METHODS IN ENZYMOLOGY EDITORS-IN-CHIEF John N. Abelson Melvin I. Simon DIVISIONOFBIOLOGY CALIFORNIAINSTITUTEOFTECHNOLOGY 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.StandishandJ.C.Watkins,J.Mol.Biol.13,238,1965).Followingearly studies on the biophysical characterization of multilamellar and unilamellar liposomes,investigators began to utilize liposomes as awell-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, whicharebeingexploredinthelaboratoryaswellasinclinicaltrials.Thefield hasprogressedenormouslyinthe38yearssince1965. This volume includes applications of liposomes in biochemistry, molecular cellbiologyandmolecularvirology.Ihopethatthesechapterswillfacilitatethe work of graduate students, post-doctoral fellows, and established scientists entering liposome research. Subsequent volumes in this series will cover add- itionalsubdisciplinesinliposomology. 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 thosewhohavecontributedtothefieldoversometime.Itisunfortunatethat 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 overlookedsomeexpertsinaparticulararea,andtotheseindividualsIextend myapologies.Theirprimarycontributionstothefieldwill,nevertheless,notgo unnoticed,inthecitationsinthesevolumesandintheheartsandmindsofthe manyinvestigatorsinliposomeresearch. xiii xiv preface I would like to express my gratitude to all the colleagues who graciously contributedtothesevolumes.IwouldliketothankShirleyLightofAcademic Pressforherencouragementforthisproject,andNoelleGracyofElsevierInc. forherhelpatthelaterstagesoftheproject. I am especially thankful to my wife Diana Flasher for her understanding, supportandloveduringtheendlesseditingprocess,andmychildrenAveryand Maxinefortheiruniquecuriosity,creativity,cheer,andlove.Iwishtodedicate thisvolumetoDiana,AveryandMaxine. Nejat Du¨zgu¨nes Contributors to Volume 372 Articlenumbersareinparenthesesandfollowingthenamesofcontributors. Affiliationslistedarecurrent. Alicia Alonso (3), Unidad de Biofisica Sue E. Delos (428), Department of and Departamento de Bioqu´ımica, Uni- Cell Biology, UVA Health System, versidad Del Pa´ıs Vasco, Aptdo. 644, School of Medicine, P.O. Box 800732, 48080Bilbao,Spain Charlottesville,Virginia22908 Bruno Antonny (151), CNRS-Institut de Arnold J. M. Driessen (86), University PharmacologieMoleculaireetCellulaire, of Groningen, Department of Microbiol- 660 Route des Lucioles, 06560 Sophia ogy, P. O. Box 14, Haren 9750AA, The Antipolis-Valbonne,France Netherlands John D. Bell (19), Department of Physi- Nejat Du¨zgu¨nes, (260), Department of ology and Developmental Biology, Brig- Microbiology, School of Dentistry, ham Young University, Provo, Utah University of the Pacific, 2155 84602 WebsterStreet,SanFrancisco,California Robert Bittman (374), Department of 94115 Medical Microbiology, Molecular Vir- Laurie J. Earp (428), Department of ology Section, University of Groningen, Cell Biology, UVA Health System, Ant.Deusinglaan1,9713AVGroningen, School of Medicine, P.O. Box 800732, TheNetherlands Charlottesville,Virginia22908 Pierre Bonnafous (408), Crucell Holland Raquel F. Epand (124), Department of BV, Archimedesweg 4, P.O. Box 2048, Biochemistry,McMasterHealthSciences Leiden,TheNetherlands Center, Hamilton, Ontario L8N 3Z5, Mauro Dalla Serra (99), CMR-ITC In- Canada stitute of Biophysics, Section at Trento, Richard M. Epand (124), Department of Via Sommarive 18, Povo, Trento 38050, Biochemistry,McMasterHealthSciences Italy Center, Hamilton, Ontario L8N 3Z5, David W. Deamer (133), Department of Canada Chemistry and Biochemistry, University of Californi-Santa Cruz, Santa Cruz, Shiroh Futaki (349), Faculty of Pharma- California95064 ceutical Sciences, The University of To- kushima, Shomachi 1-78-1, 770–8505 Pietro De Camilli (248), Department of Tokushima,Japan Cell Biology, Howard Hughes Medical Institute,YaleUniversitySchoolofMedi- YvesGaudin(392),LaboratoiredeGenet- cine, 295 Congress Avenue, New Haven, iquiedesVirusduCNRS, GifsurYvette Connecticut06510 Cedex91198,France Jeanine De Keyzer (86), University of Re´my Gibrat (166), Plant Biochemistry Groningen, Department of Microbiology, and Molecular Biology, Agro-M/CNRS/ P. O. Box 14, Haren 9750AA, The ONRA/UMII,ENSA-INRA,Montpellier, Netherlands 34060Cedex1,France ix x contributors to volume 372 Fe´lixM.Gon˜i(3),UnidaddeBiofisicaand Daniel Le´vy (65), Institut Curie, UMR- Departamento de Bioqu´ımica, Universi- CNRS 168 and LRC-CEA 34V, 11 Rue dad Del Pa´ıs Vasco, Aptdo. 