OCCURRENCE OF A PARA-NITROPHENYL PHOSPHATE-PHOSPHATASE WITH CALCINEURIN-LIKE CHARACTERISTICS IN PARAMECIUM-TETRAURELIA

Using para-nitrophenyl phosphate (pNPP) as a substrate for enzymatic a ctivity, we sought to identify CaN in Paramecium. We isolated three di fferent pNPP-phosphatases from the soluble fraction of Paramecium cell s by anion-exchange and affinity column chromatographies. One, pNPP-ph osphatase Peak I, is very similar to mammalian CaN, Divalent cation de pendency, inhibition by calmodulin (CaM) antagonists (trifluoperazine, calmidazolium), and insensitivity to various phosphatase inhibitors ( heparin, okadaic acid, sodium vanadate, etc.) show similarity to mamma lian CaN rather than to any other Paramecium pNPP-hydrolyzing enzymes tested. Polyclonal antibodies against bovine brain CaN recognizing sub units A (61 or 58 kDa) and B (17 kDa) of brain CaN cross-reacted with a 63-kDa protein in fractions containing Peak I pNPP-phosphatase activ ity and coeluted calmodulin. Overlay assays using biotinylated brain c almodulin 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-cellul ose chromatography. This finding strongly suggests that, in the absenc e of Ca2+, both subunits, A and B, were separated either before or dur ing chromatographic processing, Our data support the existence of both subunits of a CaN-like phosphatase in Paramecium cells. (C) 1997 Acad emic Press.