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Chromogenic assay for phospholipase D from Streptomyces chromofuscus: Application to the evaluation of substrate analogs


A rapid and convenient chromogenic assay for phospholipase D from Streptomyces chromofuscus (PLDSc) has been developed that converts the choline generated from the enzyme-catalyzed hydrolysis of phospholipids into a chromogenic dye. By quenching the reaction with EDTA at defined times, an initial rate curve is produced from which a k cat and K m can be readily derived. This assay has been applied to the biological evaluation of several substrate analogs, all of which appear to be activators rather than substrates or inhibitors of this enzyme. Performing the assay in 96-well microtiter plates allows for the easy screening of potential effectors of this enzyme.

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critical micelle concentration




phosphatidic acid






phospholipase D


phospholipase D from Streptomyces chromofuscus


  1. Exton, J.H. (1994) Phosphatidylcholine Breakdown and Signal Transduction, Biochim. Biophys. Acta 1212, 26–42.

    PubMed  CAS  Google Scholar 

  2. Liscovitch, M. (1992) Crosstalk Among Multiple Signal-Activated Phospholipases, Trends Biochem. Sci. 17, 393–399.

    PubMed  CAS  Article  Google Scholar 

  3. Asaoka, Y., Nakamura, S., Yoshida, K., and Nishizuka, Y. (1992) Protein Kinase C, Calcium and Phospholipid Degradation, Trends Biochem. Sci. 17, 414–417.

    PubMed  CAS  Article  Google Scholar 

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

    PubMed  CAS  Google Scholar 

  5. Nakanishi, H., and Exton, J.H. (1992) Purification and Characterization of the ξ Isoform of Protein Kinase C from Bovine Kidney, J. Biol. Chem. 267, 16347–16354.

    PubMed  CAS  Google Scholar 

  6. Kanaho, Y., Nakai, Y., Masayasu, K., and Nozawa, Y. (1993) The Phosphatase Inhibitor 2,3-Diphosphoglycerate Interferes with Phospholipase D Activation in Rabbit Peritoneal Neutrophils, J. Biol. Chem. 268, 12492–12497.

    PubMed  CAS  Google Scholar 

  7. Liscovitch, M., Chalifa, V., Danin, M., and Eli, Y. (1991) Inhibition of Neural Phospholipase D Activity by Aminoglycoside Antibiotics, Biochem. J. 279, 319–321.

    PubMed  CAS  Google Scholar 

  8. Kessels, G.C.R., Gervaix, A., Lew, P.D., and Verhoeven, A.J. (1991) The Chymotrypsin Inhibitor Carbobenzyloxy-leucine-tyrosine-chloromethylketone Interferes with Phospholipase D Activation Induced by Formyl-methionyl-leucyl-phenylalanine in Human Neutrophils, J. Biol. Chem. 266, 15870–15875.

    PubMed  CAS  Google Scholar 

  9. McNamara, P.J., Cuevas, W.A., and Songer, J.G. (1995) Toxic Phospholipases D of Corynebacterium pseudotuberculosis, C. ulcerans, and Arcanobacterium haemolyticum: Cloning and Sequence Homology, Gene 156, 113–118.

    PubMed  CAS  Article  Google Scholar 

  10. Pointing, C.P., and Kerr, I.D. (1996) A Novel Family of Phospholipase D Homologues That Includes Phospholipid Synthases and Putative Endonucleases: Identification of Duplicated Repeats and Potential Active Site Residues, Protein Sci. 5, 914–922.

    Article  Google Scholar 

  11. Carrea, G., D’Arrigo, P., Piergianni, V., Roncaglio, S., Secundo, F., and Servi, S. (1995) Purification and Properties of Two Phospholipases D from Streptomyces sp. Biochem. Biophys. Acta 1255, 273–279.

    PubMed  Google Scholar 

  12. Wang, P., Schuster, M., Wang, Y.-M., and Wong, C.-H. (1993) Synthesis of Phospholipid-Inhibitor Conjugates by Enzymatic Transphosphatidylation with Phospholipase D, J. Am. Chem. Soc. 115, 10487–10491.

    CAS  Article  Google Scholar 

  13. Imamura, S., and Horiuti, Y. (1979) Purification of Streptomyces chromofuscus Phospholipase D by Hydrophobic Affinity Chromatography on Palmitoyl Cellulose, J. Biochem. 85, 79–95.

