Reconstitution of the vertebrate visual cascade using recombinant heterotrimeric transducin purified from Sf9 cells.
Protein Expr Purif
; 20(3): 514-26, 2000 Dec.
Article
em En
| MEDLINE
| ID: mdl-11087692
ABSTRACT
For reconstitution studies with rhodopsin and cGMP phosphodiesterase (PDE), all three subunits of heterotrimeric transducin (T alpha beta gamma) were simultaneously expressed in Sf9 cells at high levels using a baculovirus expression system and purified to homogeneity. Light-activated rhodopsin catalyzed the loading of purified recombinant T alpha with GTP gamma S. In vitro reconstitution of rhodopsin, recombinant transducin, and PDE in detergent solution resulted in cGMP hydrolysis upon illumination, demonstrating that recombinant transducin was able to activate PDE. The rate of cGMP hydrolysis by PDE as a function of GTP gamma S-loaded recombinant transducin (T(*)) concentration gave a Hill coefficient of approximately 2, suggesting that the activation of PDE by T(*) was cooperatively regulated. Furthermore, the kinetic rate constants for the activation of PDE by T(*) suggested that only the complex of PDE with two T(*) molecules, PDE. T(2)(*), was significantly catalytically active under the conditions of the assay. We conclude that the model of essential coactivation best describes the activation of PDE by T(*) in a reconstituted vertebrate visual cascade using recombinant heterotrimeric transducin.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Visão Ocular
/
Transducina
Limite:
Animals
Idioma:
En
Revista:
Protein Expr Purif
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2000
Tipo de documento:
Article
País de afiliação:
Estados Unidos