Measurement of G protein-coupled receptor-stimulated phospholipase D activity in intact cells.
Mammalian phospholipase D (PLD) activity hydrolyzes phosphatidylcholine (PC) into phosphatidic acid (PA) and free choline. This activity can be stimulated by a wide variety of extracellular agonists, including those for G protein-coupled receptors (GPCRs). This chapter outlines a protocol for the measurement of PLD activity in intact cells following stimulation by an extracellular agonist. The protocol takes advantage of a unique property of mammalian PLDs--the ability to substitute a primary alcohol for water in the hydrolytic reaction. This transphosphatidylation reaction results in the formation of a phosphatidylalcohol, which is a specific and unique marker for PLD activity. This protocol is highly sensitive for the detection of PLD activity following the stimulation of intact cells, being a valuable method for studying the regulation of PLD activity in vivo.
D000818:Animals; D002855:Chromatography, Thin Layer / Q000379:methods*; D016716:PC12 Cells; D010739:Phospholipase D / Q000378:metabolism*; D051381:Rats; D043562:Receptors, G-Protein-Coupled / Q000378:metabolism*; D015398:Signal Transduction
D043562:Receptors, G-Protein-Coupled; D010739:Phospholipase D