Assay of Phospholipase D Activity in Cell-Free Systems

  • Shankar S. Iyer
  • David J. Kusner
Part of the Methods in Molecular Biology™ book series (MIMB, volume 332)

Summary

Phospholipase D (PLD) enzymes are present in all animal and plant species and have been linked to many critical cellular processes, including proliferation, differentiation, motility, and secretion. The functional significance of PLD derives from its generation of phosphatidic acid, which has both direct signaling properties via activation of numerous kinases, phosphatases, phopspholipases, and other enzymes, as well as via its conversion to diglycerides, the endogenous activators of protein kinase C. The two mammalian PLD isoforms, PLD1 and PLD2, are peripheral membrane proteins that exhibit important physical and functional interactions with the actin cytoskeleton. We outline a cell-free system for the characterization of mammalian PLDs and their activation by physiologic stimuli or pharmacologic agonists for guanine triphosphate-binding proteins. This assay system is used to illustrate the interactions of PLD1 with specific membrane domains and their associated filamentous and monomeric actin components.

Key Words

Phospholipase signal transduction enzyme membrane phospholipids phosphatidic acid macrophage phagocyte leukocyte monocyte human inflammation infection innate immunity actin cytoskeleton GTP-binding protein phagocytosis caveolae membrane raft 

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Copyright information

© Humana Press Inc. 2006

Authors and Affiliations

  • Shankar S. Iyer
    • 1
  • David J. Kusner
    • 2
  1. 1.Inflammation Program, Department of Internal MedicineUniversity of Iowa Carver College of MedicineIowa City
  2. 2.Inflammation Program, Department of Internal MedicineUniversity of Iowa Carver College of MedicineIowa City

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