BMB Reports

Korean Society for Biochemistry and Molecular Biology (생화학분자생물학회)
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Purification and Characterization of Membrane-Bound Phosphatidylinositol 4-Kinase from Mouse Brain

  • Lee, Sang-Min (Department of Biochemistry and Molecular Biology, College of Sciences, Hanyang University) ;
  • Son, Hyeog-Gin (Department of Biochemistry and Molecular Biology, College of Sciences, Hanyang University) ;
  • Lee, Young-Seek (Department of Biochemistry and Molecular Biology, College of Sciences, Hanyang University) ;
  • Lee, Kang-Suk (Laboratory of Radiation Biology, The Atomic Energy Research Institute) ;
  • Rhee, Sue-Goo (Laboratory of Biochemistry, National Heart, Lung, and Blood Institute, National Institutes of Health) ;
  • Cho, Key-Seung (Department of Biochemistry and Molecular Biology, College of Sciences, Hanyang University)
  • Received : 1996.09.18
  • Published : 1996.11.30
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Abstract

A membrane-bound phosphatidylinositol 4-kinase (PI 4-kinase) was separated in a sucrose gradient and solubilized with 1% Triton X-100 from mouse brain. The enzyme was purified 2,952-fold by various chromatographic techniques including DEAE-cellulose, PI-Sepharose and Sephacryl S-200 gel filtration. The molecular weight of PI 4-kinase was approximately 76 kDa by gel filtration and 70.8 kDa by SDS-polyacrylamide gel electrophoresis. The purified enzyme exhibited specific activity of 11.2 nmol/min/mg protein and pi value of 4.7. Kinetic analysis of the PI 4-kinase indicated apparent $K_m$, values of 190 ${\mu}M$ and 120 ${\mu}M$ for phosphatidylinositol and ATP, respectively. The maximal activity of this purified enzyme was observed at pH 7.4 at an incubation temperature of $37^{\circ}C$. The enzyme activity was significantly activated by $Mg^{2+}$, $Mn^{2+}$ and $Fe^{2+}$, and inhibited severely by $Ca^{2+}$. PI 4-kinase was proved to be pure in its immunoblot test by polyclonal antibody prepared from immunized rabbit sera. By this test, we were able to detect the existence of the same type of PI 4-kinase from other mouse organ tissues, such as liver, heart, kidney and spleen. Furthermore, similar immunoblot analysis with the same antisera recognized the different epitopes of PI 4-kinase proteins from various organs of rabbit, chinese hamster and rat.

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