Skip to main content
SpringerLink
Log in

Phospholipase D hydrolyzes short-chain analogs of phosphatidylcholine in the absence of detergent

  • Published:
Lipids

Abstract

Phospholipase D is an important enzyme in signal transduction in neuronal tissue. A variety of assays have been used to measure phospholipase D activity in vitro. The most typical measure of phospholipase D activity is the production of phosphatidylethanol in the presence of ethanol. Phosphatidylethanol is a product of transphosphatidylation activity that is considered a unique property of phospholipase D. To support transphosphatidylation activity, high concentrations of ethanol may be required. Furthermore, most assays in the literature utilize a detergent. These extreme conditions, detergent and ethanol, may alter phospholipase D and hinder the study of its regulation. In this manuscript we describe an assay that eliminates these potentially confounding conditions. It utilizes high specific activity [3H]butanol as a nucleophilic receptor. This eliminates the need for high concentrations of alcohol. The substrate is an analog of phosphatidylcholine that contains short-chain fatty acids, 1,2-dioctanoyl-sn-glycero-3-phosphocholine. Phospholipase D readily hydrolyzes this substrate in the absence of detergent. This novel assay should be useful in the further characterization of phospholipase D.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

CMC:

critical micellar concentration

K M :

Michaelis constant

PC8:

1,2-dioctanoyl-sn-glycero-3-phosphocholine

References

  1. Billah, M.M., and Anthes, J.C. (1990) The Regulation and Cellular Functions of Phosphatidylcholine Hydrolysis, Biochem. J. 269, 181–291.

    Google Scholar 

  2. Exton, J.H. (1990) Signaling Through Phosphatidylcholine Breakdown, J. Biol. Chem. 265, 1–4.

    PubMed  CAS  Google Scholar 

  3. Shukla, S., and Halenda, S. (1991) Phospholipase D in Cell Signaling and Its Relationship to Phospholipase C, Life Sci. 48, 851–866.

    Article  PubMed  CAS  Google Scholar 

  4. Holbrook, P.G., Pannell, L.K., Mirata, Y., and Daly, J.W. (1992) Molecular Species Analysis of a Product of Phospholipase D Activation. Phosphatidylethanol Is Formed from Phosphatidylcholine in Phorbol Ester- and Bradykinin-Stimulated Cells, J. Biol. Chem. 267, 16834–16840.

    PubMed  CAS  Google Scholar 

  5. Horwitz, J., and Davis, L.L. (1993) The Substrate Specificity of Brain Microsomal Phospholipase D, Biochem. J. 295, 793–798.

    PubMed  CAS  Google Scholar 

  6. Kobayashi, M., and Kanfer, J.N. (1987) Phosphatidylethanol Formation via Transphosphatidylation by Rat Brain Synaptosomal Phospholipase D, J. Neurochem. 48, 1597–1603.

    Article  PubMed  CAS  Google Scholar 

  7. Mohn, H., Chalifa, V., and Liscovitch, M. (1992) Substrate Specificity of Neutral Phospholipase D from Rat Brain Studied by Selective Labeling of Endogenous Synaptic Membrane Phospholipid In Vitro, J. Biol. Chem. 267, 11131–11136.

    PubMed  CAS  Google Scholar 

  8. Wang, P., Anthes, J.C., Siegel, M.I., Egan, R.W., and Billah, M.M. (1991) Existence of Cytosolic Phospholipase D. Identification and Comparison with Membrane Bound Enzyme, J. Biol. Chem. 266, 14877–14880.

    PubMed  CAS  Google Scholar 

  9. Nishida, A., Shimizu, M., Kanaho, Y., Nozawa, Y., and Yamawaki, S. (1992) Characterization of Phospholipase D in a Cell-Free System of Cultured Cells Derived from Rat Frontal Cortex, Brain Res. 595, 12–16.

    Article  PubMed  CAS  Google Scholar 

  10. Heller, M. (1978) Phospholipase D, Adv. Lipid Res. 16, 267–326.

    PubMed  CAS  Google Scholar 

  11. Liscovitch, M. (1989) Phosphatidylethanol Biosynthesis in Ethanol-Exposed NG108-15 Neuroblastoma X Glioma Hybrid Cells, J. Biol. Chem. 254, 1450–1456.

    Google Scholar 

  12. Horwitz, J. (1991) Bradykinin Activates a Phospholipase D That Hydrolyzes Phosphatidylcholine in PC12 Cells, J. Neurochem. 56, 509–517.

