Purification of CDPdiacylglycerol synthase from Escherichia coli.

Publisher Summary This chapter discusses the purification of CDPdiacylglycerol synthase from Escherichia coli (E.coli). CDPdiacylglycerol synthase catalyzes the activation of phosphatidic acid by CTP to form CDPdiacylglycerol and inorganic pyrophosphate. This activity is essential for all phospholipid biosynthesis in E.coli and for the biosynthesis of acidic phospholipids in mammalian cells. The enzymes of phospholipid synthesis are usually membrane bound and present in relatively low levels, making purification difficult. The purification of E. coli CDPdiacylglycerol synthase is greatly aided by enzyme overproduction driven by an expression plasmid. Starting with cells that overproduce the enzyme 50-fold, essentially homogeneous enzyme can be obtained after chromatographic procedures that yield 160-fold purification. The enzyme can be partially purified from wild-type cells using the same procedures. The assay follows the conversion of [α- 32 P]dCTP to chloroform-soluble material, dependent on phosphatidic acid.