A Gel-electrophoretic Analysis for Improved Sensitivity and Specificity of DNA-dependent Protein Kinase Activity
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概要
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DNA-dependent protein kinase (DNA-PK) is considered a critical enzyme in the repair and/or signal transduction of DNA double-strand breaks. DNA-PK activity has been mostly measured through "DNAplus- minus" or "DNA-pull-down" procedures using synthetic peptide as substrate followed by filter-binding analysis, i.e. liquid scintillation counting of acid-insoluble radioactivity bound to phosphocellulose filter. Considering that non-specific phosphorylation of other cellular proteins in filter-bound acid-insoluble count could interfere with the detection of specific phosphorylation of peptide substrate, we examined the specificity and characteristics of these assay procedures by SDS gel-electrophoresis of the reaction mixture. The electrophoretic pattern showed phosphorylation in wide range of non-specific protein bands other than the specific substrate. The very low DNA-PK activity shown by murine L5178Y or FSA1233 cells was unambiguously detectable as the count in substrate band. Even following DNA-pull-down procedure, which would separate DNA-PK from most of other protein kinases, substantial amount of phosphorylation of other cellular proteins were still contaminated. Thus by selectively counting the particular bands, small amount of specific phosphorylation of peptide substrate was reliably quantified. These results indicated that the DNA-PK activity through filter-binding analysis was, as suspected, contaminated by non-specific phosphorylation of other cellular proteins and also that the gel-electrophoretic analysis would improve detectability of specific phosphorylation by DNA-PK of synthetic peptide substrate and, therefore, would improve the kinase assay in both sensitivity and specificity.
- 日本放射線影響学会の論文
著者
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Suzuki N
Dept. Radiat. Oncol. Grad. Sch. Med. Univ. Tokyo
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Suzuki Norio
Department of Biological Sciences, Graduate School of Science, The University of Tokyo
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Sakai K
Resarch Center For Radiation Protection National Institute Of Radiological Sciences
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Suzuki Norio
東京大学 医系研究 救急医
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MATSUMOTO Yoshihisa
Department of Mechanical Engineering, Oita National College of Technology
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HIRANO Kazuya
Lab. Public Health. Sch. Pharmacy and Life Science.
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HIRANO KAZUYA
Department of Radiation Oncology, Graduate School of Medicine, University of Tokyo
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UMEDA NORIKO
Department of Radiation Oncology, Graduate School of Medicine, University of Tokyo
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SAKAI KAZUO
Department of Radiation Oncology, Graduate School of Medicine, University of Tokyo
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Matsumoto Yukiko
Radiat. Biol. Kyoto Univ.
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Matsumoto Yoshihisa
Department Of Anesthesiology And Intensive Care Medicine Graduate School Of Medical Science Kanazawa
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Matsumoto Yoshihisa
Res. Lab. For Nuclear Reactors And Dep. Of Nuclear Engineering Tokyo Inst. Of Technol.
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Umeda Noriko
Depts. Radiat. Res. And Radiat. Oncol. Grad. Sch. Med. Univ. Tokyo:tokyo Univ. Pharm. Life Sci.
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Sakata Koh-ichi
Department Of Radiology Sapporo Medical University School Of Medicine
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Suzuki Norio
Department Of Biological Sciences Graduate School Of Science Hokkaido University
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SAKAI Kazuo
School of Law, Meiji University
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Sakai Kazuo
Department Of Physics Faculty Of Science Science University Of Tokyo
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Matsumoto Yoshihisa
Atomic Bomb Disease Institute Graduate School Of Biochemical Sciences Nagasaki University
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Suzuki Norio
Department Of Agricultural Chemistry The University Of Tokyo
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Sakai Kazuo
Department of Computer Science and Media Engineering, Faculty of Engineering, Yamanashi University
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