644, 48080 PierreetMarieCurie,75231ParisCedex Bilbao,Spain 05,France Ckayde Grignon (166), Plant Biochemis- SongLiu(274),DepartmentofBiochemis- try and Molecular Biology, Agro-M/ try and Cell Biology, Rice University, CNRS/ONRA/UMII, ENSA-INRA, Houston,Texas77005 Montpellier,34060Cedex1,France Manas Mandal (319), Department of Hideyoshi Harashima (349), Faculty of PharmaceuticalSciences,CollegeofPhar- Pharmaceutical Sciences, The University macy,UniversityofMichigan,428Church of Tokushima, Shomachi 1-78-1, 770– Street,AnnArbor,Michigan48109 8505Tokushima,Japan Elizabeth Mathew (319), Department of Theodore L. Hazlett (19), Laboratory Pharmaceutical Sciences, College of forFluorescenceDynamics,Universityof Pharmacy, University of Michigan, 428 Illinois at Urbana-Champaign, Urbana, Church Street, Ann Arbor, Michigan Illinois61801 48109 Lorraine D. Hernandez (428), Depart- James A. McNew (274), Department ment of Cell Biology, UVA Health of Biochemistry and Cell Biology, Rice System, School of Medicine, P.O. University,Houston,Texas77005 Box 800732, Charlottesville, Virginia 22908 ThomasJ.Melia(274),CellularBiochem- istry and Biophysics Program, Memorial AndreasHoffman(186),Macromolecular Sloan-Kettering Cancer Center, New Crystallography Laboratory, NCI at York,NewYork10021 Frederick, 539 Boyles Street, Frederick, Maryland21702 GianfrancoMenestrina(99),CMR-ITC InstituteofBiophysics,SectionatTrento, RobertHuber(186),InstituteofCelland Via Sommarive 18, Povo, Trento 38050, Molecular Biology, University of Edin- Italy burgh, Michael Swann Building, The King’s Building Mayfield Road, EH9 Pierre-Alain Monnard (133), Depart- 3JREdinburgh,Scotland ment of Chemistry and Biochemistry, UniversityofCaliforni-SantaCruz,Santa HiroshiKiwada(349),FacultyofPharma- Cruz,California95064 ceutical Sciences, The University of Tokushima, Shomachi 1-78-1, 770–8505 Jose´ L.Nieva(3,235),UnidaddeBiofisica Tokushima,Japan and Departamento de Bioqu´ımica, Uni- versidad Del Pa´ıs Vasco, Aptdo, 644, Kyung-Dall Lee (319), Department of 48080Bilbao,Spain Pharmaceutical Sciences, College of Pharmacy, University of Michigan, 428 ShlomoNir(235),SeagramCenterforSoil Church Street, Ann Arbor, Michigan and Water Sciences, Faculty of Agricul- 48109 tural, Food and Environmental Quality Sciences,Rehovot76100,Israel TatianaS.Levchenko(339),Department ofPharmaceuticalSciences,Northeastern Olivier Nosjean (216), Pharmacology University, 360 Huntington Avenue, Moleculaire et Cellulaire, Institut de Re- Boston,Massachusetts02115 cherchesServier,Crossy-sur-Seine,France contributors to volume372 xi ChristianOker-Blom(418),Universityof Brenton L. Scott (274), Department of Jvaskyla, Department of Biological and BiochemistryandCellBiology,RiceUni- Environmental Sciences, P.O. Box 35, versity,Houston,Texas77005 FIN40351Jyvaskyla,Finland Yechiel Shai (361), Department of Bio- Frank Opitz (48), University of Leipzig, logicalChemistry,WeigmannInstituteof Institute for Medical Physics and Bio- Science,Rehovot76100,Israel physics, Liebigstrasse 27, Leipzig D- 04103,Germany Jolanda M. Smit (374), Department of Medical Microbiology, Molecular Vir- Sergio Gerardo Peisajovich (361), De- ology Section, University of Groningen, partment of Biological Chemistry, Weig- Ant.Deusinglaan1,9713AVGroningen, mannInstituteofScience,Rehovot76100, TheNetherlands