liposomes, part b

METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISION OF BIOLOGY CALIFORNIA INSTITUTE OF TECHNOLOGY PASADENA, CALIFORNIA FOUNDING EDITORS Sidney P. Colowick and Nathan O. Kaplan Preface The origins of liposome research can be traced to the contributions by Alec Bangham and colleagues in the mid 1960s. The description of lecithin dispersions as containing ‘‘spherulites composed of concentric lamellae’’ (A. D. Bangham and R. W. Horne, J. Mol. Biol. 8, 660, 1964) was followed by the observation that ‘‘the diffusion of univalent cations and anions out of spontaneously formed liquid crystals of lecithin is remarkably similar to the diffusion of such ions across biological membranes (A. D. Bangham, M. M. Standish and J. C. Watkins, J. Mol. Biol. 13, 238, 1965). Following early studies on the biophysical characterization of multilamellar and unilamellar liposomes, investigators began to utilize liposomes as a well-defined model to understand the structure and function of biological membranes. It was also recognized by pioneers including Gregory Gregoriadis and Demetrios Papa- hadjopoulos that liposomes could be used as drug delivery vehicles. It is gratifying that their efforts and the work of those inspired by them have lead to the development of liposomal formulations of doxorubicin, daunorubicin and amphotericin B now utilized in the clinic. Other medical applications of liposomes include their use as vaccine adjuvants and gene delivery vehicles, which are being explored in the laboratory as well as in clinical trials. The field has progressed enormously in the 38 years since 1965. This volume includes applications of liposomes in biochemistry, molecular cell biology and molecular virology. I hope that these chapters will facilitate the work of graduate students, post-doctoral fellows, and established scientists entering liposome research. Subsequent volumes in this series will cover add- itional subdisciplines in liposomology. The areas represented in this volume are by no means exhaustive. I have tried to identify the experts in each area of liposome research, particularly those who have contributed to the field over some time. It is unfortunate that I was unable to convince some prominent investigators to contribute to the volume. Some invited contributors were not able to prepare their chapters, despite generous extensions of time. In some cases I may have inadvertently overlooked some experts in a particular area, and to these individuals I extend my apologies. Their primary contributions to the field will, nevertheless, not go unnoticed, in the citations in these volumes and in the hearts and minds of the many investigators in liposome research. xiii I would like to express my gratitude to all the colleagues who graciously contributed to these volumes. I would like to thank Shirley Light of Academic Press for her encouragement for this project, and Noelle Gracy of Elsevier Inc. for her help at the later stages of the project. I am especially thankful to my wife Diana Flasher for her understanding, support and love during the endless editing process, and my children Avery and Maxine for their unique curiosity, creativity, cheer, and love. I wish to dedicate this volume to Diana, Avery and Maxine. Nejat Du ¨ zgu ¨ nes xiv preface Contributors to Volume 372 Article numbers are in parentheses and following the names of contributors. Affiliations listed are current. Alicia Alonso (3), Unidad de Biofisica and Departamento de Bioquı ´ mica, Uni- versidad Del Paı ´ s Vasco, Aptdo. 