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To establish a non-radioactive, cell-free detection system for protein N-myristoylation, metabolic labeling in a cell-free protein synthesis system using bioorthogonal myristic acid analogues was performed. After Cu(I)-catalyzed azide-alkyne cycloaddition (CuAAC) with a biotin tag, the tagged proteins were separated by sodium dodecyl sulfate-polyacrylamide(More)
N-myristoylation of eukaryotic cellular proteins has been recognized as a modification that occurs mainly on cytoplasmic proteins. In this study, we examined the membrane localization, membrane integration, and intracellular localization of four recently identified human N-myristoylated proteins with predicted transmembrane domains. As a result, it was(More)
The subcellular localization of 13 recently identified N-myristoylated proteins and the effects of overexpression of these proteins on cellular morphology were examined with the aim of understanding the physiological roles of the protein N-myristoylation that occurs on these proteins. Immunofluorescence staining of HEK293T cells transfected with cDNAs(More)
To establish a strategy to identify dually fatty acylated proteins from cDNA resources, seven N-myristoylated proteins with cysteine (Cys) residues within the 10 N-terminal residues were selected as potential candidates among 27 N-myristoylated proteins identified from a model human cDNA resource. Seven proteins C-terminally tagged with FLAG tag or EGFP(More)
To identify physiologically important human N-myristoylated proteins, 90 cDNA clones predicted to encode human N-myristoylated proteins were selected from a human cDNA resource (4,369 Kazusa ORFeome project human cDNA clones) by two bioinformatic N-myristoylation prediction systems, NMT-The MYR Predictor and Myristoylator. After database searches to exclude(More)
An amino acid-sensing system with absorption spectrophotometric detection was developed. To ensure specific recognition of each amino acid, aminoacyl-tRNA synthetases were employed and the concentration of NADH produced by way of several enzymatic reactions was measured. Using this detection system, from 1.5 to 55 μM of histidine and from 15 to 95 μM of(More)
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