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Ecto-alkaline phosphatase activity identified at physiological pH range on intact P19 and HL-60 cells is induced by retinoic acid

: Scheibe, R.J.; Kuehl, H.; Krautwald, S.; Meissner, J.D.; Mueller, W.H.


Journal of cellular biochemistry 76 (2000), Nr.3, S.420-436
ISSN: 0730-2312
ISSN: 1097-4644
Fraunhofer ITEM ()

The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 myeloblastic leukemia cells was studied at physiological pH using p- nitrophenylphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations of 30 min and was optimal at micromolar substrate concentrations over the pH range 7.4-8.5. The value of apparent K(m) for pNPP in P19 and HL- 60 cells was 120 M. Hydrolytic activity of the ecto-enzyme at physiological pH decreased by the addition of levamisole, a specific and noncompetitive inhibitor of ALP (K(i) P19 = 57 M; K(i) HL-60 = 50 M). Inhibition of hydrolysis was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamisole-sensitive ectophosphohydrolase activity at pH 7.4. After its autophosphorylation by e cto-kinase activity, a 98-kDa membrane protein in P19 cells was found to be sensitive to ecto-ALP, and protein dephosphorylation increased after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an inhibitor of all phosphatase activities, blocked the levamisole-sensitive dephosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstrate that the levamisole-sensitive phosphohydrolase activity on the cell surface is consistent with ecto-ALP activity degrading both physiological concentrations of exogenously added substrate and endogenous surface phosphoproteins under physiological pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiating P19 teratocarcinoma and HL-60 myeloblastic leukemia cells.