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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
Author(s) -
Scheibe Renate J.,
Kuehl Henning,
Krautwald Stefan,
Meissner Joachim D.,
Mueller Walter H.
Publication year - 2000
Publication title -
journal of cellular biochemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 1.028
H-Index - 165
eISSN - 1097-4644
pISSN - 0730-2312
DOI - 10.1002/(sici)1097-4644(20000301)76:3<420::aid-jcb10>3.0.co;2-f
Subject(s) - dephosphorylation , biochemistry , levamisole , alkaline phosphatase , chemistry , substrate (aquarium) , phosphatase , retinoic acid , enzyme , microbiology and biotechnology , biology , ecology , gene , immunology
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 ( p NPP) 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 p NPP in P19 and HL‐60 cells was 120 μM. 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 μM; K i HL‐60 = 50 μM). 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. J. Cell. Biochem. 76:420–436, 2000. © 2000 Wiley‐Liss, Inc.