Nucleoside diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii: purification and characterization.
An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23,000) and of the oligomer determined by sedimentation equilibrium experiments (125,000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [gamma-32P]ATP, and Mg2+ was required for the incorporation of phosphate. The kinase preserved the ability to transfer gamma-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms.
Institute of Protein Research, Russian Academy of Sciences, Moscow Region, Russia.
Polosina YYu
K F
KF
Jarrell
O V
OV
Fedorov
A S
AS
Kostyukova
D000595:Amino Acid Sequence; D000818:Animals; D004777:Environment; D006801:Humans; D006863:Hydrogen-Ion Concentration; D008969:Molecular Sequence Data; D008970:Molecular Weight; D019633:Natronobacterium / Q000201:enzymology* / Q000235:genetics; D009701:Nucleoside-Diphosphate Kinase / Q000235:genetics / Q000302:isolation & purification* / Q000378:metabolism; D011487:Protein Conformation; D017386:Sequence Homology, Amino Acid; D012965:Sodium Chloride
D012965:Sodium Chloride; D009701:Nucleoside-Diphosphate Kinase