Skip to main content
PMC home page
Biophysical Journal logoLink to Biophysical Journal
. 2002 Aug;83(2):932–943. doi: 10.1016/S0006-3495(02)75219-3

Macroscopic consequences of the action of phospholipase C on giant unilamellar liposomes.

Juha M Holopainen 1, Miglena I Angelova 1, Tim Söderlund 1, Paavo K J Kinnunen 1
PMCID: PMC1302197  PMID: 12124275

Abstract

Macroscopic consequences of the formation of diacylglycerol by phospholipase C (PC-PLC) in giant 1-stearoyl-2-oleoyl-sn-glycero-3-phosphocholine (SOPC) unilamellar vesicles (GUVs, diameter 10-100 microm) were studied by phase contrast and fluorescence microscopy. PC-PLC caused a series of fast stepwise shrinkages of fluid SOPC GUVs, continuing until the vesicle disappeared beyond the optical resolution of the microscope. The presence of N-palmitoyl-sphingomyelin (mole fraction X = 0.25) in the GUVs did not affect the outcome of the PC-PLC reaction. In addition to hydrolysis, PC-PLC induced adhesion of vicinal vesicles. When multilamellar SOPC vesicles were used only a minor decrease in their diameter was evident suggesting that PC-PLC can exert its hydrolytic activity only in the outer monolayer. A series of stepwise shrinkages was observed also for 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) GUVs above their main phase transition temperature, T(m), i.e., when the bilayer is in the liquid crystalline state. However, this process was not observed for DMPC GUVs in the gel state, below T(m). These results are supported by the enhanced activity of PC-PLC upon exceeding T(m) of DMPC large unilamellar vesicles (diameter approximately 0.1 microm) used as a substrate. Studies on SOPC monolayers revealed that PC-PLC can exert its hydrolytic activity only at surface pressures below approximately 30 mN/m. Accordingly, the lack of changes in the gel state DMPC GUVs could be explained by the equilibrium lateral pressure in these vesicles exceeding this critical value.

Full Text

The Full Text of this article is available as a PDF (813.8 KB).

Selected References

These references are in PubMed. This may not be the complete list of references from this article.

