Purification and characterization of nucleoside diphosphate kinase from the brain of Bombyx mori.
Nucleoside diphosphate kinase in the brain of Bombyx mori was purified by ammonium sulfate fractionation, and a sequence of chromatographies on DEAE-Cellulofine, hydroxyapatite, Mono-S, and Mono-Q column. The purified enzyme preparation was found to be electrophoretically homogeneous on SDS-PAGE, and its molecular mass was determined to be 18 kDa. The purified protein was digested and the amino acid sequences of resulting peptides were determined. The enzyme showed high similarity to the amino acid sequences of the Drosophila NDP kinase. The enzyme showed NDP kinase activity and mediated the phosphorylation of myelin basic protein. Gel filtration and Hill plot analysis indicate that the purified NDP kinase forms a tetramer and shows little interaction among substrates. Dephosphorylation of NDP kinase by bacterial alkaline phosphatase increased NDP kinase activity. This result indicates that phosphorylation of NDP kinase represses NDP kinase activity.
Laboratory of Biological Chemistry, Department of Biofunctional Chemistry, Faculty of Agriculture, Kobe University, Nada-ku, Kobe Hyogo, Japan. unotom@kobe-u.ac.jp
Tomohide
T
Uno
Mayumi
M
Ueno
Michiko
M
Kikuchi
Yasuo
Y
Aizono
D000595:Amino Acid Sequence; D000818:Animals; D012831:Bombyx / Q000201:enzymology*; D001921:Brain / Q000201:enzymology*; D004331:Drosophila melanogaster; D008969:Molecular Sequence Data; D009701:Nucleoside-Diphosphate Kinase / Q000032:analysis*; D010766:Phosphorylation; D020539:Sequence Analysis, Protein; D017386:Sequence Homology, Amino Acid
D009701:Nucleoside-Diphosphate Kinase