Reconstitution System Based on Cytosol-Depleted Cells to Study the Regulation of Phospholipase D

  • Amanda Fensome-Green
  • Shamshad Cockcroft
Part of the Methods in Molecular Biology™ book series (MIMB, volume 332)


Phospholipase D (PLD) hydrolyzes phosphatidylcholine to produce the membraneassociated second messenger, phosphatidic acid (PA) and choline. Two phospholipase D enzymes—PLD1 and PLD2—have been identified, although their regulatory mechanisms are yet to be fully understood. To study the regulation of PLD, we established a reconstitution system that allows the study of the PLD enzymes in their native environment while enabling the cytosol to be manipulated. Cells are permeabilized with a bacterial cytolysin (streptolysin O), which produces lesions in the plasma membrane, resulting in the release of cytosolic proteins. With increasing permeabilization times, guanosine 5′-[γ-thio]triphosphate and receptor-activated PLD activity diminishes. Once the conditions for the run-down of the response is established, cellular factors, such as cytosol and purified proteins, can be added to these cells to restore activity. In addition to examining PLD activity, this reconstitution system allows the study of potential cellular targets of PA, such as phosphatidylinositol 4-phosphate (PIP) 5-kinase activity by monitoring PIP2 synthesis, and also functional outputs, such as exocytosis.

Key Words

ARF phosphatidylcholine phosphatidate streptolysin O alcohols permeabilization 


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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Amanda Fensome-Green
    • 1
  • Shamshad Cockcroft
    • 2
  1. 1.Department of Physiology, Rockefeller BuildingUniversity College LondonLondonUK
  2. 2.Department of Physiology, Rockefeller BuildingUniversity College LondonLondonUK

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