Abstract
Phospholipases are a diverse group of lipolytic enzymes with specificities for different sites on the phospholipid molecule (1). Based on the site of their hydrolytic attack, they can be classified into several groups, among which phospholipase A2 (PLA2), phospholipase C (PLC), and phospholipase D (PLD) have been the most extensively studied. Because of their critical involvement in many extra- and intracellular processes, a sensitive continuous kinetic assay for phospholipase is an essential tool for many biological studies.
This is a preview of subscription content, log in via an institution.
Buying options
Tax calculation will be finalised at checkout
Purchases are for personal use only
Learn about institutional subscriptionsSpringer Nature is developing a new tool to find and evaluate Protocols. Learn more
References
Waite, M. (1987) The Phospholipases, Plenum, New York.
Reynolds, L. J., Washburn, W. N., Deems, R. A. and Dennis, E. A. (1991) Assay strategies and methods for phospholipases. Methods Enzymol. 197, 3–23.
Radvanyi, F., Jordan, L., Russo-Marie, F., and Bon, C. (1989) A sensitive and continuous fluorometric assay for phospholipase A2 using pyrene-labeled phospholipids in the presence of serum albumin. Anal. Biochem. 177, 103–109.
Thuren, T., Virtanen, J. A., Vainio, P., and Kinnunen, P. K. J. (1983) Hydrolysis of 1-triacontanyl-2-pyrene-1-yl)hexanoyl-sn-glycero-3-phosphocholine by human pancreatic phospholipase A2. Chem. Phy. Lipids 33, 283–292.
Hendrikson, H. S. and Rauk, P. N. (1981) Continous fluorometric assay for phospholipase A2 with pyrene-labeled lecithins as a substrate. Anal. Biochem. 116, 553–558.
Kinkaid, A. R. and Wilton, D. C. (1993) A continuous fluorescence diplacement assay for phospholipase A2 using albumin and medium chain phospholipid substrates. Anal. Biochem. 212, 65–70.
Wu, S.-K. and Cho, W. (1994) A continuous fluorometric assay for phospholipases using polymerized mixed liposomes. Anal. Biochem. 221, 152–159.
Wu, S.-K. and Cho, W. (1993) Use of polymerized liposomes to study interactions of phospholipase A2 with biological membranes. Biochemistry 32, 13,902–13,908.
Sadownik, A., Stefely, J., and Regen, S. L. (1986) Polymerized liposomes formed under extremely mild conditions. J. Amer. Chem. Soc. 108, 7789–7792.
Clark, J. D., Lin, L.-L., Kriz, R. W., Ramesha, C. S., Sultzman, L. A., Lin, A. Y., Milona, N., and Knopf, J. L. (1991) A novel arachidonic acid-selective cytosolic PLA2 contains a Ca2+-dependent translocation domain with homology to PKC and GAP. Cell 65, 1043–1051.
Sharp, J. D., White, D. L., Chiou, X. G., Gooden, T., Gamboa, G. C, McClure, D., Burgett, S., Hoskin, J., Skatrud, P. L., Sportsman, J. R., Becker, G. W., Kang, L. H., Roberts, E. F., and Kramer, R. M. (1991) Molecular cloning and expression of human Ca2+-sensitive cytosolic phospholipase A2. J. Biol. Chem. 266, 14,850–14,853.
Kates, M. (1986) Techniques of Lipidology, Elsevier, Amsterdam.
Jain, M. K. and Berg, O. G. (1989) The kinetics of interfacial catalysis by phospholipase A2 and regulation of interfacial activation: hopping versus scooting. Biochim. Biophys. Ada 1002, 127–156.
Snitko, Y., Yoon, E. T., and Cho, W. (1997) High Specificity of Human Secretory Class II Phospholipase A2 for Phosphatidic Acid. Biochem. J. 321, 737–741.
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 1999 Humana Press Inc, Totowa, NJ
About this protocol
Cite this protocol
Cho, W., Wu, SK., Yoon., E., Lichtenbergova, L. (1999). Fluorometric Phospholipase Assays Based on Polymerized Liposome Substrates. In: Doolittle, M., Reue, K. (eds) Lipase and Phospholipase Protocols. Methods in Molecular Biology™, vol 109. Humana Press. https://doi.org/10.1385/1-59259-581-2:7
Download citation
DOI: https://doi.org/10.1385/1-59259-581-2:7
Publisher Name: Humana Press
Print ISBN: 978-0-89603-546-1
Online ISBN: 978-1-59259-581-5
eBook Packages: Springer Protocols