细胞生物学教学课件：Chapter 7 Mitochondria and Peroxisome

CHAPTER7

MitochondriaandPeroxisome线粒体和过氧化物酶体线粒体和过氧化物酶体Theresearchof

mitochondrialfunction◆Earlyin1850,KÖlliker:

osmoticphenomenon.◆In1900，Michaelis,JanusgreenB(詹纳斯绿),livercells:oxygen:Indicatorblue◆In1913，Warburgseparatedmitochondriafromcellhomogenate(细胞匀浆)anddiscoveredthatitcouldconsumeo2.◆Earlyin1940s，Claudeinventedtheisolationtechnique,obtainedmitochondriausingsaltextractiontechniques.◆In1948,GeorgeHogeboom,WalterSchneiderandGeorgePalade

obtainedactivated

mitochondriausingsugarextractiontechniques.◆KenedyandLehninger:

tricarboxylicacidcycle（三羧酸循环），electron-transport，oxidativephosphorylation（氧化磷酸化），fattyacidoxidation(脂肪酸氧化).◆in1950s，G.Palade,K.Porter

andF.Sjostrand等--electronmicroscope:ultrastructure(超微结构)OUTLINE●

Mitochondria

structure●Mitochondriaproteinlocalization●Mitochondriafunction---oxidativephosphorylation●Mitochondria

proliferation

●Peroxisomes1.StructureofMitochondria(1)Shape:Granular(粒状),bacilliform(杆状)orelongatedcylinderswithadiameterof0.5～1.0µm.(2)Thenumberanddistribution◆Number:obviousdifferenceindifferenttypesofcells:1000--2000Miinlivercellsandno

Miinmammalmatureredbloodcells.◆

Distribution:theregionsrequiringenergy:

fatdropletandspermtail.鞭毛轴丝肌原纤维(3)UltrastructureofMitochondria（外室）（内室）Outermembrane◆Lipid/protein(6nmthick):1:1--richporins（孔蛋白）

forminglargewater-filledchannels:likeasievepermeabletoallhydrophilic

moleculesof

lessthan5kDa.◆TOMcomplexes（translocatorcomplexesofoutermembrane）andsomespecialenzymes.◆Markerenzyme----Monoamineoxidase(单胺氧化酶):

catalyzingoxidativedeaminization(氧化脱氨)ofmonoamine(单胺).Innermembrane（6-8nmthick）

◆Lipid/protein:---highproportionofcardiolipin(心磷脂):themembraneimpermeabletoions.◆usuallyhighlyconvoluted--formingaseriesofinfoldings(cristae-嵴)

projectingintothematrixandcoveredwithmanygranum(基粒：基本颗粒,elementaryparticle).双磷脂酰甘油◆manytypesofproteinsforenergyconversion:electron-transportchain,ATPsynthase.

◆Markerenzyme--cytochromeoxidase(细胞色素氧化酶)◆Manytypesoftransportproteins:metabolitesintoandoutofthematrix，TIMcomplexes（translocatorcomplexesofinnermembrane-内膜转运蛋白复合体）

Intermenbranespace（6-8nmwide）◆manysoluble

enzymes,substratesandcofactors----setupelectrochemicalgradient.

◆Markerenzyme----adenylatekinase

(腺苷酸激酶):FormationofADPAMP+ATP→2ADPMatrix

◆Highlyconcentrated

mixtureofhundredsofenzymes----theoxidationofpyruvate,fattyacidsandaminoacids.◆Markerenzyme--malatedehydrogenase

(苹果酸脱氢酶):苹果酸草酰乙酸.◆mitochondrialgenome,ribosomes,tRNAsandvariousenzymesforgeneexpression.◆densegranulardepositsofcalciumphosphate(磷酸钙)--Ca2+storage线粒体组分的分离毛地黄苷

线粒体质体(芦布若尔：膜蛋白的增溶剂)(4)ChemicalcomponentofMi◆

proteins(65%-70%)andLipids(25%-30%---Phospholipids(Principalingredient)).----Innermembrane:P/L(80/20),

