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 microM. 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 microM; K(i) HL-60 = 50 microM). 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 ecto-
phosphohydrolase activity at pH 7.4. After its autophosphorylation by ecto-
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.