Characterization of Ca2+- or Mg2+-ATPase of the excitable ciliary membrane from Paramecium tetraurelia: comparison with a soluble Ca2+-dependent ATPase.
We have characterized divalent-cation-stimulated nucleoside triphosphate hydrolase activity of the excitable ciliary membrane and compared it with a soluble Ca2+-ATPase released upon deciliation of Paramecium. The membrane-bound activity is strongly dependent on a divalent cation; calcium stimulates the basal activity of this enzyme at least 10-fold; magnesium and manganese stimulate less well, and strontium and barium, although less effective, also give measurable stimulation. This membrane-bound activity prefers ATP and GTP as substrates but also hydrolyzes UTP and CTP at measurable rates. The maximum velocity at saturating ATP concentrations and optimal calcium concentrations is 0.3 mumol/min per mg. The pH optimum for the membrane-bound activity is broad and centers around pH 7. From the temperature dependence of ATP hydrolysis, we calculate activation energies of 14 and 11 kcal/mol for the Ca2+- and Mg2+-stimulated activities, respectively. The Arrhenius plot is linear over the temperature range of 4 to 25 degrees C. The membrane ATPase is relatively insensitive to ouabain, oligomycin, N,N'-dicyclohexylcarbodiimide, vanadate, Ruthenium red and two calmodulin antagonists. Polyclonal antisera raised against the purified soluble ATPase from the deciliation supernatant show low reactivity with the membrane-bound ATPase. We conclude from the comparison of properties of the two activities that the ciliary membrane-bound ATPase is distinct from the soluble ATPase released by deciliation.
D000818:Animals; D017301:Ca(2+) Mg(2+)-ATPase / Q000032:analysis*; D002118:Calcium / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000032:analysis*; D002147:Calmodulin / Q000037:antagonists & inhibitors; D002923:Cilia / Q000201:enzymology*; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D009705:Nucleosides / Q000378:metabolism; D010247:Paramecium / Q000201:enzymology; D012430:Ruthenium Red / Q000494:pharmacology; D012995:Solubility; D013696:Temperature; D014638:Vanadates; D014639:Vanadium / Q000494:pharmacology
D002147:Calmodulin; D009705:Nucleosides; D014639:Vanadium; D012430:Ruthenium Red; D014638:Vanadates; D017301:Ca(2+) Mg(2+)-ATPase; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium