RESUMEN
This section provides detailed protocols for the analysis of a mammalian diacylglycerol kinase, DGKθ, including an activity assay, a kinetic analysis, preparation of small unilamellar vesicles, and a vesicle pulldown assay. The goal of this section is to provide an overview of the unique challenges inherent in the study of an interfacial enzyme such as DGKθ and to outline methods useful for analysis. We include a short tutorial on selecting lipids for forming the interface since this is critical for a successful in vitro assay, and lipids are important regulators of this enzyme. The general principles can be applied to the study of other interfacial enzymes.
Asunto(s)
Adenosina Trifosfato/química , Diacilglicerol Quinasa/química , Pruebas de Enzimas , Membranas Intracelulares/química , Liposomas Unilamelares/química , Animales , Diacilglicerol Quinasa/aislamiento & purificación , Diglicéridos/química , Isoenzimas/química , Isoenzimas/aislamiento & purificación , Cinética , Mamíferos , Fosfatidilcolinas/química , Fosfatidiletanolaminas/química , Fosfatidilserinas/química , Fosforilación , Unión Proteica , Saccharomyces cerevisiae/química , Saccharomyces cerevisiae/enzimología , Propiedades de SuperficieRESUMEN
The story of nuclear diacyglycerol is proving to be a complex one. Sub-pools of nuclear diglyceride that differ in their metabolism, nuclear localization and temporal regulation have been identified, suggesting potentially diverse signaling functions. One of the great remaining challenges is to assign functional roles to these diverse populations. In the last twenty years great strides have been made toward understanding the character and composition of nuclear DAG. Determining the functions of this nuclear lipid should make the next twenty years interesting indeed.
