Intestinal-type alkaline phosphatase produced by human hepatoblastoma cell line HUH-6 clone 5.
Two enzyme forms of alkaline phosphatase have been partially purified from the medium spent for the culture of HUH-6 clone 5 cells, which were originally derived from hepatoblastoma tissue. The purification methods used are ammonium sulfate precipitation, ethanol precipitation, diethylaminoethyl cellulose chromatography, Affi-Gel Blue chromatography, and Sephadex G-200 gel filtration. These alkaline phosphatases have been characterized by thermostability, inhibition, and immunological and electrophoretic studies. Both are L-phenylalanine and L-tryptophan sensitive and L-homoarginine and L-leucylglycylglycine insensitive, and both react with an antiserum against intestinal alkaline phosphatase. The major enzyme form is a neuraminidase-cleavable, moderately thermostable isoenzyme which on polyacrylamide gel shows an electrophoretic mobility similar to that of liver alkaline phosphatase. The minor enzyme form is a neuraminidase-uncleavable, thermolabile isoenzyme which shows an intermediate electrophoretic mobility between liver and hepatoma alkaline phosphatases. The molecular weights of the major and minor enzymes have been estimated by gel filtration to be 170,000 and 110,000, respectively. These results support the conclusion that the two enzyme forms of HUH-6 alkaline phosphatase are intestinal in type, with the major enzyme form closely resembling hepatoma and oncoamnionic alkaline phosphatases, and the minor enzyme form resembling "intestine-like liver alkaline phosphatase." HUH-6 clone 5 cell line may be a useful in vitro model to study the regulatory mechanism for phenotypic expression of intestinal-type alkaline phosphatase isoenzymes in liver cancer cells.
D000469:Alkaline Phosphatase / Q000302:isolation & purification / Q000378:metabolism*; D006528:Carcinoma, Hepatocellular / Q000201:enzymology*; D002460:Cell Line; D002999:Clone Cells; D006801:Humans; D007422:Intestines / Q000201:enzymology*; D007527:Isoenzymes / Q000302:isolation & purification / Q000378:metabolism*; D007700:Kinetics; D008113:Liver Neoplasms / Q000201:enzymology*
D007527:Isoenzymes; D000469:Alkaline Phosphatase