A gel-electrophoretic analysis for improved sensitivity and specificity of DNA-dependent protein kinase activity.
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 "DNA-plus-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.
Matsumoto
Y
Y
Department of Radiation Oncology, University of Tokyo, Japan.
Umeda
N
N
Suzuki
N
N
Sakai
K
K
Hirano
K
K
D000595:Amino Acid Sequence; D000818:Animals; D004247:DNA / Q000378:metabolism / Q000528:radiation effects; D004249:DNA Damage; D004260:DNA Repair; D051747:DNA-Activated Protein Kinase; D004268:DNA-Binding Proteins; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D006801:Humans; D051379:Mice; D008969:Molecular Sequence Data; D009687:Nuclear Proteins; D010455:Peptides / Q000737:chemistry; D017346:Protein Serine-Threonine Kinases / Q000032:analysis*; D012680:Sensitivity and Specificity; D013379:Substrate Specificity; D014407:Tumor Cells, Cultured
D004268:DNA-Binding Proteins; D009687:Nuclear Proteins; D010455:Peptides; D004247:DNA; D051747:DNA-Activated Protein Kinase; C497263:PRKDC protein, human; D017346:Protein Serine-Threonine Kinases