Use of diacylclycerol kinase to quantitate picomole levels of 1,2-diacylglycerol.

An extremely sensitive and specific analytic procedure is described for quantitating 1,2-diacylglycerol (DAG). Escherichia coli DAG kinase (EC 2.7.1.-) catalyzed the formation of ³²P]phosphatidic acid by the transfer of ³²PO₄ from γ-³²P]adenosine 5′-triphosphate to DAG in linear proportion to the quantity of added DAG from 10 to 1000 pmol. This technique allowed reliable detection of as little as 2 pmol of added DAG. To assess levels of DAG in tissue lipid extracts a miniaturized method for silicic acid column chromatography was developed to separate DAG from triglycerides and phospholipids. When these procedures were applied to erythrocytes, lysis in the presence of Ca²⁺ caused a 10.6-fold rise in cellular DAG confirming not only the results obtained in an earlier investigation (1), but also the utility of this technique in the analysis of exceedingly small quantities of cellular DAG.