Divalent cation-dependent ATPase activities in ciliary membranes and other surface structures in Paramecium tetraurelia: Comparative in vitro studies

Cilia membrane preparations from axenically grown Paramecium contain ATPase activities with distinct electrophoretic mobilities on Triton-polyacrylamide gels M. J. Doughty and E. S. Kaneshiro (1983) J. Protozool.30, 569–575]. Such gel analyses also show additional ATPase activity bands associated with ciliary axonemes (dyneins), cell pellicles, exocytotic trichocysts, and the external cell surface (ectoenzyme). In the present report, the in vitro properties of these activities in various cell fractions were compared. The activity in ciliary membranes was stimulated by Ca²⁺ > Mg²⁺, in pellicles by Ca²⁺ > Mg²⁺, and in trichocysts by Ca²⁺ = Mg²⁺. The ecto-ATPase was strictly Ca²⁺ dependent. Determination of the affinities for various phosphate-containing substrates showed that the activities in all fractions were nucleoside triphosphate phosphohydrolases. Unlike the axonemal dynein ATPases, all other fractions were vanadate- and p-chloromercuribenzoate-insensitive. Activities in all cell fractions were sensitive to ruthenium red, the ciliary membrane being the most sensitive (K_i = 4 μm). The ciliary membrane Ca²⁺ ATPase activity exhibited an apparent affinity for CaATP²⁻ of 9 μm and was inhibited by other divalent cations, La³⁺, and phosphate, but not by ADP or AMP. The kinetic properties of the ciliary membrane Ca²⁺ ATPase activity in wild type and several behavioral mutants were similar except for those in the pawn mutant, d₄95, and the paranoiac mutant, d₄90, both of which had lower specific activities. These studies support the finding that the ciliary membrane ATPase activity of Paramecium is a specific Ca²⁺-dependent ATPase distinct from other divalent cation-dependent ATPase activities found in either the cilia or other cell surface structures.