A review of phosphatidate phosphatase assays

Abstract

Phosphatidate phosphatase (PAP) catalyzes the penultimate step in the synthesis of triacylglycerol and regulates the synthesis of membrane phospholipids. There is much interest in this enzyme because it controls the cellular levels of its substrate, phosphatidate (PA), and product, DAG; defects in the metabolism of these lipid intermediates are the basis for lipid-based diseases such as obesity, lipodystrophy, and inflammation. The measurement of PAP activity is required for studies aimed at understanding its mechanisms of action, how it is regulated, and for screening its activators and/or inhibitors. Enzyme activity is determined through the use of radioactive and nonradioactive assays that measure the product, DAG, or P_i However, sensitivity and ease of use are variable across these methods. This review summarizes approaches to synthesize radioactive PA, to analyze radioactive and nonradioactive products, DAG and P_i, and discusses the advantages and disadvantages of each PAP assay.

Keywords: Pah1; diacylglycerol; enzyme assays; lipid metabolism; lipin; nonradioactive assays; radioactive assays; triacylglycerol.

Fig. 1.

Role of PAP in the synthesis of TAG and membrane phospholipids in yeast and mammals. The structures of CDP-DAG, PA, DAG, and TAG are shown with the C16:0 and C18:1 fatty acyl groups. PAP plays a major role in governing whether cells utilize PA for the synthesis of TAG via DAG or for the synthesis of membrane phospholipids via CDP-DAG. The DAG produced in the PAP reaction is also used for the synthesis of phospholipids. More detailed pathways for the synthesis of phospholipids in yeast (38) and mammals (19) are found elsewhere. Cho, choline; Etn, ethanolamine; PGP, hosphatidylglycerophosphate; PIPs, phosphoinositides; Ser, serine.

Fig. 2.

Synthesis of radioactive PA. The figure outlines the enzymatic reactions used to synthesize PA with label in the phosphate moiety (reaction 1), glycerol backbone (reaction 2), and fatty acyl groups (reactions 3 and 4). The radioactive moieties in PA are depicted in red color for emphasis; the phosphorous atom is labeled in [³²P]PA and the first carbon atom in the acyl moiety is labeled in [¹⁴C]PA.