Chemical and immunological characterization of rat ascites hepatoma alkaline phosphatase: a comparison with the liver enzyme.
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.
D000469:Alkaline Phosphatase / Q000276:immunology / Q000302:isolation & purification / Q000378:metabolism*; D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002241:Carbohydrates / Q000032:analysis; D002462:Cell Membrane / Q000201:enzymology; D004591:Electrophoresis, Polyacrylamide Gel; D008099:Liver / Q000201:enzymology*; D008114:Liver Neoplasms, Experimental / Q000201:enzymology*; D008297:Male; D011233:Precipitin Tests; D051381:Rats; D012794:Sialic Acids / Q000032:analysis
D000596:Amino Acids; D002241:Carbohydrates; D012794:Sialic Acids; D000469:Alkaline Phosphatase