    PubMed  CAS  Google Scholar 

  14. Martin, S.F., Wong, Y.-L., and Wagman, A.S. (1994) Design, Synthesis, and Evaluation of Phospholipid Analogues as Inhibitors of the Bacterial Phospholipase C from Bacillus cereus, J. Org. Chem. 59, 4821–4831.

    CAS  Article  Google Scholar 

  15. Rawyler, A., and Siegenthaler, P.A. (1989) A Single and Continuous Spectrophotometric Assay for Various Lipolytic Enzymes, Using Natural, Non-Labelled Lipid Substrates, Biochim. Biophys. Acta 1004, 337–344.

    PubMed  CAS  Google Scholar 

  16. D’Arrigo, P., Piergianni, V., Scarcelli, D., and Servi, S. (1995) A Spectrophotometric Assay for Phospholipase D, Anal. Chim. Acta 304, 249–254.

    CAS  Article  Google Scholar 

  17. Takayama, M., Itoh, S., Nagosaki, T., and Tanimizu, I. (1977) A New Enzymatic Method for Determination of Serum Choline-Containing Phospholipids, Clin. Chim. Acta 79, 93–98.

    PubMed  CAS  Article  Google Scholar 

  18. Hergenrother, P.J., Spaller, M.R., Haas, M.K., and Martin, S.F. (1995) Chromogenic Assay for Phospholipase C from Bacillus cereus, Anal. Biochem. 229, 313–316.

    PubMed  CAS  Article  Google Scholar 

  19. Martin, S.F., Spaller, M.R., and Hergenrother, P.J. (1996) Expression and Site-Directed Mutagenesis of the Phosphatidylcholine-Preferring Phospholipase C of Bacillus cereus: Probing the Role of the Active Site Glu146, Biochemistry 35, 12970–12977.

    PubMed  CAS  Article  Google Scholar 

  20. Furton, K.G., and Norelus, A. (1993) Determining the Critical Micelle Concentration of Aqueous Surfactant Solutions, J. Chem. Ed. 70, 254–257.

    CAS  Article  Google Scholar 

  21. El-Sayed, M.Y., DeBose, C.D., Coury, L.A., and Roberts, M.F. (1985) Sensitivity of Phospholipase C (Bacillus cereus) Activity to Phosphatidylcholine Structural Modifications, Biochim. Biophys. Acta 837, 325–335.

    PubMed  CAS  Google Scholar 

  22. Orr, G.A., Brewer, C.F., and Heney, G. (1982) Synthesis of the Diastereoisomers of 1,2-Dipalmitoyl-sn-glycero-3-thiophosphorylethanolamine and Their Stereospecific Hydrolysis by Phospholipases A2 and C, Biochemistry 21, 3202–3206.

    PubMed  CAS  Article  Google Scholar 

  23. Bruzik, K., Gupte, S.M., and Tsai, M.-D. (1982) Phospholipids Chiral at Phosphorus. 2. Preparation, Property, and Application of Chiral Thiophospholipids, J. Am. Chem. Soc. 104, 4682–4684.

    CAS  Article  Google Scholar 

  24. Jiang, R.-T., Shyy, Y.-J., and Tsai, M.-D. (1984) Phospholipids Chiral at Phosphorus. Absolute Configuration of Chiral Thiophospholipids and Stereospecificity of Phospholipase D, Biochemistry 23, 1661–1667.

    PubMed  CAS  Article  Google Scholar 

  25. Burns, R.A., El-Sayed, M.Y., and Roberts, M.F. (1982) Kinetic Model for Surface-Active Enzymes Based on the Langmuir Adsorption Isotherm: Phospholipase C (Bacillus cereus) Activity Toward Dimyristoyl Phosphatidylcholine/Detergent Micelles, Proc. Natl. Acad. Sci. USA 79, 4902–4906.

    PubMed  CAS  Article  Google Scholar 

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Correspondence to Stephen F. Martin.

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Hergenrother, P.J., Haas, M.K. & Martin, S.F. Chromogenic assay for phospholipase D from Streptomyces chromofuscus: Application to the evaluation of substrate analogs. Lipids 32, 783–788 (1997).

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  • Choline
  • Streptomyces
  • Critical Micelle Concentration
  • Bacillus Cereus
  • Phosphatidic Acid