Occurrence of a para-nitrophenyl phosphate-phosphatase with calcineurin-like characteristics in Paramecium tetraurelia.
Using para-nitrophenyl phosphate (pNPP) as a substrate for enzymatic activity, we sought to identify CaN in Paramecium. We isolated three different pNPP-phosphatases from the soluble fraction of Paramecium cells by anion-exchange and affinity column chromatographies. One, pNPP-phosphatase Peak I, is very similar to mammalian CaN. Divalent cation dependency, inhibition by calmodulin (CaM) antagonists (trifluoperazine, calmidazolium), and insensitivity to various phosphatase inhibitors (heparin, okadaic acid, sodium vanadate, etc.) show similarity to mammalian CaN rather than to any other Paramecium pNPP-hydrolyzing enzymes tested. Polyclonal antibodies against bovine brain CaN recognizing subunits A (61 or 58 kDa) and B (17 kDa) of brain CaN cross-reacted with a 63-kDa protein in fractions containing Peak IpNPP-phosphatase activity and coeluted calmodulin. Overlay assays using biotinylated brain calmodulin indicated Ca2+-dependent CaM-binding by the 63-kDa protein. A Ca2+-binding protein with the same electrophoretic mobility as CaN B (17 kDa) was also present, though in other fractions from DEAE-cellulose chromatography. This finding strongly suggests that, in the absence of Ca2+, both subunits, A and B, were separated either before or during chromatographic processing. Our data support the existence of both subunits of a CaN-like phosphatase in Paramecium cells.
Kissmehl
R
R
Faculty of Biology, University of Konstanz, Germany. Roland.Kissmehl@uni-konstanz.de
Treptau
T
T
Kottwitz
B
B
Plattner
H
H
D009597:4-Nitrophenylphosphatase / Q000302:isolation & purification / Q000378:metabolism*; D000818:Animals; D015153:Blotting, Western; D001921:Brain / Q000201:enzymology; D019703:Calcineurin; D002118:Calcium / Q000494:pharmacology; D002135:Calcium-Binding Proteins / Q000378:metabolism; D002147:Calmodulin / Q000494:pharmacology; D002148:Calmodulin-Binding Proteins / Q000302:isolation & purification / Q000378:metabolism*; D002413:Cations, Divalent / Q000494:pharmacology; D002417:Cattle; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D004791:Enzyme Inhibitors / Q000494:pharmacology; D008345:Manganese / Q000494:pharmacology; D008670:Metals / Q000494:pharmacology; D009596:Nitrophenols / Q000378:metabolism*; D009943:Organophosphorus Compounds / Q000378:metabolism*; D016805:Paramecium tetraurelia / Q000201:enzymology*; D010749:Phosphoprotein Phosphatases / Q000302:isolation & purification / Q000378:metabolism*
D002135:Calcium-Binding Proteins; D002147:Calmodulin; D002148:Calmodulin-Binding Proteins; D002413:Cations, Divalent; D004791:Enzyme Inhibitors; D008670:Metals; D009596:Nitrophenols; D009943:Organophosphorus Compounds; C008644:nitrophenylphosphate; D008345:Manganese; D019703:Calcineurin; D010749:Phosphoprotein Phosphatases; D009597:4-Nitrophenylphosphatase; D002118:Calcium