    Article  PubMed  CAS  Google Scholar 

  13. Kobayashi, M., McCartney, D.G., and Kanfer, J.N. (1988) Developmental Changes and Regional Distribution of Phospholipase D and Base Exchange Enzyme Activities in Rat Brain, Neurochem. Res. 13, 771–776.

    Article  PubMed  CAS  Google Scholar 

  14. Tausk, R.J., Oudshoorn, C., and Overbrook, J.T.G. (1974) Physical Chemical Studies of Short-Chain Lecithin Homologues. 3. Phase Separation and Light Scattering Studies on Aqueous Dioctanoyllecithin Solutions. Biophys. Chem. 2, 53–63.

    Article  PubMed  CAS  Google Scholar 

  15. Roberts, M.F., Bothner-By, A.A., and Dennis, E.A. (1978) Magnetic Nonequivalence Within Fatty Acyl Chains of Phospholipids in Membrane Models: 1H Nuclear Magnetic Resonance Studies of the Alpha-Methylene Groups, Biochemistry 17, 935–942.

    Article  PubMed  CAS  Google Scholar 

  16. Burns, R.A., Donovan, J.M., and Roberts, M.F. (1983) Structural Analysis of Short-Chain Lecithin/Triglyceride Micellar Particles, Biochemistry 22, 964–973.

    Article  PubMed  CAS  Google Scholar 

  17. Chalifa, V., Mohn, H., and Liscovitch, M. (1990) A Neutral Phospholipase D Activity from Rat Brain Synaptic Plasma Membranes. Identification and Partial Characterization. J. Biol. Chem. 265, 17512–17519.

    PubMed  CAS  Google Scholar 

  18. Kanoh, H., Kanaho, Y., and Nozawa, Y. (1991) Activation and Solubilization by Triton X-100 of Membrane-Bound Phospholipase D of Rat Brain, Lipids 26, 426–430.

    PubMed  CAS  Google Scholar 

  19. Kanfer, J.N., and McCartney, D. (1994) Phospholipase D Activity of Isolated Rat Brain Plasma Membranes, FEBS Lett. 337, 251–254.

    Article  PubMed  CAS  Google Scholar 

  20. Liscovitch, M., and Eli, Y. (1991) Ca2+ Inhibits Guanine Nucleotide-Activated Phospholipase D in Neural-Derived NG108-15 Cells, Cell Reg. 2, 1011–1019.

    CAS  Google Scholar 

  21. Okamura, S., and Yamashita, S. (1994) Purification and Characterization of Phosphatidylcholine Specific Phospholipase D from Pig Lung, J. Biol. Chem. 269, 31207–31213.

    PubMed  CAS  Google Scholar 

  22. Liscovitch, M., Chalifa, V., Pertiles, P., Chen, C.-S., and Cantley, L.C. (1994) Novel Function of Phosphatidylinositol 4,5-Bisphosphate as a Cofactor for Brain Membrane Phospholipase D, J. Biol. Chem. 269, 21403–21406.

    PubMed  CAS  Google Scholar 

  23. Waksman, M., Eli, Y., Liscovitch, M., and Gerst, J.E. (1996) Identification and Characterization of a Gene Encoding Phospholipase D Activity in Yeast, J. Biol. Chem. 271, 2361–2364.

    Article  PubMed  CAS  Google Scholar 

  24. Vinggard, A.N., Jensen, T., Morgan, C.P., Cockcroft, S., and Hansen, H.S. (1996) Didecanoyl Phosphatidylcholine Is a Superior Substrate for Assaying Mammalian Phospholipase D, Biochem. J. 319, 861–864.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. Department of Pharmacology MCP Hahnemann School of Medicine, Allegheny University of the Health Sciences, 3200 Henry Ave, 19129, Philadelphia, PA

    Leslee L. Davis, Jeffrey J. Maglio & Joel Horwitz

Authors
  1. Leslee L. Davis
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jeffrey J. Maglio
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Joel Horwitz
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Joel Horwitz.

About this article

Cite this article

Davis, L.L., Maglio, J.J. & Horwitz, J. Phospholipase D hydrolyzes short-chain analogs of phosphatidylcholine in the absence of detergent. Lipids 33, 223–227 (1998). https://doi.org/10.1007/s11745-998-0199-5

Download citation

  • Received:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s11745-998-0199-5

Keywords

Search

Navigation