Israel James E. Smolen (300), Department of Jens Pittler (48), University of Leipzig, Pediatrics, Baylor College of Medicine, Institute for Medical Physics and Bio- 1100Bates,Room6014,Houston,Texas physics, Liebigstrasse 27, Leipzig D- 77030 04103,Germany Chester Provoda (319), Department of Toon Stegmann (408), Crucell Holland BV, Archimedesweg 4, P.O. Box 2048, Pharmaceutical Sciences, College of Leiden,TheNetherlands Pharmacy, University of Michigan, 428 Church Street, Ann Arbor, Michigan Reiko Tachibani (349), Faculty of 48109 Pharmaceutical Sciences, The University Ram Rammohan (339), Department of of Tokushima, Shomachi 1-78-1, 770- Pharmaceutical Sciences, Northeastern 8505Tokushima,Japan University, 360 Huntington Avenue, VladimirP.Torchilin(339),Department Boston,Massachusetts02115 ofPharmaceuticalSciences,Northeastern Jean-Louis Rigaud (65), Institut Curie, University, 360 Huntington Avenue, UMR-CNRS 168 and LRC-CEA 34V, Boston,Massachusetts02115 11 Rue Pierre et Marie Curie, 75231 ParisCedex05,France Chris Van der Does (86), University of Groningen, Department of Microbiology, Karine Robbe (151), CNRS-Institut de P. O. Box 14, Haren 9750AA, The PharmacologieMoleculaireetCellulaire, Netherlands 660 Route des Lucioles, 06560 Sophia Antipolis-Valbonne,France MartinVanderLaan(86),Universityof Groningen, Department of Microbiology, Ste´phane Roche (392), Laboratoire de P.O. Box 14, Haren 9750AA, The Genetiquie des Virus du CNRS, Gif sur Netherlands YvetteCedex91198,France Bernard Roux (216), Physico-Chemie JeffreyS.VanKomen(274),Department of Biochemistry and Cell Biology, Rice Biologique,UniversiteCBernard-Lyon1, University,Houston,Texas77005 Villeurbanne,France Susana A. Sanchez (19), Laboratory for AnaV.Villar(3),UnidaddeBiofisicaand Fluorescence Dynamics, University of Departamento de Bioqu´ımica, Universi- Illinois at Urbana-Champaign, Urbana, dad Del Pa´ıs Vasco, Aptdo. 644, 48080 Illinois61801 Bilbao,Spain xii contributors to volume 372 Natalia Volodina (339), Department of Markus R. Wenk (248), Department of Pharmaceutical Sciences, Northeastern Cell Biology, Howard Hughes Medical University, 360 Huntington Avenue, Institute,YaleUniversitySchoolofMedi- Boston,Massachusetts02115 cine, 295 Congress Avenue, New Haven, Connecticut06510 MattiVuento(418),UniversityofJvasky- la,DepartmentofBiologicalandEnviron- Judith M. White (428), Department of mentalSciences,P.O.Box35,FIN40351 Cell Biology, UVA Health System, Jyvaskyla,Finland School of Medicine, P.O. Box 800732, Barry-Lee Waarts (374), Department of Charlottesville,Virginia22908 Medical Microbiology, Molecular Vir- JanWilschut(374),DepartmentofMed- ology Section, University of Groningen, ical Microbiology, Molecular Virology Ant.Deusinglaan1,9713AVGroningen, Section, University of Groningen, Ant. TheNetherlands Deusinglaan 1, 9713 AV Groningen, The ThomasWeber(274),DepartmentofMo- Netherlands lecular, Cell, and Developmental Biology andCarlC.IcahnInstituteforGeneTher- Olaf Zscho¨rnig (48), University of Leip- apy and Molecular Medicine, Mount zig, Institute for Medical Physics and Sinai School of Medicine, New York, Biophysics, Liebigstrasse 27, Leipzig D- NewYork10029 04103,Germany [1] phospholipase–liposome interactions 3 [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 Theconventionalclassificationofmembraneproteinsasintrinsicorin- tegral,andasextrinsicorperipheral,hasonthewholebeensupersededby amorecomplexpatterninwhichacontinuumofpossibilitiesisconsidered, fromtheintegralproteinfirmlyembeddedinthebilayertothesolublepro- tein that contacts the membrane only transiently for a specific function. Phospholipasesstandinaclassoftheirownasmembraneproteinsbecause, 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 oftheirinteraction 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. Morerecently,we