Innermembraneproteins(21%Miproteins

)----Outermembrane:P/L(50/50),outermembraneproteins(6%Miproteins

)----Intermenbranespace:6%Miproteins

----Matrix:

67%Miproteins(enzymes,Coenzyme,vitamins,andmetalions，DNA，RNAandribosomes）2.MitochondriaproteintargetingCelladdresstarget(地址签)1)Translocation

—MiproteintargetLeadingpeptide（导肽或引导肽）:位于新生肽链N-末端的一段氨基酸序列，能引导肽链进入线粒体。(1)Characteristics(特征)●Sequencecharacteristics----attheN-terminus----about20-80aminoacidsequenceinlength----richalkalineaminoacids(Arginineandlysine)----fewacidicaminoacids----positivelychargedaminoacidsalternatewithhydrophobiconesinsignalsequences

●anamphiphilicahelix(hydrophobicandhydrophilic):specificreceptor

recognizesthisconfigurationratherthanthepreciseaminoacidsequence.

subunitⅣofcytochromeoxidase●Intheleadingpeptide,differentsequencescontaindifferentinformation.双导向序列●Theleadingpeptidehasnospecific

claimontheguidedproteins.◆实验设计●分离线粒体●与具有线粒体基质定位信号的前体蛋白温育●胰蛋白酶处理导肽如何引导蛋白质进入线粒体?●

Precursorproteinsunfolded●

Manytypesoftranslocatorsinvolvedintheprocess:(2)TranslocationofmitochondrialproteinsTranslocaseoftheouterandinnermembranesThematrixproteintranslocationTheinnermembraneproteintranslocationOXA负责将基质中线粒体DNA编码的内膜蛋白和被运进基质的内膜蛋白插到内膜上twoRoutes●Afamilyofmetabolite-specifictransportersforATP,ADP,andphosphate,whicharemultipasstransmembraneproteins.●TheTIM22complexmediatestheinsertionofthesetypesofproteins..

Theintermembranespaceproteintranslocation(a)conservativesortingtransientlybindspecializedchaperoneproteinsbothinsertsproteinsintotheoutermembraneandhelpsthemfoldproperly.

FirstintotheintermembranespaceacrosstheTOMcomplexTheoutermembraneproteintranslocation●

TheN-terminalaminoacidsoftheproteinsintheoutermembranecanrecognizereceptorintheoutermembrane.(3)Therequisite

factorfortranslocationofMiproteins●leadingpeptide（导肽）

●chaperoneproteins(分子伴侣)●receptor（受体）●thecontactpoint（接触点）betweentheoutermembraneandinnermembrane●energy(ATPandMembranePotential)●transitpeptidase(转运肽酶)●translocators（膜转运复合体）3.MiFunction●Oxidativephosphorylation(氧化磷酸化)—产生ATP●Regulationof

cytosolicCa2+concentration●Regulationofthecellapoptosis①Aerobicoxidationofglucose:◆Glycolysis(糖酵解:细胞质)◆pyruvate(丙酮酸)→acetylCoA(pyruvatedehydrogenase):mitochondrialmatrix)◆Tricarboxylicacidcycle(

(三羧酸循环--TCAcycle).乳酸（1)oxidation（生物氧化）：细胞质中的多糖、脂肪和蛋白质分解成简单的可溶性产物）--细胞氧化Oxidativephosphorylation视频Krebs:英国生化学家:32岁--鸟氨酸循环;37岁–

TCA:三种主要有机物代谢的联系枢纽,

1953获诺贝尔生理医学奖.TCAcycle:tricarboxylicacidcycle(三羧酸循环)Krebscycle柠檬酸循环柠檬酸有3个羧基◆Oxidationofreductivecoenzyme--oxidationofglucoseproducedNADHandFADH2.黄素腺嘌呤二核苷酸烟酰胺腺嘌呤二核苷酸NAD+→NADHFAD+→FADH2NADH+1/2O2→NAD++能量FADH2+1/2O2→FAD++能量

