive phosphatase activity in the presence of K +, it produced a small stimulation of the activity in the absence of K +. Approximately 0.1 mM Ca 2+ gave the maximum stimulation. 6. 6. The observed Ca 2+− and Mg 2+-dependent phosphatase activity was inhibited strongly by ouabain and by spermine. The half-maximal inhibition concentrations of ouabain and spermine were 0.1 and 63 μM, respectively. It is likely that Mg 2+, Ca 2+ and spermine bind to the same site as does K +.