644, 48080 Bilbao, Spain Bruno Antonny (151), CNRS-Institut de Pharmacologie Moleculaire et Cellulaire, 660 Route des Lucioles, 06560 Sophia Antipolis-Valbonne, France John D. Bell (19), Department of Physi- ology and Developmental Biology, Brig- ham Young University, Provo, Utah 84602 Robert Bittman (374), Department of Medical Microbiology, Molecular Vir- ology Section, University of Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands Pierre Bonnafous (408), Crucell Holland BV, Archimedesweg 4, P.O. Box 2048, Leiden, The Netherlands Mauro Dalla Serra (99), CMR-ITC In- stitute of Biophysics, Section at Trento, Via Sommarive 18, Povo, Trento 38050, Italy David W. Deamer (133), Department of Chemistry and Biochemistry, University of Californi-Santa Cruz, Santa Cruz, California 95064 Pietro De Camilli (248), Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medi- cine, 295 Congress Avenue, New Haven, Connecticut 06510 Jeanine De Keyzer (86), University of Groningen, Department of Microbiology, P. O. Box 14, Haren 9750AA, The Netherlands Sue E. Delos (428), Department of Cell Biology, UVA Health System, School of Medicine, P.O. Box 800732, Charlottesville, Virginia 22908 Arnold J. M. Driessen (86), University of Groningen, Department of Microbiol- ogy, P. O. Box 14, Haren 9750AA, The Netherlands Nejat Du ¨ zgu ¨ nes, (260), Department of Microbiology, School of Dentistry, University of the Pacific, 2155 Webster Street, San Francisco, California 94115 Laurie J. Earp (428), Department of Cell Biology, UVA Health System, School of Medicine, P.O. Box 800732, Charlottesville, Virginia 22908 Raquel F. Epand (124), Department of Biochemistry, McMaster Health Sciences Center, Hamilton, Ontario L8N 3Z5, Canada Richard M. Epand (124), Department of Biochemistry, McMaster Health Sciences Center, Hamilton, Ontario L8N 3Z5, Canada Shiroh Futaki (349), Faculty of Pharma- ceutical Sciences, The University of To- kushima, Shomachi 1-78-1, 770–8505 Tokushima, Japan Yves Gaudin (392), Laboratoire de Genet- iquie des Virus du CNRS, Gif sur Yvette Cedex 91198, France Re ´ my Gibrat (166), Plant Biochemistry and Molecular Biology, Agro-M/CNRS/ ONRA/UMII,ENSA-INRA,Montpellier, 34060 Cedex 1, France ix Fe ´ lix M. Gon ˜ i (3), Unidad de Biofisica and Departamento de Bioquı ´ mica, Universi- dad Del Paı ´ s Vasco, Aptdo. 644, 48080 Bilbao, Spain Ckayde Grignon (166), Plant Biochemis- try and Molecular Biology, Agro-M/ CNRS/ONRA/UMII, ENSA-INRA, Montpellier, 34060 Cedex 1, France Hideyoshi Harashima (349), Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1-78-1, 770– 8505 Tokushima, Japan Theodore L. Hazlett (19), Laboratory for Fluorescence Dynamics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Lorraine D. Hernandez (428), Depart- ment of Cell Biology, UVA Health System, School of Medicine, P.O. Box 800732, Charlottesville, Virginia 22908 Andreas Hoffman (186), Macromolecular Crystallography Laboratory, NCI at Frederick, 539 Boyles Street, Frederick, Maryland 21702 Robert Huber (186), Institute of Cell and Molecular Biology, University of Edin- burgh, Michael Swann Building, The King’s Building Mayfield Road, EH9 3JR Edinburgh, Scotland Hiroshi Kiwada (349), Faculty of Pharma- ceutical Sciences, The University of Tokushima, Shomachi 1-78-1, 770–8505 Tokushima, Japan Kyung-Dall Lee (319), Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109 Tatiana S. Levchenko (339), Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115 Daniel Le ´ vy (65), Institut Curie, UMR- CNRS 168 and LRC-CEA 34V, 11 Rue Pierre et Marie Curie, 75231 Paris Cedex 05, France Song Liu (274), Department of Biochemis- try and Cell Biology, Rice University, Houston, Texas 77005 Manas Mandal (319), Department of Pharmaceutical Sciences, College of Phar- macy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109 Elizabeth Mathew (319), Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109 James A. McNew (274), Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005 Thomas J. Melia (274), Cellular Biochem- istry and Biophysics Program, Memorial Sloan-Kettering Cancer Center, New York, New York 10021 Gianfranco Menestrina (99), CMR-ITC Institute of Biophysics, Section at Trento, Via Sommarive 18, Povo, Trento 38050, Italy Pierre-Alain Monnard (133), Depart- ment of Chemistry and Biochemistry, University of Californi-Santa Cruz, Santa Cruz, California 95064 Jose ´ L. Nieva (3, 235), Unidad de Biofisica and Departamento de Bioquı ´ mica, Uni- versidad Del Paı ´ s Vasco, Aptdo, 