Identification of glycogen phosphorylase and creatine kinase as calpain substrates in skeletal muscle.
Int J Biochem Cell Biol
; 33(5): 531-40, 2001 May.
Article
en En
| MEDLINE
| ID: mdl-11331208
The goal of this study was to identify calpain substrates in muscle cells. Our hypothesis was that the yeast two-hybrid method could be used to identify novel calpain substrates. To accomplish this, native mu- and m-calpains, as well as a variety of calpain DNA fragments, were expressed in yeast cells and used to screen for binding proteins in a human skeletal muscle cDNA library. Calpain constructs that were used in the screening process included native mu- and m-calpains, a dominant negative (DN) m-calpain (i.e. active site modified), N-terminal truncated DN m-calpain (i.e. autolyzed DN-m-calpain) and, finally, an N- and C-terminal truncated m-calpain (i.e. autolyzed DN-m-calpain lacking a calcium-binding domain). Yeast cells were transformed using yeast two-hybrid expression vectors containing the different calpain constructs as "baits". Beta-galactosidase activity was assayed as an index of interaction between calpain and its potential target proteins. From this analysis, four clones (Ca2+-ATPase, novel nebulin-related protein (N-RAP), creatine kinase and glycogen phosphorylase) were recovered. Two of these, creatine kinase and glycogen phosphorylase, were selected for further study. In in-vitro assays, calpain was able to partially digest both proteins, suggesting that both creatine kinase and glycogen phosphorylase are natural calpain substrates.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Calpaína
/
Músculo Esquelético
/
Creatina Quinasa
/
Fosforilasas
Tipo de estudio:
Diagnostic_studies
Límite:
Humans
Idioma:
En
Revista:
Int J Biochem Cell Biol
Asunto de la revista:
BIOQUIMICA
Año:
2001
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Países Bajos