title: Measuring Diacylglycerol Kinase-θ Activity and Binding.
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.
authors.affiliation: Johns Hopkins University, Baltimore, MD, United States.
authors.forename: B
authors.initials: B
authors.lastname: Tu-Sekine
authors.affiliation: Johns Hopkins University, Baltimore, MD, United States. Electronic address: draben@jhmi.edu.
authors.forename: D M
authors.initials: DM
authors.lastname: Raben
mesh_terms: D000255:Adenosine Triphosphate / Q000737:chemistry*; D000818:Animals; D019852:Diacylglycerol Kinase / Q000737:chemistry* / Q000302:isolation & purification; D004075:Diglycerides / Q000737:chemistry; D057075:Enzyme Assays; D007425:Intracellular Membranes / Q000737:chemistry*; D007527:Isoenzymes / Q000737:chemistry / Q000302:isolation & purification; D007700:Kinetics; D008322:Mammals; D010713:Phosphatidylcholines / Q000737:chemistry; D010714:Phosphatidylethanolamines / Q000737:chemistry; D010718:Phosphatidylserines / Q000737:chemistry; D010766:Phosphorylation; D011485:Protein Binding; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000201:enzymology; D013499:Surface Properties; D053835:Unilamellar Liposomes / Q000737:chemistry*
chemical_list: D004075:Diglycerides; D007527:Isoenzymes; D010713:Phosphatidylcholines; D010714:Phosphatidylethanolamines; D010718:Phosphatidylserines; D053835:Unilamellar Liposomes; C057561:1-palmitoyl-2-oleoylphosphatidylethanolamine; C050294:1-palmitoyl-2-oleoylglycero-3-phosphoserine; D000255:Adenosine Triphosphate; D019852:Diacylglycerol Kinase; C028694:1-palmitoyl-2-oleoylphosphatidylcholine; C013965:diolein
keywords: Diacylglycerol kinase; Interfacial binding; Interfacial enzymology; Liposomes