Synergistic activation of protein kinase C by arachidonic acid and diacylglycerols in vitro: generation of a stable membrane-bound, cofactor-independent state of protein kinase C activity.
Biochim Biophys Acta
; 1291(2): 167-76, 1996 Oct 24.
Article
em En
| MEDLINE
| ID: mdl-8898879
ABSTRACT
The present study examines the synergistic activation of PKC by arachidonic acid and diacylglycerols in phospholipid vesicles and demonstrates that this combination of activators leads to the formation of a constitutively active, phospholipid-bound form of the enzyme. Activation of PKC was almost entirely calcium-dependent with vesicles containing dioleoylglycerol alone. In contrast, considerable calcium-independent activity was observed when vesicles contained both a diacylglycerol and free arachidonic acid. High-affinity association of enzyme activity with diacylglycerol-containing vesicles was calcium dependent and reversible. However, addition of arachidonic acid to diacylglycerol-containing vesicles resulted in irreversible PKC binding in the absence of calcium. Immunoblot analysis indicated that the calcium-independent binding was not isozyme-specific. The activity of the vesicle-associated PKC, bound to vesicles in the absence of calcium, was predominantly calcium-dependent. On the other hand, when the binding and isolation of vesicle-bound enzyme was conducted in the presence of calcium, the subsequent activity was almost entirely resistant to calcium chelation. This vesicle-associated form of the enzyme, when detergent extracted and recombined with phospholipid vesicles, maintained significant 'constitutive' activity (activity in the absence of both diacylglycerol and calcium). The data from this in vitro system provide the basis for a model of the physiological regulation of PKC in which the combined actions of arachidonate and diacylglycerol facilitate the stable formation of a tightly membrane-associated, intrinsically active form of PKC.
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Coleções:
01-internacional
Base de dados:
MEDLINE
Assunto principal:
Proteína Quinase C
/
Ácido Araquidônico
/
Diglicerídeos
Tipo de estudo:
Prognostic_studies
Limite:
Animals
Idioma:
En
Ano de publicação:
1996
Tipo de documento:
Article