ECTO-ALKALINE PHOSPHATASE CONSIDERED AS LEVAMISOLE-SENSITIVE PHOSPHOHYDROLASE AT PHYSIOLOGICAL PH RANGE DURING MINERALIZATION IN CULTURED FETAL CALVARIA CELLS

Alkaline phosphatase (ALP) activity expressed on the external surface of cultured fetal rat calvaria cells and its relationship with mineral deposition were investigated under pH physiological conditions. After replacement of culture medium by assay buffer and addition of p-nitro phenyl phosphate (pNPP), the rate of substrate hydrolysis catalyzed by whole cells remained constant for up to seven successive incubations of 10 min and was optimal over the pH range 7.6-8.2. It was decreased by levamisole by a 90% inhibition at 1 mM which was reversible within 10 min, dexamisole having no effect. Values of apparent Km for pNPP we re close to 0.1 mM, and inhibition of pNPP hydrolysis by levamisole wa s uncompetitive (K-i = 45 mu M). Phosphatidylinositol-specific phospho lipase C (PI-PLC) produced the release into the medium of a p-nitrophe nyl phosphatase (pNPPase) sensitive to levamisole at pH 7.8. The relea sed activity whose rate was constant up to 75 min represented after 15 min 60% of the value of ecto-pNPPase activity. After 75 min of PI-PLC treatment the ecto-pNPPase activity remained unchanged despite the 30 % decrease in Nonidet P-40-extractable ALP activity. High levels of Ca -45 incorporation into cell layers used as index of mineral deposition were decreased by levamisole in a stereospecific manner after 4 h, an effect which was reversed within 4 h after inhibitor removal, in acco rdance with ecto-pNPPase activity variations. These results evidenced the levamisole-sensitive activity of a glycosylphosphatidylinositol-an chored pNPPase consistent with ALP acting as an ecto-enzyme whose func tioning under physiological conditions was correlated to Ca-45 incorpo ration and permit the prediction of the physiological importance of th e enzyme dynamic equilibrium at the cell surface in cultured fetal cal varia cells. (C) 1996 Wiley-Liss, Inc.