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Alkaline phosphatase (ALP) expressed on the surface of MC 3 T 3-E l cells cultured in 96-well plates was assayed directly with 10mM P-nitrophenyl phosphate (pNPP) on the same plate using a microplate reader, after replacement of culture medium with isotonic alkaline carbonate-bicarbonatebuffered saline. The rate of substrate hydrolysis catalyzed at 24°Cby the ecto-enzyme was maximal at pH 9.8-10.2. The ecto-ALP activity at pH 10 was 43-fold greater than that observed at pH 7.4 and was approximately 80%of the total activity in cell lysates.Furthermore, the ecto-enzyme activity could be measured accurately in proportion to the enzyme level on MC 3 T 3-E 1 cells during 40 min-incubation. The Km of ecto-ALP for pNPP in the carbonate-bicarbonatebuffered saline at pH 10 was 613μM, which was close to the value of the total ALP composed of soluble forms in the cell lysates. The alkaline carbonate-bicarbonate-buffered saline used for the assay did not induce the release of ALP or lactate dehydrogenase from cells, or a change in membrane permeability and shape of cells.Levamisole proved to be an uncompetitive inhibitor for both the ecto-and total ALP activities. There was no difference between the Ki values of these enzyme forms. In contrast, eugeno1, a dental medicament, inhibited only the ecto-ALP activity on intact cells, but did not alter the total ALP activity in disintegrated cells.This study showed a sensitive and simple method for measurement of ecto-enzyme in 96-well plates which is useful for observations of slight alterations in ALP activity in osteoblasts.

Micromethod for the Detection of Ecto-Alkaline Phosphatase Activity on an Osteoblastic Cell Line(MC3T3-E1) in 96-Well Plates.