◆NADH和FADH2必须被氧化才能releaseenergy◆NADH和FADH2如何被氧化及被氧化时释放的H+、电子和能量如何安置?●

Eachcomplexhasanabsorptionspectrum，whichcanabsorbvisiblelightandchangecolorwhentheyareoxidizedorreduced.●Manycomplexeswereisolatedandidentifiedfrominnermembrane.●Eachofthesepurifiedcomplexescanbeinsertedintolipidbilayervesicles.离子交换色谱法◆Function:●H+andelectron

transfer

manycomplexes◆

Threelargeenzymecomplexesembeddedintheinnermembraneandonesmallcomplex.Thecomplexesareasymmetricallyorientedintheinnermembrane.

复合体ǁ的晶体结构被中国的饶子和院士团队,Ⅲ和Ⅳ分别为美国和日本解析，复合体I还不清楚Electron-transport

(电子传递）◆Electron-transportchain(电子传递链)--Respiratorychain(呼吸链)：Aseriesofproteincomplexesarrangedinorder(按顺序排列)intheMiinnermembrane,canreversiblyacceptorreleaseH+andelectrons.◆功能是参与对还原型辅酶的氧化(A)NADHdehydrogenasecomplex(NADH脱氢酶复合体):thelargestcomplex(morethan40polypeptidechains),aflavinprostheticgroup(辅基),atleastseveniron-sulfurcentersComplexI◆

ComplexI

catalyzesNADH

oxidationbyacceptingtwohigh-energyelectronsfromNADHandpassingthemtoubiquinone,transferringH+fromthematrixtotheintermembranespace.NADH-coenzymeQreductaseComplexI黄素单核苷酸(B)Succinatedehydrogenase(琥珀酸脱氢酶):

aFADprostheticgroup,twoiron-sulfurproteins.Complexǁ◆Complex

ǁ

transferstwolow-energyelectronstoCoQfromsuccinate(琥珀酸+Q→延胡索酸+QH2).Succinate-coenzymeQreductase(琥珀酸-coenzymeQ还原酶)

黄素腺嘌呤二核苷酸(C)Cytochromeb-c1complex(细胞色素b-c1复合体):

atleast11differentpolypeptidechains，adimer.Eachmonomer：onecytochromeb(twohemes),onecytochromec1(oneheme),aniron-sulfurprotein.ComplexⅢ◆

ComplexⅢ

acceptselectronsfromubiquinoneandpassesthemontocytochromec，andpumpsprotonfromthematrixtotheintermembranespace.CoQ-cytochromecreductase（D）cytochromecoxidasecomplex（细胞色素c氧化酶复合体):adimer,eachmonomer:l3differentpolypeptidechains,twocytochromes（cytaandcyta3）andtwocopper(Cu)atoms.

ComplexⅣ◆

It

acceptsoneelectronatatimefromcytochromecandpassesfourelectronsatatimetooxygen,Itisherethatnearlyalloftheoxygen(90%)webreatheisused.4cyt(还原型)+8HN++O2→4cyt(氧化型)+4Hp++2H2O

OrderofRespiratoryChainComplexes线粒体内膜主次呼吸链NADHrespiratorychainSuccinaterespiratorychain◆

NADHrespiratorychain--主呼吸链◆

Succinate(琥珀酸)respiratorychain--次呼吸链不经ⅠElectroncarriers（递电子体）◆Fourtypesofelectroncarriers：●

flavinprotein(黄素蛋白)●

cytochromes(细胞色素)●

iron-sulfurproteins(Fe-S蛋白)●

ubiquinone(泛醌)orcoenzymeQ(辅酶Q).--TheflavinproteinsandcoenzymeQalsotransferH+●Flavinprotein：

①NADH-coenzymeQreductasewithaprostheticgroupsofFMN;②succinate-coenzymeQreductasewithaprostheticgroupsofFAD

◆Acceptingtwoelectronsandtwoprotons.黄素单核苷酸黄素腺嘌呤二核苷酸H+H+●Cytochromes(细胞色素)

