The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide

The ATPase activities were studied in rat erythrocytes permeabilized with saponin. The concentrations of calcium and magnesium ions were varied within the range of 0.1-60 microM and 50-370 microM, respectively, by using EGTA-citrate buffer. The maximal activity of Ca2(+)-ATPase of permeabilized erythrocytes was by one order of magnitude higher, whereas the Ca2(+)-binding affinity was 1.5-2 times higher than that in erythrocyte ghosts washed an isotonic solution containing EGTA. Addition of the hemolysate restored the kinetic parameters of ghost Ca2(+)-ATPase practically completely, whereas in the presence of exogenous calmodulin only part of Ca2(+)-ATPase activity was recovered. Neither calmodulin nor R24571, a highly potent specific inhibitor of calmodulin-dependent reactions, influenced the Ca2(+)-ATPase activity of permeabilized erythrocytes. At Ca2+ concentrations below 0.7 microM, ouabain (0.5-1 mM) activated whereas at higher Ca2+ concentrations it inhibited the Ca2(+)-ATPase activity. Taking this observation into account the Na+/K(+)-ATPase was determined as the difference of between the ATPase activities in the presence of Na+ and K+ and in the presence of K+ alone. At physiological concentration of Mg2+ (370 microM), the addition of 0.3-1 microM Ca2+ increased Na+/K(+)-ATPase activity by 1.5-3-fold. Higher concentrations of this cation inhibited the enzyme. At low Mg2+ concentration (e.g., 50 microM) only Na+/K(+)-ATPase inhibition by Ca2+ was seen. It was found that at NaCl] less than 20 mM furosemide was increased ouabain-inhibited component of ATPase in Ca2(+)-free media. This activating effect of furosemide was enhanced with a diminution of Na+] upto 2 mM and did not reach the saturation level unless the 2 mM of drug was used. The activating effect of furosemide on Na+/K(+)-ATPase activity confirmed by experiments in which the ouabain-inhibited component was measured by the 86Rb+ influx into intact erythrocytes.