  1. Akashi K., Miyata H., Itoh H., Kinosita K., Jr Preparation of giant liposomes in physiological conditions and their characterization under an optical microscope. Biophys J. 1996 Dec;71(6):3242–3250. doi: 10.1016/S0006-3495(96)79517-6. [DOI] [PMC free article] [PubMed] [Google Scholar]
  2. Allan D., Billah M. M., Finean J. B., Michell R. H. Release of diacylglycerol-enriched vesicles from erythrocytes with increased intracellular (Ca2+). Nature. 1976 May 6;261(5555):58–60. doi: 10.1038/261058a0. [DOI] [PubMed] [Google Scholar]
  3. Allan D., Michell R. H. Calcium ion-dependent diacylglycerol accumulation in erythrocytes is associated with microvesiculation but not with efflux of potassium ions. Biochem J. 1977 Sep 15;166(3):495–499. doi: 10.1042/bj1660495. [DOI] [PMC free article] [PubMed] [Google Scholar]
  4. Allan D., Thomas P., Michell R. H. Rapid transbilayer diffusion of 1,2-diacylglycerol and its relevance to control of membrane curvature. Nature. 1978 Nov 16;276(5685):289–290. doi: 10.1038/276289a0. [DOI] [PubMed] [Google Scholar]
  5. Bai J., Pagano R. E. Measurement of spontaneous transfer and transbilayer movement of BODIPY-labeled lipids in lipid vesicles. Biochemistry. 1997 Jul 22;36(29):8840–8848. doi: 10.1021/bi970145r. [DOI] [PubMed] [Google Scholar]
  6. Bangham A. D., Standish M. M., Watkins J. C. Diffusion of univalent ions across the lamellae of swollen phospholipids. J Mol Biol. 1965 Aug;13(1):238–252. doi: 10.1016/s0022-2836(65)80093-6. [DOI] [PubMed] [Google Scholar]
  7. Basáez G., Nieva J. L., Goñi F. M., Alonso A. Origin of the lag period in the phospholipase C cleavage of phospholipids in membranes. Concomitant vesicle aggregation and enzyme activation. Biochemistry. 1996 Dec 3;35(48):15183–15187. doi: 10.1021/bi9616561. [DOI] [PubMed] [Google Scholar]
  8. Basáez G., Ruiz-Argüello M. B., Alonso A., Goñi F. M., Karlsson G., Edwards K. Morphological changes induced by phospholipase C and by sphingomyelinase on large unilamellar vesicles: a cryo-transmission electron microscopy study of liposome fusion. Biophys J. 1997 Jun;72(6):2630–2637. doi: 10.1016/S0006-3495(97)78906-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  9. Blume A. A comparative study of the phase transitions of phospholipid bilayers and monolayers. Biochim Biophys Acta. 1979 Oct 19;557(1):32–44. doi: 10.1016/0005-2736(79)90087-7. [DOI] [PubMed] [Google Scholar]
  10. Brockman H. Lipid monolayers: why use half a membrane to characterize protein-membrane interactions? Curr Opin Struct Biol. 1999 Aug;9(4):438–443. doi: 10.1016/S0959-440X(99)80061-X. [DOI] [PubMed] [Google Scholar]
  11. Cunningham B. A., Tsujita T., Brockman H. L. Enzymatic and physical characterization of diacylglycerol-phosphatidylcholine interactions in bilayers and monolayers. Biochemistry. 1989 Jan 10;28(1):32–40. doi: 10.1021/bi00427a006. [DOI] [PubMed] [Google Scholar]
  12. Daniele J. J., Maggio B., Bianco I. D., Goñi F. M., Alonso A., Fidelio G. D. Inhibition by gangliosides of Bacillus cereus phospholipase C activity against monolayers, micelles and bilayer vesicles. Eur J Biochem. 1996 Jul 1;239(1):105–110. doi: 10.1111/j.1432-1033.1996.0105u.x. [DOI] [PubMed] [Google Scholar]
  13. Dawson R. M., Irvine R. F., Bray J., Quinn P. J. Long-chain unsaturated diacylglycerols cause a perturbation in the structure of phospholipid bilayers rendering them susceptible to phospholipase attack. Biochem Biophys Res Commun. 1984 Dec 14;125(2):836–842. doi: 10.1016/0006-291x(84)90615-6. [DOI] [PubMed] [Google Scholar]
  14. Dorovska-Taran V., Wick R., Walde P. A 1H nuclear magnetic resonance method for investigating the phospholipase D-catalyzed hydrolysis of phosphatidylcholine in liposomes. Anal Biochem. 1996 Aug 15;240(1):37–47. doi: 10.1006/abio.1996.0328. [DOI] [PubMed] [Google Scholar]
  15. Fulford A. J., Peel W. E. Lateral pressures in biomembranes estimated from the dynamics of fluorescent probes. Biochim Biophys Acta. 1980 May 23;598(2):237–246. doi: 10.1016/0005-2736(80)90002-4. [DOI] [PubMed] [Google Scholar]
  16. Gabriel N. E., Agman N. V., Roberts M. F. Enzymatic hydrolysis of short-chain lecithin/long-chain phospholipid unilamellar vesicles: sensitivity of phospholipases to matrix phase state. Biochemistry. 1987 Nov 17;26(23):7409–7418. doi: 10.1021/bi00397a032. [DOI] [PubMed] [Google Scholar]
  17. Goñi F. M., Alonso A. Structure and functional properties of diacylglycerols in membranes. Prog Lipid Res. 1999 Jan;38(1):1–48. doi: 10.1016/s0163-7827(98)00021-6. [DOI] [PubMed] [Google Scholar]
  18. Goñi F. M., Basáez G., Begoña Ruiz-Argüello M., Alonso A. Interfacial enzyme activation, non-lamellar phase formation and membrane fusion. Is there a conducting thread? Faraday Discuss. 1998;(111):55–78. doi: 10.1039/a806352d. [DOI] [PubMed] [Google Scholar]
