title: The ATPase activity of saponin-treated rat erythrocytes: regulation by monovalent cations, calcium, ouabain, and furosemide.
abstract: 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.
authors.affiliation: Institute of Biological Physics, USSR Academy of Sciences, Pushchino, Moscow Region.
authors.forename: V V
authors.initials: VV
authors.lastname: Petrunyaka
authors.forename: E A
authors.initials: EA
authors.lastname: Panyushkina
authors.forename: E P
authors.initials: EP
authors.lastname: Severina
authors.forename: S N
authors.initials: SN
authors.lastname: Orlov
mesh_terms: D000251:Adenosine Triphosphatases / Q000378:metabolism*; D000818:Animals; D002021:Buffers; D017301:Ca(2+) Mg(2+)-ATPase / Q000378:metabolism; D002118:Calcium / Q000494:pharmacology*; D000252:Calcium-Transporting ATPases / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D002414:Cations, Monovalent / Q000494:pharmacology*; D002463:Cell Membrane Permeability; D004305:Dose-Response Relationship, Drug; D004848:Epithelium / Q000187:drug effects / Q000201:enzymology; D005665:Furosemide / Q000494:pharmacology*; D007093:Imidazoles / Q000494:pharmacology; D007668:Kidney / Q000187:drug effects / Q000201:enzymology*; D007700:Kinetics; D008297:Male; D010042:Ouabain / Q000494:pharmacology*; D051381:Rats; D012503:Saponins / Q000494:pharmacology*; D000254:Sodium-Potassium-Exchanging ATPase / Q000378:metabolism
chemical_list: D002021:Buffers; D002147:Calmodulin; D002414:Cations, Monovalent; D007093:Imidazoles; D012503:Saponins; C031938:calmidazolium; D010042:Ouabain; D005665:Furosemide; D000251:Adenosine Triphosphatases; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D000254:Sodium-Potassium-Exchanging ATPase; D002118:Calcium