Measurement of Mammalian Diacylglycerol Kinase Activity In Vitro and in Cells Chapter uri icon

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abstract

  • Diacylglycerol kinase (DGK) catalyzes the conversion of diacylglycerol to phosphatidic acid. Because both the lipid substrate and the product are important in regulation, this enzyme plays an important role in signal transduction. In mammals there are several isoforms of diacylglycerol kinase. Their activities can be evaluated in vitro as well as in intact cells. In vitro assays are based on measuring the incorporation of (32)P from ATP into diacylglycerol, resulting in the formation of labeled phosphatidic acid. Diacylglycerol with long acyl chains is insoluble in water and must be dispersed with detergent or incorporated into liposomes. Detergent-based assays are easier to perform and generally more precise; however, liposomes more closely resemble the organization of biological membranes and also allow for the testing of the modulation of enzyme activity by changes in the physical or chemical properties of the membrane. The micelle assay can also be used to measure DGK activity in cellular organelles after stimulation of intact cells to activate particular DGK isoforms. This will assess the translocation of DGK among different subcellular compartments. In this regard the plasma membrane and nucleus appear to be particularly important for the regulatory actions of these enzymes. Finally, one can also measure the DGK activity in whole cells that have been prelabeled with [(32)P]phosphate and determine the incorporation of label into phosphatidic acid that can be extracted from the whole cell or from cellular organelles.

publication date

  • 2007