644, 48080 Bilbao, Spain Shlomo Nir (235), Seagram Center for Soil and Water Sciences, Faculty of Agricul- tural, Food and Environmental Quality Sciences, Rehovot 76100, Israel Olivier Nosjean (216), Pharmacology Moleculaire et Cellulaire, Institut de Re- cherches Servier, Crossy-sur-Seine, France x contributors to volume 372 Christian Oker-Blom (418), University of Jvaskyla, Department of Biological and Environmental Sciences, P.O. Box 35, FIN 40351 Jyvaskyla, Finland Frank Opitz (48), University of Leipzig, Institute for Medical Physics and Bio- physics, Liebigstrasse 27, Leipzig D- 04103, Germany Sergio Gerardo Peisajovich (361), De- partment of Biological Chemistry, Weig- mann Institute of Science, Rehovot 76100, Israel Jens Pittler (48), University of Leipzig, Institute for Medical Physics and Bio- physics, Liebigstrasse 27, Leipzig D- 04103, Germany Chester Provoda (319), Department of Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan 48109 Ram Rammohan (339), Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115 Jean-Louis Rigaud (65), Institut Curie, UMR-CNRS 168 and LRC-CEA 34V, 11 Rue Pierre et Marie Curie, 75231 Paris Cedex 05, France Karine Robbe (151), CNRS-Institut de Pharmacologie Moleculaire et Cellulaire, 660 Route des Lucioles, 06560 Sophia Antipolis-Valbonne, France Ste ´ phane Roche (392), Laboratoire de Genetiquie des Virus du CNRS, Gif sur Yvette Cedex 91198, France Bernard Roux (216), Physico-Chemie Biologique, Universite C Bernard-Lyon 1, Villeurbanne, France Susana A. Sanchez (19), Laboratory for Fluorescence Dynamics, University of Illinois at Urbana-Champaign, Urbana, Illinois 61801 Brenton L. Scott (274), Department of Biochemistry and Cell Biology, Rice Uni- versity, Houston, Texas 77005 Yechiel Shai (361), Department of Bio- logical Chemistry, Weigmann Institute of Science, Rehovot 76100, Israel Jolanda M. Smit (374), Department of Medical Microbiology, Molecular Vir- ology Section, University of Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands James E. Smolen (300), Department of Pediatrics, Baylor College of Medicine, 1100 Bates, Room 6014, Houston, Texas 77030 Toon Stegmann (408), Crucell Holland BV, Archimedesweg 4, P.O. Box 2048, Leiden, The Netherlands Reiko Tachibani (349), Faculty of Pharmaceutical Sciences, The University of Tokushima, Shomachi 1-78-1, 770- 8505 Tokushima, Japan Vladimir P. Torchilin (339), Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115 Chris Van der Does (86), University of Groningen, Department of Microbiology, P. O. Box 14, Haren 9750AA, The Netherlands Martin Van der Laan (86), University of Groningen, Department of Microbiology, P.O. Box 14, Haren 9750AA, The Netherlands Jeffrey S. Van Komen (274), Department of Biochemistry and Cell Biology, Rice University, Houston, Texas 77005 Ana V. Villar (3), Unidad de Biofisica and Departamento de Bioquı ´ mica, Universi- dad Del Paı ´ s Vasco, Aptdo. 644, 48080 Bilbao, Spain contributors to volume 372 xi Natalia Volodina (339), Department of Pharmaceutical Sciences, Northeastern University, 360 Huntington Avenue, Boston, Massachusetts 02115 Matti Vuento (418), University of Jvasky- la, Department of Biological and Environ- mental Sciences, P.O. Box 35, FIN 40351 Jyvaskyla, Finland Barry-Lee Waarts (374), Department of Medical Microbiology, Molecular Vir- ology Section, University of Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands Thomas Weber (274), Department of Mo- lecular, Cell, and Developmental Biology and Carl C. Icahn Institute for Gene Ther- apy and Molecular Medicine, Mount Sinai School of Medicine, New York, New York 10029 Markus R. Wenk (248), Department of Cell Biology, Howard Hughes Medical Institute, Yale University School of Medi- cine, 295 Congress Avenue, New Haven, Connecticut 06510 Judith M. White (428), Department of Cell Biology, UVA Health System, School of Medicine, P.O. Box 800732, Charlottesville, Virginia 22908 Jan Wilschut (374), Department of Med- ical Microbiology, Molecular Virology Section, University