Alkaline phosphatase was purified from plasma membrane of rat
ascites hepatoma AH-130 cells by chromatographies on
DEAE-cellulose and
Affi-Gel Blue and preparative
polyacrylamide gel electrophoresis. The yield of the purified
enzyme, finally as the denatured subunits, was about three times better than that obtained previously [J. Biochem. (1978) 83, 1471-1483]. The purified
enzyme, a
glycoprotein, was analyzed for
amino acid and
carbohydrate compositions. The composition of the
carbohydrate moiety, which amounted to about 18% by weight, indicated that both N- and O-glycosidic
sugar chains were contained in the
protein. The purified
alkaline phosphatase (as the denatured subunits) was used to produce monospecific antibody, which was found to have the same immunological reaction with the native
antigen as the antibody raised against the partially purified native
enzyme. Immunological analysis by Ouchterlony double gel diffusion and quantitative immunoprecipitation demonstrated that the
hepatoma enzyme was identical with that of the liver. In standard
polyacrylamide gel electrophoresis at pH 8.9, the
hepatoma alkaline phosphatase migrated a little more slowly than the liver
enzyme. Both
enzymes, however, showed identical mobility after complete removal of
sialic acid from them with
neuraminidase. These results suggest that the only difference between the two
enzymes was in the
carbohydrate moiety, especially in the
sialic acid content.