Characterization of a putative Ca2(+)-transporting Ca2(+)-ATPase in the pellicles of Paramecium tetraurelia.
In Paramecium, no Ca2(+)-ATPases with the properties of Ca2+ pumps have been identified. Here we report a pellicle associated Ca2(+)-ATPase activity and a corresponding phosphoprotein intermediate characteristic of a pump. The Ca2(+)-ATPase activity requires 3 mM Mg for optimal Ca2+ stimulation (KCa = 90 nM) and is specific for ATP as substrate (Km = 75 microM). Vanadate and calmidazolium inhibit Ca2(+)-stimulated activity with an EC50 of about 2 microM and 0.5 microM, respectively. Likewise, 10 microM trifluoperazine inhibits 80% of Ca2(+)-ATPase activity, but bovine calmodulin fails to stimulate. The Ca2(+)-ATPase is not inhibited by sodium azide (10 mM), oligomycin (10 micrograms/ml) or ouabain (0.2 mM). Incubation of pellicles with [gamma-32P]ATP specifically labels a 133 kDa protein in a Ca2(+)-dependent, hydroxylamine-sensitive manner, and the level of phosphorylation is increased by 100 microM La3+. Phosphorylation of an endoplasmic reticulum-enriched fraction labels a Ca2(+)-dependent protein different from the pellicle protein, being lower in molecular mass and unaffected by La3+. Ca2+ uptake by the alveolar sacs, integral components of the pellicle membrane complex, is poorly coupled to Ca2(+)-stimulated ATP hydrolysis (Ca2+ transported/ATP hydrolysed less than 0.2) and is much less sensitive to vanadate inhibition (EC50 approx. 20 microM) compared to the total Ca2(+)-ATPase activity. Therefore, the majority of the Ca2(+)-ATPase activity is likely to be plasma membrane associated.
Wright
M V
MV
Department of Zoology, University of Vermont, Burlington 05405.
van Houten
J L
JL
D000255:Adenosine Triphosphate / Q000378:metabolism; D000818:Animals; D002118:Calcium / Q000378:metabolism* / Q000494:pharmacology; D000252:Calcium-Transporting ATPases / Q000032:analysis* / Q000037:antagonists & inhibitors; D004721:Endoplasmic Reticulum / Q000201:enzymology; D006863:Hydrogen-Ion Concentration; D008274:Magnesium / Q000494:pharmacology; D010247:Paramecium / Q000201:enzymology*; D013379:Substrate Specificity
D000255:Adenosine Triphosphate; D000252:Calcium-Transporting ATPases; D008274:Magnesium; D002118:Calcium