Extremophiles

, Volume 2, Issue 3, pp 333–338

Nucleoside diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii: purification and characterization

  • Yaroslava Y. Polosina
  • Ken F. Jarrell
  • Oleg V. Fedorov
  • A. S. Kostyukova

Abstract

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 γ-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 [γ-32P]ATP, and Mg2+ was required for the incorporation of phosphate. The kinase preserved the ability to transfer γ-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.

Key words Nucleoside diphosphate kinase Archaea Haloalkaliphiles 

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Copyright information

© Springer-Verlag Tokyo 1998

Authors and Affiliations

  • Yaroslava Y. Polosina
    • 1
  • Ken F. Jarrell
    • 2
  • Oleg V. Fedorov
    • 1
  • A. S. Kostyukova
    • 1
  1. 1.Institute of Protein Research, Russian Academy of Sciences, 142292 Pushchino, Moscow Region, Russia Tel./Fax +7-095-9240493 e-mail: supra@sun.ipr.serpukhov.suRU
  2. 2.Department of Microbiology and Immunology, Queen's University, Kingston, Ontario K7L 3N6, CanadaCA

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