of Groningen, Ant. Deusinglaan 1, 9713 AV Groningen, The Netherlands Olaf Zscho ¨ rnig (48), University of Leip- zig, Institute for Medical Physics and Biophysics, Liebigstrasse 27, Leipzig D- 04103, Germany xii contributors to volume 372 [1] Interaction of Phospholipases C and Sphingomyelinase with Liposomes By Fe ´ lix M. Gon ˜ i,Ana V. Villar,Jose ´ L. Nieva, and Alicia Alonso Introduction The conventional classification of membrane proteins as intrinsic or in- tegral, and as extrinsic or peripheral, has on the whole been superseded by a more complex pattern in which a continuum of possibilities is considered, from the integral protein firmly embedded in the bilayer to the soluble pro- tein that contacts the membrane only transiently for a specific function. Phospholipases stand in a class of their own as membrane proteins because, irrespective of their more or less ‘‘peripheral’’ location, they perturb the physical properties of the membrane through chemical modification of its lipid components. Thus it is not their mere binding and/or insertion into the bilayer, but the chemical reactions they catalyze, that determines ultimately the nature of their interaction with the membrane. In this laboratory we have examined the membrane interactions of phosphatidylcholine (PC)-preferring phospholipase C (PC-PLC), and of the sphingomyelin-specific phospholipase C usually known as sphingomy- elinase. More recently, we have explored the effects of a phosphatidylino- sitol (PI)-specific phospholipase C (PI-PLC). The effects of these enzymes occur essentially through their lipid end-products, diacylglycerol or cera- mide. Depending on the enzyme, and on the bilayer lipid compositions, a variety of effects can be observed. Enzyme activity is commonly followed by vesicle–vesicle aggregation, and, under certain conditions, by interve- sicular lipid mixing, and by mixing of vesicular aqueous contents. Observa- tion of intervesicular contents mixing is always accompanied by detection of mixing of lipid inner monolayers, indicative of vesicle–vesicle fusion. Moreover, efflux of vesicle contents, whether or not accompanied by other effects, is observed often as a result of phospholipase C treatment. All of the above-described phenomena can be monitored conveniently through the use of fluorescence spectroscopy techniques, as detailed below. A summary of the results obtained by these methods in our laboratory is presented in a review. 1 1 F. M. Gon ˜ i and A. Alonso, Biosci. Rep. 20, 443 (2000). [1] phospholipase–liposome interactions 3 Copyright 2003, Elsevier Inc. All rights reserved. METHODS IN ENZYMOLOGY, VOL. 372 0076-6879/03 $35.00 Materials Enzymes Phospholipase C (EC 3.1.4.3) from Bacillus cereus (MW, $23,000) is usually obtained from Roche Molecular Biochemicals (Indianapolis, IN) and used without further purification. Routine sodium dodecyl sulfate- polyacrylamide gel electrophoresis (SDS–PAGE) controls reveal that the enzyme preparations supplied by this company are !90% pure. The enzyme shows broad specificity (see below) and is active on glycero- phospholipids in a variety of aggregational states, for example, monomeric in solution, dispersed in detergent-mixed micelles, and in model bilayers. Roche Molecular Biochemicals has discontinued the sale of this enzyme. Other suppliers provide equivalent enzymes, but they have not been tested thoroughly in our laboratory. Phosphatidylinositol-specific phospholipase C (EC 4.6.1.13) from B. cereus is supplied by Molecular Probes (Eugene, OR) and used without further purification. Sphingomyelinase (EC 3.1.4.12) from B. cereus is pur- chased from Sigma (St. Louis, MO). As indicated by the manufacturer, preparations of this enzyme often contain significant phospholipase C con- tamination, in amounts that vary from batch to batch. We have been unable to separate the PC-PLC impurity from sphingomyelinase, using a variety of chromatographic methods. In our case, and with the exception of those experiments in which the simultaneous