haveexploredtheeffectsofaphosphatidylino- sitol(PI)-specificphospholipaseC(PI-PLC).Theeffectsoftheseenzymes 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- sicularlipidmixing,andbymixingofvesicularaqueouscontents.Observa- tion of intervesicular contents mixing is always accompanied by detection of mixing of lipid inner monolayers, indicative of vesicle–vesicle fusion. Moreover,effluxofvesiclecontents,whetherornotaccompaniedbyother 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 1F.M.Gon˜iandA.Alonso,Biosci.Rep.20,443(2000). Copyright2003,ElsevierInc. Allrightsreserved. METHODSINENZYMOLOGY,VOL.372 0076-6879/03$35.00 4 liposomes in biochemistry [1] Materials Enzymes Phospholipase C (EC 3.1.4.3) from Bacillus cereus (MW, (cid:1)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 (cid:2)90% pure. The enzyme shows broad specificity (see below) and is active on glycero- phospholipidsinavarietyofaggregationalstates,forexample,monomeric in solution, dispersed in detergent-mixed micelles, and in model bilayers. Roche Molecular Biochemicals has discontinued the sale of this enzyme. Othersuppliersprovideequivalentenzymes,buttheyhavenotbeentested thoroughlyinour laboratory. Phosphatidylinositol-specific phospholipase C (EC 4.6.1.13) from B.cereusissuppliedbyMolecularProbes(Eugene,OR)andusedwithout furtherpurification.Sphingomyelinase(EC3.1.4.12)fromB.cereusispur- chased from Sigma (St. Louis, MO). As indicated by the manufacturer, preparationsofthisenzymeoftencontainsignificantphospholipaseCcon- 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 otherthansphingomyelin,forexample,ceramidephosphorylethanolamine, hasnotbeentested. Substrates Egg phosphatidylcholine (PC), egg phosphatidylethanolamine (PE), andwheatgermphosphatidylinositol(PI)aregradeIfromLipidProducts (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 thatislaterrevealedbycharringinanovenunderconditionsthatallowde- tection of1 (cid:1)goflipid.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 (cid:3)20(cid:4) and used within [1] phospholipase–liposome interactions 5 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 outwith phospholipid vesicles(liposomes). Forliposomeproduction,phospholipiddispersionsarepreparedbyre- hydrating lipid films dried from organic solvents. Solvents are evaporated thoroughlyundera current of N , and then leftfor atleast 2 h underhigh 2 vacuum to remove solvent traces. Small unilamellar vesicles are prepared by sonication3 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)with10-to12-(cid:1)mpulsesfor30 min,alternat- ing on and off periods every 10 s. Probe debris and MLV remains are pelleted by centrifugation at6000 g and 4(cid:4) for 10 min. Large unilamellar vesicles (LUVs) are prepared by the extrusion method.4Toobtainthesevesiclesaqueouslipidsuspensions(MLVs)areex- truded10timesthroughtwostackedNuclepore(Pleasanton,CA)polycar- bonatefilters(porediameter,0.1 (cid:1)m).TheextruderissuppliedbyNorthern Lipids(Vancouver, BC,Canada).Extrusiontakes place atroom tempera- ture,exceptforLUVsconsistingofpureSM,inwhichcasetheextruderis equilibrated at 42(cid:4) with a temperature regulation accessory. Average ves- iclediametersaremeasuredbyquasi-elasticlightscattering(QELS),using a Zetasizer instrument (Malvern Instruments, Malvern, Worcestershire, UK).LUVmeandiametersare(cid:1)100–115and(cid:1)160–190 nmforPC-based liposomesandSM-basedliposomes,respectively. Toascertainthat theextrusion proceduredoesnotalterthelipidcom- 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 organicphaseisconcentratedandseparatedonthin-layerchromatography (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 2I. Varela-Nieto, L. Alvarez, and J. M. Mato, ‘‘Handbook of Endocrine Research Techniques,’’p.391.AcademicPress,SanDiego,CA,1993. 3A.Alonso,R.Sa´ez,A.Villena,andF.M.Gon˜i,J.Membr.Biol.67,55(1982). 4L.D.Mayer,M.H.Hope,andP.R.Cullis,Biochim.Biophys.Acta858,161(1986).