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

The activity of membrane-bound alkaline phosphatase (ALP) expressed on the external surface of cultured murine P19 teratocarcinoma and human HL-60 mye loblastic leukemia cells was studied at physiological pn using p-nitropheny lphosphate (pNPP) as substrate. The rate of substrate hydrolysis catalyzed by intact viable cells remained constant for eight successive incubations o f 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 w as 120 mu M. Hydrolytic activity of the ecto-enzyme at physiological pH dec reased by the addition of levamisole, a specific and noncompetitive inhibit or of ALP (K-i P19 = 57 mu M; K-i HL-60 = 50 mu M). Inhibition of hydrolysi s was reversed by removal of levamisole within 30 min. Retinoic acid (RA), which promotes the differentiation of P19 and HL-60 cells, induced levamiso le-sensitive ecto-phosphohydrolase activity at pH 7.4. After its autophosph orylation by ecto-kinase activity, a 98-kDa membrane protein in P19 cells w as found to be sensitive to ecto-ALP, and protein dephosphorylation increas ed after incubation of cells with RA for 24 h and 48 h. Orthovanadate, an i nhibitor of all phosphatase activities, blocked the levamisole-sensitive de phosphorylation of the membrane phosphoproteins, while (R)-(-)-epinephrine reversed the effect by complexation of the inhibitor. The results demonstra te that the levamisole-sensitive phosphohydrolase activity on the cell surf ace is consistent with ecto-ALP activity degrading both physiological conce ntrations of exogenously added substrate and endogenous surface phosphoprot eins under physioiogical pH conditions. The dephosphorylating properties of ecto-ALP are induced by RA, suggesting a specific function in differentiat ing P19 teratocarcinoma and HL-GO myeloblastic leukemia cells. (C) 2000 Wil ey-Liss, Inc.