activities of PC-PLC and sphingomyelinase are required, the PC-PLC inhibitor o-phenanthroline is used routinely in sphingomyelinase assays (see below). In the absence of PLC activity, sphingomyelinase is found to cleave specifically sphingomy- elin, and not any glycerophospholipid. Activity on sphingophospholipids other than sphingomyelin, for example, ceramide phosphorylethanolamine, has not been tested. Substrates Egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), and wheat germ phosphatidylinositol (PI) are grade I from Lipid Products (South Nutfield, Surrey, UK). Egg sphingomyelin (SM) is from Avanti Polar Lipids (Alabaster, AL). The purity of the above-described lipids is checked by running 0.1 mg of lipid on a thin-layer chromatography plate that is later revealed by charring in an oven under conditions that allow de- tection of 1 g of lipid. Dihexanoylphosphatidylcholine (DHPC) and cho- lesterol are supplied by Sigma. All these lipids are used without further purification. Glycosylphosphatidylinositol (GPI) is purified from rat liver according to Varela-Nieto et al. 2 GPI is stored at À20  and used within 4 liposomes in biochemistry [1] the following 2 weeks. Oxidation or other forms of degradation are detected after long-term storage. DHPC is used below its critical micellar concentration (i.e., below 10 mM) to obtain dispersions of monomeric phospholipid. However, enzyme assays on defined substrates are usually carried out with phospholipid vesicles (liposomes). For liposome production, phospholipid dispersions are prepared by re- hydrating lipid films dried from organic solvents. Solvents are evaporated thoroughly under a current of N 2 , and then left for at least 2 h under high vacuum to remove solvent traces. Small unilamellar vesicles are prepared by sonication 3 from aqueous phospholipid dispersions, consisting mainly of multilamellar vesicles (MLVs). Samples on ice are treated in a Soniprep 150 probe sonicator (MSE, Crawley, Surrey, UK) with 10- to 12-m pulses for 30 min, alternat- ing on and off periods every 10 s. Probe debris and MLV remains are pelleted by centrifugation at 6000 g and 4  for 10 min. Large unilamellar vesicles (LUVs) are prepared by the extrusion method. 4 To obtain these vesicles aqueous lipid suspensions (MLVs) are ex- truded 10 times through two stacked Nuclepore (Pleasanton, CA) polycar- bonate filters (pore diameter, 0.1 m). The extruder is supplied by Northern Lipids (Vancouver, BC, Canada). Extrusion takes place at room tempera- ture, except for LUVs consisting of pure SM, in which case the extruder is equilibrated at 42  with a temperature regulation accessory. Average ves- icle diameters are measured by quasi-elastic light scattering (QELS), using a Zetasizer instrument (Malvern Instruments, Malvern, Worcestershire, UK). LUV mean diameters are $ 100–115 and $160–190 nm for PC-based liposomes and SM-based liposomes, respectively. To ascertain that the extrusion procedure does not alter the lipid com- position of the systems under study, the lipid mixtures are quantitated oc- casionally after the extrusion treatment. For that purpose, the resulting LUV suspensions are extracted with chloroform–methanol (2:1, v/v). The organic phase is concentrated and separated on thin-layer chromatography (TLC) Silica Gel 60 plates, using successively in the same direction the solvents chloroform–methanol–water (60:30:5, v/v/v) for the first 10 cm and petroleum ether-ethyl ether-acetic acid (60:40:1, v/v/v) for the whole plate. After charring with a sulfuric acid reagent, the spot intensities are quantified with a dual-wavelength TLC scanner (CS-930; Shimadzu, Tokyo, Japan). The results of these studies have shown that, under our 2 I. Varela-Nieto, L. Alvarez, and J. M. Mato, ‘‘Handbook of Endocrine Research Techniques,’’ p. 391. Academic Press, San Diego, CA, 1993. 3 A. Alonso, R. Sa ´ ez, A. Villena, and F. M. Gon ˜ i, J. Membr. Biol. 67, 55 (1982). 4 L. D. Mayer, M. H. Hope, and P. R. Cullis, Biochim. Biophys. Acta 858, 161 (1986). [1] phospholipase–liposome interactions 5 [...]