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Biochem Biophys Res Commun ; 659: 29-33, 2023 06 04.
Artigo em Inglês | MEDLINE | ID: mdl-37031591

RESUMO

Calmodulin (CaM) is known to function as a central signal transducer in calcium-mediated intracellular pathways. In this study, a fusion molecule of a recently developed proximity biotinylation enzyme (AirID) with rat CaM (AirID-CaM) was expressed and purified to near homogeneity using an E. coli expression system to examine the physical interactions between CaM and its target proteins by converting the interaction to biotinylation of CaM targets under nondenatured conditions. AirID-CaM catalyzed a Ca2+-dependent biotinylation of a target protein kinase (Ca2+/CaM-dependent protein kinase kinase α/1, CaMKKα/1) in vitro, which was suppressed by the addition of excess amounts of CaM, and AirID alone did not catalyze the biotinylation of CaMKKα/1, indicating that the biotinylation of CaMKKα/1 by AirID-CaM likely occurs in an interaction-dependent manner. Furthermore, we also observed the Ca2+-dependent biotinylation of GST-CaMKIα and GST-CaMKIV by AirID-CaM, suggesting that AirID-CaM can be useful for the rapid detection of CaM/target interactions with relatively high sensitivity.


Assuntos
Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina , Calmodulina , Ratos , Animais , Calmodulina/metabolismo , Quinase da Proteína Quinase Dependente de Cálcio-Calmodulina/metabolismo , Biotinilação , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas Quinases Dependentes de Cálcio-Calmodulina/metabolismo , Fosforilação , Cálcio/metabolismo
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