Epand Richard M, Topham Matthew K
Department of Biochemistry and Biomedical Sciences, McMaster University Health Sciences Centre, Hamilton, Ontario, Canada.
Methods Enzymol. 2007;434:293-304. doi: 10.1016/S0076-6879(07)34016-0.
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.
二酰基甘油激酶（DGK）催化二酰基甘油转化为磷脂酸。由于脂质底物和产物在调节过程中都很重要，因此该酶在信号转导中发挥着重要作用。在哺乳动物中，存在几种二酰基甘油激酶同工型。它们的活性可以在体外以及完整细胞中进行评估。体外测定基于测量（32）P从ATP掺入二酰基甘油，从而形成标记的磷脂酸。具有长酰基链的二酰基甘油不溶于水，必须用去污剂分散或掺入脂质体中。基于去污剂的测定更容易进行，通常也更精确；然而，脂质体更类似于生物膜的组织结构，并且还允许通过膜的物理或化学性质的变化来测试酶活性的调节。在刺激完整细胞以激活特定的DGK同工型后，胶束测定也可用于测量细胞器中的DGK活性。这将评估DGK在不同亚细胞区室之间的转运。在这方面，质膜和细胞核似乎对这些酶的调节作用尤为重要。最后，还可以测量用[（32）P]磷酸盐预标记的全细胞中的DGK活性，并确定标记掺入可从全细胞或细胞器中提取的磷脂酸中的情况。