... temperatures 22 S D Brown, B L Baker, and J D Bell, Biochim Biophys Acta 1168, 13 (1993) M Menashe, G Romero, R L Biltonen, and D Lichtenberg, J Biol Chem 261, 5328 (1986) 24 D Lichtenberg, G Romero, M Menashe, and R L Biltonen, J Biol Chem 261, 5334 (1986) 25 J D Bell, M L Baker, E D Bent, R W Ashton, D J Hemming, and L D Hansen, Biochemistry 34, 11551 (1995) 23 24 liposomes in biochemistry [2] below the main... 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L Biltonen, J Biol Chem 256, 4541 (1981) 28 J D Bell and R L Biltonen, J Biol Chem 267, 11046 (1992) 29 T Honger, K Jorgensen, R L Biltonen, and O G Mouritsen, Biochemistry 35, 9003 (1996) 30 W R Burack, M E Gadd, and R L Biltonen, Biochemistry 34, 14819 (1995) 31 J D Bell, M Burnside, J A Owen, M L Royall, and M L Baker, Biochemistry 35, 4945 (1996) 32 S P White, D L Scott, Z Otwinowski, M H Gelb,... mixing, based on FRET between NBD-PE and Rh-PE.12 Vesicles composed of PI–PE–PC–cholesterol (Ch) (40:30:15:15, mole ratio), containing 0.6 mol% of each probe, are prepared as described above Fluorescence probes are thus located in both membrane layers Fluorescence from the outer monolayer is quenched by addition of 0.2% (w/v) bovine serum albumin (BSA) and 10 mM dithiothreitol (DTT) Addition of BSA and... concentration was 0.3 mM NBD-PE was present at 0.6 mol% in the bilayer, and its fluorescence emission intensity was considered 100% (A) Effect of 10 mM DTT (B) Effect of 0.1% (w/v) BSA (C) Combined effect of 0.2% (w/v) BSA and 10 mM DTT (D) Effect of 0.2% (w/v) BSA (A V Villar, unpublished data, 2002) activity by DTT, which reduces BSA disulfide bonds, decreasing BSA lipid extraction capacity The BSA:DTT mole ratio... influence on the susceptibility of the bilayer to sPLA2 The relationship between membrane phases and the action of sPLA2 are complicated by effects on both steps in Fig 1 The adsorption of sPLA2 to the surface of PC SUVs in the absence of calcium requires the membrane to be in the gel phase.13 Alternatively, for LUVs, the rate of turnover of substrate by bound enzyme is higher when the membrane is near the... Mandel, P B Sigler, and B Honig, Biophys J 67, 493 (1994) 17 F Ghomashchi, Y Lin, M S Hixon, B Z Yu, R Annand, M K Jain, and M H Gelb, Biochemistry 37, 6697 (1998) 18 W R Burack and R L Biltonen, Chem Phys Lipids 73, 209 (1994) 19 M K Jain, J Rogers, D V Jahagirdar, J F Marecek, and F Ramirez, Biochim Biophys Acta 860, 435 (1986) 20 O G Berg, B Z Yu, J Rogers, and M K Jain, Biochemistry 30, 7283 (1991)... complex between the bound enzyme and a phospholipid monomer from the membrane Evidence from X-ray diffraction experiments and studies using 1 I Kudo, M Murakami, S Hara, and K Inoue, Biochim Biophys Acta 1170, 217 (1993) G Lambeau and M Lazdunski, Trends Pharmacol Sci 20, 162 (1999) 3 E A Dennis, J Biol Chem 269, 13057 (1994) 4 M F Roberts, FASEB J 10, 1159 (1996) 5 B K Lathrop and R L Biltonen, J Biol... means that experiments must be 47 Y Snitko, R S Koduri, S K Han, R Othman, S F Baker, B J Molini, D C Wilton, M H Gelb, and W Cho, Biochemistry 36, 14325 (1997) 48 S K Han, E T Yoon, and W Cho, Biochem J 331, 353 (1998) 49 S Bezzine, R S Koduri, E Valentin, M Murakami, I Kudo, F Ghomashchi, M Sadilek, G Lambeau, and M H Gelb, J Biol Chem 275, 3179 (2000) 50 J D Bell and R L Biltonen, Methods Enzymol . 1-aminonaphthalene-3,6,8-trisulfonic acid (ANTS), and N,N 0 -p-xylene-bis(pyridinium bromide) (DPX) are provided by Molecular Probes. 6-Carboxyfluorescein (6-CF) is supplied by Eastman Kodak (Burnaby, BC, Canada). Fluorescein isothiocyanate- derivatized. sub- strates. Ghost membranes are obtained from erythrocyte concentrate, as supplied by a blood bank, using the procedure of Steck and Kant. 5 Fluorescent probes The following fluorescent probes. allows GPI to bind the membrane and become stabi- lized there. The incorporation does not disrupt membrane stability. 6 Once the symmetric LUV liposomes are prepared as described above, they are diluted