Synthesis and use of a biotinylated 3-azidophenothiazine to photolabel both amino- and carboxyl-terminal sites in calmodulin.
Bioconjug Chem
; 6(5): 558-66, 1995.
Article
en En
| MEDLINE
| ID: mdl-8974454
The biotinylated probe, 3-azido-10-(4-(4-biotinyl-1-piperazinyl)butyl)phenothiazine, was used to examine the phenothiazine binding domains in calmodulin (CaM) by photolabeling. This phenothiazine, synthesized from 3-azido-10-(4-(1-piperazinyl)butyl)phenothiazine and d-biotinyl tosylate, inhibited the CaM-mediated activation of phosphodiesterase (PDE) with an I50 of 12.5 (+/- 2.8) microM. Photolabeling of CaM produced covalent adducts in excellent yield (32%) in a light- and Ca2+-dependent manner. Studies performed over a range of drug concentrations suggested a 2:1 stoichiometry for the binding of the phenothiazine probe to CaM. Limited trypsin digestion and purification of the resulting fragments by either SDS-PAGE or HPLC provided two principal phenothiazine-containing peptides. Amino acid composition and sequence analyses performed on these two peptides established that both the N- and C-terminal domains in CaM, particularly the regions amino terminal to Ca2+-binding loops 1 and 3, were modified by the photoactivated phenothiazine derivative. These data, particularly for the C-terminal domain, are in excellent agreement with the X-ray structure analysis of a 1:1 CaM-trifluoperazine complex.
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Colección:
01-internacional
Base de datos:
MEDLINE
Asunto principal:
Fenotiazinas
/
Calmodulina
/
Marcadores de Afinidad
Tipo de estudio:
Prognostic_studies
Límite:
Animals
Idioma:
En
Revista:
Bioconjug Chem
Asunto de la revista:
BIOQUIMICA
Año:
1995
Tipo del documento:
Article
País de afiliación:
Estados Unidos
Pais de publicación:
Estados Unidos