  19. Hodgkin M. N., Pettitt T. R., Martin A., Michell R. H., Pemberton A. J., Wakelam M. J. Diacylglycerols and phosphatidates: which molecular species are intracellular messengers? Trends Biochem Sci. 1998 Jun;23(6):200–204. doi: 10.1016/s0968-0004(98)01200-6. [DOI] [PubMed] [Google Scholar]
  20. Holopainen J. M., Angelova M. I., Kinnunen P. K. Vectorial budding of vesicles by asymmetrical enzymatic formation of ceramide in giant liposomes. Biophys J. 2000 Feb;78(2):830–838. doi: 10.1016/S0006-3495(00)76640-9. [DOI] [PMC free article] [PubMed] [Google Scholar]
  21. Holopainen J. M., Medina O. P., Metso A. J., Kinnunen P. K. Sphingomyelinase activity associated with human plasma low density lipoprotein. J Biol Chem. 2000 Jun 2;275(22):16484–16489. doi: 10.1074/jbc.275.22.16484. [DOI] [PubMed] [Google Scholar]
  22. Hønger T., Jørgensen K., Stokes D., Biltonen R. L., Mouritsen O. G. Phospholipase A2 activity and physical properties of lipid-bilayer substrates. Methods Enzymol. 1997;286:168–190. doi: 10.1016/s0076-6879(97)86011-9. [DOI] [PubMed] [Google Scholar]
  23. Hübner S., Couvillon A. D., Käs J. A., Bankaitis V. A., Vegners R., Carpenter C. L., Janmey P. A. Enhancement of phosphoinositide 3-kinase (PI 3-kinase) activity by membrane curvature and inositol-phospholipid-binding peptides. Eur J Biochem. 1998 Dec 1;258(2):846–853. doi: 10.1046/j.1432-1327.1998.2580846.x. [DOI] [PubMed] [Google Scholar]
  24. Lehtonen J. Y., Kinnunen P. K. Phospholipase A2 as a mechanosensor. Biophys J. 1995 May;68(5):1888–1894. doi: 10.1016/S0006-3495(95)80366-8. [DOI] [PMC free article] [PubMed] [Google Scholar]
  25. Moolenaar W. H. Lysophosphatidic acid signalling. Curr Opin Cell Biol. 1995 Apr;7(2):203–210. doi: 10.1016/0955-0674(95)80029-8. [DOI] [PubMed] [Google Scholar]
  26. Mountford C. E., Wright L. C. Organization of lipids in the plasma membranes of malignant and stimulated cells: a new model. Trends Biochem Sci. 1988 May;13(5):172–177. doi: 10.1016/0968-0004(88)90145-4. [DOI] [PubMed] [Google Scholar]
  27. Rao N. M. Differential susceptibility of phosphatidylcholine small unilamellar vesicles to phospholipases A2, C and D in the presence of membrane active peptides. Biochem Biophys Res Commun. 1992 Jan 31;182(2):682–688. doi: 10.1016/0006-291x(92)91786-p. [DOI] [PubMed] [Google Scholar]
  28. Rao N. M., Sundaram C. S. Sensitivity of phospholipase C (Bacillus cereus) activity to lipid packing in sonicated lipid mixtures. Biochemistry. 1993 Aug 24;32(33):8547–8552. doi: 10.1021/bi00084a022. [DOI] [PubMed] [Google Scholar]
  29. Roberts M. F. Phospholipases: structural and functional motifs for working at an interface. FASEB J. 1996 Aug;10(10):1159–1172. doi: 10.1096/fasebj.10.10.8751718. [DOI] [PubMed] [Google Scholar]
  30. Ruiz-Argüello M. B., Basáez G., Goñi F. M., Alonso A. Different effects of enzyme-generated ceramides and diacylglycerols in phospholipid membrane fusion and leakage. J Biol Chem. 1996 Oct 25;271(43):26616–26621. doi: 10.1074/jbc.271.43.26616. [DOI] [PubMed] [Google Scholar]
  31. Ruiz-Argüello M. B., Goñi F. M., Alonso A. Phospholipase C hydrolysis of phospholipids in bilayers of mixed lipid compositions. Biochemistry. 1998 Aug 18;37(33):11621–11628. doi: 10.1021/bi980615x. [DOI] [PubMed] [Google Scholar]
  32. Ruiz-Argüello M. B., Goñi F. M., Alonso A. Vesicle membrane fusion induced by the concerted activities of sphingomyelinase and phospholipase C. J Biol Chem. 1998 Sep 4;273(36):22977–22982. doi: 10.1074/jbc.273.36.22977. [DOI] [PubMed] [Google Scholar]
  33. Schnorf M., Potrykus I., Neuhaus G. Microinjection technique: routine system for characterization of microcapillaries by bubble pressure measurement. Exp Cell Res. 1994 Feb;210(2):260–267. doi: 10.1006/excr.1994.1038. [DOI] [PubMed] [Google Scholar]
  34. Sundler R., Alberts A. W., Vagelos P. R. Phospholipases as probes for membrane sideness. Selective analysis of the outer monolayer of asymmetric bilayer vesicles. J Biol Chem. 1978 Aug 10;253(15):5299–5304. [PubMed] [Google Scholar]
  35. Vandenbranden M., De Gand G., Brasseur R., Defrise-Quertain F., Ruysschaert J. M. Hydrolysis of phosphatidylcholine liposomes by lysosomal phospholipase A is maximal at the phase transition temperature of the lipid substrate. Biosci Rep. 1985 Jun;5(6):477–482. doi: 10.1007/BF01116946. [DOI] [PubMed] [Google Scholar]
  36. Wick R., Angelova M. I., Walde P., Luisi P. L. Microinjection into giant vesicles and light microscopy investigation of enzyme-mediated vesicle transformations. Chem Biol. 1996 Feb;3(2):105–111. doi: 10.1016/s1074-5521(96)90286-0. [DOI] [PubMed] [Google Scholar]
  37. Zidovetzki R., Lester D. S. The mechanism of activation of protein kinase C: a biophysical perspective. Biochim Biophys Acta. 1992 Apr 7;1134(3):261–272. doi: 10.1016/0167-4889(92)90185-e. [DOI] [PubMed] [Google Scholar]

Articles from Biophysical Journal are provided here courtesy of The Biophysical Society

RESOURCES