H. Alex Brown
Principle Investigator; Professor of Pharmacology, Chemistry, and Biochemistry; Associate Director, VICB;
Last active: 2/12/2015

Measurement of G protein-coupled receptor-stimulated phospholipase D activity in intact cells.

Walker SJ, Brown HA, Molecular and Cellular Biology, Cornell University, Ithaca, NY, USA
Methods Mol Biol. 2004 237: 89-97

PMID: 14501041 · DOI:10.1385/1-59259-430-1:89

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.

MeSH Terms (7)

Animals Chromatography, Thin Layer PC12 Cells Phospholipase D Rats Receptors, G-Protein-Coupled Signal Transduction

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