The structure of chicken liver xanthine dehydrogenase. cDNA cloning and the domain structure.
The amino acid sequence of chicken liver xanthine dehydrogenase (EC 1.1.1.204) was determined by cDNA cloning and partial amino acid sequencing of the purified enzyme. The enzyme consisted of 1358 amino acids with calculated molecular mass of 149,633 Da. In order to compare the structure of the chicken and rat enzymes, limited proteolysis was performed with the purified chicken liver xanthine dehydrogenase. When the enzyme was digested with subtilisin, it was not converted from the NAD-dependent dehydrogenase type to the O2-dependent oxidase type, in contrast with the mammalian enzyme. However, the enzyme was cleaved mainly into three fragments in a manner similar to that for the rat enzyme at pH 8.2 (20, 37, and 84 kDa) and retaining a full complement of redox centers. The cleavage sites were identified by determination of amino-terminal sequences of the produced fragments. It was concluded that the 20-kDa fragment was amino-terminal, the 84-kDa fragment carboxyl-terminal, and the 37-kDa fragment an intermediate portion in the enzyme protein. On the other hand, when the enzyme was digested with the same protease at pH 10.5, the sample contained only the 20- and 84-kDa portions and lacked the 37-kDa portion. The resultant sample possessed xanthine dichlorophenol indophenol reductase activity, indicating that the molybdenum center remained intact. The absorption spectrum showed the sample was very similar to deflavo-enzyme. From these results and sequence analyses, the domain structure of the enzyme is discussed.
Department of Biochemistry, Yokohama City University School of Medicine, Japan.
A
A
Sato
T
T
Nishino
K
K
Noda
Y
Y
Amaya
T
T
Nishino
D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D002645:Chickens; D003001:Cloning, Molecular; D018076:DNA, Complementary; D006868:Hydrolysis; D007506:Iron-Sulfur Proteins / Q000737:chemistry; D008099:Liver / Q000201:enzymology*; D008969:Molecular Sequence Data; D008982:Molybdenum / Q000737:chemistry; D010446:Peptide Fragments / Q000737:chemistry; D017386:Sequence Homology, Amino Acid; D013379:Substrate Specificity; D013381:Subtilisins / Q000737:chemistry; D014968:Xanthine Dehydrogenase / Q000737:chemistry* / Q000235:genetics
D018076:DNA, Complementary; D007506:Iron-Sulfur Proteins; D010446:Peptide Fragments; D008982:Molybdenum; D014968:Xanthine Dehydrogenase; D013381:Subtilisins