Assay of Phospholipase D as a Neuronal Receptor-Effector Mechanism

Abstract

Phospholipase D is a commonly encountered but poorly understood member of the phospholipase family of cell signaling enzymes. Until recently, its study was inhibited by the lack of a simple and adaptable assay in intact cells that is not complicated by the presence of phospholipase C activity. Here, we review the various methods used to measure phospholipase D in whole cells in culture and with disrupted neuronal preparations, and we introduce the use of transphosphatidylation as a method of measuring the activity of phospholipase D in the presence of millimolar concentrations of alcohol. We then describe in detail the use of transphosphatidylation by butanol with ³²P-labeled neuron-like cells in culture. Alternative radiolabeling procedures, using [³H]glycerol and ³H-labeled fatty acids, with these cells are discussed. Finally, the application of procedures such as these to brain preparations, in particular, to intact synaptosomal preparations, is described.