Characterization of a putative Ca 2+-transporting Ca 2+-ATPase in the pellicles of Paramecium tetraurelia

Abstract

In Paramecium, no Ca 2+-ATPases with the properties of Ca 2+ pumps have been identified. Here we report a pellicle associated Ca 2+-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca 2+-ATPase activity requires 3 mM Mg for optimal Ca 2+ stimulation ( K Ca = 90 nM) and is specific for ATP as substrate ( K m = 75 μM). Vanadate and calmidazolium inhibit Ca 2+-stimulated activity with an EC 50 of about 2 μM and 0.5 μM, respectively. Likewise, 10 μM trifluoperazine inhibits 80% of Ca 2+-ATPase activity, but bovine calmodulin fails to stimulate. The Ca 2+-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 μg/ml) or ouabain (0.2 mM). Incubation of pellicles with [γ- 32P]ATP specifically labels a 133 kDa protein in a Ca 2+-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 μM La 3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca 2+-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La 3+. Ca 2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca 2+-stimulated ATP hydrolysis (Ca 2+ transported/ATP hydrolysed < 0.2) and is much less sensitive to vanadate inhibition (EC 50 approx. 20 μM) compared to the total Ca 2+-ATPase activity. Therefore, the majority of the Ca 2+-ATPase activity is likely to be plasma membrane associated.