【线粒体】Mitochondria2nded

MethodsinCellBiologyVOLUME80Mitochondria,2ndEditionSeriesEditorsLeslieWilsonDepartmentofMolecular,CellularandDevelopmentalBiologyUniversityofCaliforniaSantaBarbara,CaliforniaPaulMatsudairaWhiteheadInstituteforBiomedicalResearchDepartmentofBiologyDivisionofBiologicalEngineeringMassachusettsInstituteofTechnologyCambridge,MassachusettsMethodsinCellBiologyVOLUME80Mitochondria,2ndEditionEditedbyLizaA.PonDepartmentofAnatomyandCellBiologyCollegeofPhysiciansandSurgeonsColumbiaUniversityNewYork,NewYorkEricA.SchonDepartmentofNeurologyandDepartmentofGeneticsandDevelopmentColumbiaUniversityNewYork,NewYorkAMSTERDAM•BOSTONHEIDELBERGLONDONNEWYORKOXFORDPARISSANDIEGOSANFRANCISCOSINGAPORESYDNEYTOKYOAcademicPressisanimprintofElsevierCoverPhotoCredit:LaserscanningconfocalimageofhumanfibroblastsstainedformitochondriausingMitoTracker(red)andmicrotubules(green)usingamonoclonalanti-tubulinantibodyandindirectimmunofluorescence.ImageprovidedbyIstvanBoldogh(ColumbiaUniversity)andMichaelYaffe(UniversityofCalifornia,SanDiego).AcademicPressisanimprintofElsevier525BStreet,Suite1900,SanDiego,California92101-4495,USA84Theobald’sRoad,LondonWC1X8RR,UKThisbookisprintedonacid-freepaper.Copyright
2007,ElsevierInc.AllRightsReserved.Nopartofthispublicationmaybereproducedortransmittedinanyformorbyanymeans,electronicormechanical,includingphotocopy,recording,oranyinformationstorageandretrievalsystem,withoutpermissioninwritingfromthePublisher.TheappearanceofthecodeatthebottomofthefirstpageofachapterinthisbookindicatesthePublisher’sconsentthatcopiesofthechaptermaybemadeforpersonalorinternaluseofspecificclients.Thisconsentisgivenonthecondition,however,thatthecopierpaythestatedpercopyfeethroughtheCopyrightClearanceCenter,Inc.(),forcopyingbeyondthatpermittedbySections107or108oftheU.S.CopyrightLaw.Thisconsentdoesnotextendtootherkindsofcopying,suchascopyingforgeneraldistribution,foradvertisingorpromotionalpurposes,forcreatingnewcollectiveworks,orforresale.Copyfeesforpre-2007chaptersareasshownonthetitlepages.Ifnofeecodeappearsonthetitlepage,thecopyfeeisthesameasforcurrentchapters.0091-679X/2007$35.00PermissionsmaybesoughtdirectlyfromElsevier’sScience&TechnologyRightsDepartmentinOxford,UK:phone:(þ44)1865843830,fax:(þ44)1865853333,E-mail:[e-mail]queston-lineviatheElsevierhomepage(),byselecting‘‘Support&Contact’’then‘‘CopyrightandPermission’’andthen‘‘ObtainingPermissions.’’ForinformationonallAcademicPresspublicationsvisitourWebsiteat:0-12-544173-8PRINTEDINTHEUNITEDSTATESOFAMERICA07080910987654321CONTENTSContributorsxviiPrefacexxiiiPARTIIsolationandSubfractionationofMitochondria1.IsolationandSubfractionationofMitochondriafromAnimalCellsandTissueCultureLinesFrancescoPallottiandGiorgioLenazI.Introduction4II.GeneralFeaturesofMitochondrialPreparations5III.MitochondriafromBeefHeart8IV.MitochondriafromRatLiver17V.MitochondriafromSkeletalMuscle23VI.SynapticandNonsynapticMitochondriafromDiVerentRatBrainRegions26VII.MitochondriafromKidneyandTestis29VIII.MitochondriafromHamsterBrownAdiposeTissue29IX.MitochondriafromInsectFlightMuscle30X.MitochondriafromPorcineAdrenalCortex30XI.MitochondriafromHumanPlatelets31XII.MitochondriafromFishLiver32XIII.MitochondriafromSeaUrchinEggs33XIV.MitochondriaandKinetoplastsfromProtozoa33XV.MitochondriafromFishErythrocytes35XVI.MitochondriafromCaenorhabditiselegans35XVII.MitochondriafromDrosophila35XVIII.MitochondriaandMitoplastsfromCulturedCells36References392.PurificationandSubfractionationofMitochondriafromtheYeastSaccharomycescerevisiaeIstvanR.BoldoghandLizaA.PonI.Introduction45II.IsolationofMitochondria46III.AnalysisofIsolatedMitochondria53vIV.IsolationofMitochondrialOuterMembranes,ContactSites,andInnerMembranes61References633.IsolationandSubfractionationofMitochondriafromPlantsA.H.Millar,A.Liddell,andC.J.LeaverI.Introduction66II.GrowthandPreparationofPlantMaterial66III.IsolationofMitochondria67IV.DensityGradientPurificationofMitochondria69V.MitochondrialYield,Purity,Integrity,Storage,andFunction73VI.SubfractionationofMitochondrialCompartments77VII.InOrganelloTranslationAnalysis83VIII.ProteomeAnalysis85IX.Conclusion87References87PARTIIBiochemicalAssaysofMitochondrialActivity4.BiochemicalAssaysofRespiratoryChainComplexActivityDeniseM.Kirby,DavidR.Thorburn,DouglassM.Turnbull,andRobertW.TaylorI.Introduction93II.MaterialsandMethods96III.InterpretingResultsofRCEnzymology114References1165.PolarographicAssaysofRespiratoryChainComplexActivityGaetanoVillaniandGiuseppeAttardiI.Introduction121II.MeasurementsofEndogenousRespirationinIntactCells122III.KCNTitrationofCOXActivityinIntactCells124IV.InSituAnalysisofMitochondrialOXPHOS128References1326.OpticalImagingTechniques(Histochemical,Immunohistochemical,andInSituHybridizationStainingMethods)toVisualizeMitochondriaKurenaiTanjiandEduardoBonillaI.Introduction135II.Histochemistry137viContentsIII.Immunohistochemistry142IV.InSituHybridizationtomtDNA148References1517.