Purification of phospholipase C beta and phospholipase C epsilon from Sf9 cells.
Methods Mol Biol
; 237: 55-64, 2004.
Article
em En
| MEDLINE
| ID: mdl-14501038
ABSTRACT
Phosphatidylinositol-specific phospholipase C (PLC) enzymes catalyze hydrolysis of phosphatidylinositol 4,5-bisphosphate (PIP2) generating diacylglycerol (DAG) and inositol 1,4,5-triphosphate (IP(3)). The PLC beta isoforms of PLCs are activated by G proteins after hormone or neurotransmitter stimulation of G protein-coupled receptors (GPCR). PLC epsilon is a recently identified PLC isoform that is activated by Ras and G beta gamma subunit although the physiological role of this enzyme is not well understood. Methods for purification of PLC beta and PLC epsilon from Sf9 cells are described. In the case of hexahistidine (6-His)-tagged PLC beta the purification involves two steps, affinity chromatography with Ni-NTA agarose followed by heparin Sepharose chromatography. 6-His-tagged PLC epsilon can be purified in a single step with nickel nitrilotriacetic acid-agarose (Ni-NTA) affinity chromatography.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Fosfolipases Tipo C
/
Cromatografia de Afinidade
/
Isoenzimas
Limite:
Animals
Idioma:
En
Revista:
Methods Mol Biol
Assunto da revista:
BIOLOGIA MOLECULAR
Ano de publicação:
2004
Tipo de documento:
Article
País de afiliação:
Estados Unidos