Measuring Diacylglycerol Kinase-θ Activity and Binding

B. Tu-Sekine, Daniel Max Raben

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

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.

Original languageEnglish (US)
Title of host publicationMethods in Enzymology
PublisherAcademic Press Inc.
Pages231-253
Number of pages23
Volume583
DOIs
StatePublished - 2017

Publication series

NameMethods in Enzymology
Volume583
ISSN (Print)00766879
ISSN (Electronic)15577988

Keywords

  • Diacylglycerol kinase
  • Interfacial binding
  • Interfacial enzymology
  • Liposomes

ASJC Scopus subject areas

  • Biochemistry
  • Molecular Biology

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  • Cite this

    Tu-Sekine, B., & Raben, D. M. (2017). Measuring Diacylglycerol Kinase-θ Activity and Binding. In Methods in Enzymology (Vol. 583, pp. 231-253). (Methods in Enzymology; Vol. 583). Academic Press Inc.. https://doi.org/10.1016/bs.mie.2016.09.002