Measurement of mammalian diacylglycerol kinase activity in vitro and in cells.

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