Studies on the induction and phosphorylation of xanthine dehydrogenase in cultured chick embryo hepatocytes.
Chick embryo hepatocytes, cultured in a chemically defined medium, were used to investigate hormonal requirements for xanthine-dehydrogenase induction and to determine whether the enzyme is phosphorylated. Triiodothyronine is found to be required to induce the synthesis of active enzyme. Inclusion of sodium tungstate in the medium resulted in the complete loss of enzyme activity but no decrease of immunochemically detectable levels of enzyme. Immunoprecipitated xanthine dehydrogenase from cell extracts migrates with enzyme purified from adult chicken liver on SDS/PAGE. Both the native 150-kDa subunit and the 130-kDa form of the enzyme is observed. N-terminal sequence analysis of the 150-kDa subunit shows the following; Ala-Pro-Pro-Glu-Thr-Gly-Asp-Glu-Leu-Val-Phe-Phe-Val-Asn-Gly-Lys-Lys-Val- Val which is similar to the published N-terminal sequences of rat, mouse and insect xanthine dehydrogenases. Autoradiography of denaturing gels of xanthine dehydrogenase isolated from 32P(i)-labeled hepatocytes demonstrates that the 150-kDa and the 130-kDa forms of the enzyme are phosphorylated. Chemical phosphate analysis of acid-precipitated, electrophoretically pure chicken liver xanthine dehydrogenase also shows the presence of covalently bound phosphate. Phosphoamino acid analysis of both 32P-labeled forms of the enzyme demonstrates the presence of phosphoserine. Thus, chicken liver xanthine dehydrogenase contains a phosphoserine residue as found previously in bovine milk xanthine oxidase [Davis, M. D., Edmondson, D. E. & Müller, F. (1984) Eur. J. Biochem. 145, 237-250].
Schieber
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Department of Biochemistry, Emory University School of Medicine, Atlanta, GA 30322.
Edmondson
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D000595:Amino Acid Sequence; D000818:Animals; D015153:Blotting, Western; D002478:Cells, Cultured; D002642:Chick Embryo; D003907:Dexamethasone / Q000494:pharmacology; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction; D007328:Insulin / Q000494:pharmacology; D008099:Liver / Q000166:cytology / Q000201:enzymology*; D008969:Molecular Sequence Data; D010766:Phosphorylation; D010768:Phosphoserine / Q000032:analysis; D016415:Sequence Alignment; D014284:Triiodothyronine / Q000494:pharmacology; D014968:Xanthine Dehydrogenase / Q000096:biosynthesis / Q000737:chemistry / Q000378:metabolism*
D007328:Insulin; D014284:Triiodothyronine; D010768:Phosphoserine; D003907:Dexamethasone; D014968:Xanthine Dehydrogenase