◆

Eachcytochromemoleculeconsistsofachromoprotein(色素蛋白)

andaprostheticgroupsofferriporphyrin(铁卟啉)thatisattachedcovalentlytotheprotein.◆

therearefivetypesofcytochromesofa,a3(a,a3除含铁外，还含铜),b,c,c1，whichtransferselectronsbytheconversionbetweenFe3+andFe2+bypassingoneelectronatatime.血红素铁Cytochromecisasmall,water-solubleproteinconsistingofa104-AApolypeptiedchain

withasinglehemegroup.◆Themoleculecontainssulfursandprostheticgroupsofnon-hemeirons(非血红素铁)

linkedwithcysteinesidechains,.

forminganiron-sulfurcenterwithintheprotein.◆Likethecytochromes,thesecenterscarryoneelectronatatimebytheconversionbetweenFe3+andFe2+.Fe3++e-Fe2+●iron-sulfurproteins(Fe-S):Twocommonforms:Fe2S2andFe4S4●CoenzymeQ–Ubiquinone(泛醌)

：

Aquinone(Q)isasmallhydrophobic(脂溶性)moleculethatcanpickupordonateeitheroneortwoelectrons.◆ATPsynthesis

iscoupledtoelectrontransport(2)ATPsynthesis线粒体内膜重建超声波破碎亚线粒体颗粒（sub-mitochondrialparticle）thesiteofATPsynthesis.thesiteofelectrontransport●Protongradient（ΔpH）●Potentialdifference(内膜两侧电位差--膜电位)①Institutionof

transmembrane

electrochemicalgradient(质子动力)主要作用TransmembraneelectrochemicalgradientdrivesATPsynthesisinthepresenceofADP,Pi

and

F0-F1-ATPase(F0-F1couplingfactor—F0-F1偶联因子).②ATPsynthase◆ATPsynthaseorF0-F1-ATPase(H+-ATPase):widelydistributedinthemembraneofmitochondria,chloroplast,heterotrophicbacterium(异养菌)andphotosyntheticbacterium,involvedinATPsynthesis.mushroom-likeATPsynthaseembeddedincristae(嵴)consistofaheadportion(F1)andatransmembraneH+carrier(F0)F1

catalyticsubunit:fivekindsofpolypeptidesα3β3γδε.EachβsubunithasacatalyticsiteofATPsynthesis.F0complex:threetypesofsubunits(1a,2b,10-14c)),mediatesprotontransport.Arotatingstalk(γandε)isfixedtoarotor.◆F1andF0arelinkedbytherotorandthestatormembraneportionheadarmrotorofcringstatorofδ,a,bStructureofATPsynthase寡霉素（oligomycin）可与F0结合，阻塞质子通道，使ATPsynthase

失去活性。1960s，美国学者PaulBoyer:binding-changemodel（结合变构模型：ATP合成酶合成ATP的机制）1、F1上有3个活性部位轮替催化合成ATP，ß亚基开始处于ß-ADP构象，可与介质中的ADP和磷酸结合2、ß亚基转变为ß-ATP构象，与ATP紧密结合。3、ß亚基最后转变为ß-排空构象。ATP离开酶表面。4、γ亚基中心轴每旋转120度，即与不同的ß亚基接触，迫使其变为ß-排空构象。ProtonflowCunitrotatesβrotatesconformationchangeATPsynthesized◆In1994,WalkerJEpublishedthecrystalstructureofbovine(牛心)mitochondrialF1-ATPaseandobtainedNobelPrizesinChemistrywithBoyerin1997.FunctionofF0F1particles◆F0F1particleisakindofreversiblemultiplexenzyme●ATPase:ATPhydrolysis●ATPsynthase(ATP合酶):ATP

synthesis

--Chemiosmoticcouplinghypothesis（化学渗透学说，1961,PeterMitchell,abritainbiochemist):

electrontransport--pumpprotonsacrossthemembrane--electrochemicalprotongradient--protonsflowsbackthroughF0F1particles--

ATP

synthesis.◆

ATPsynthesis◆Thehypothesismayexplaintherelationshipbetweenelectrontransport,generationofprotonelectrochemicalgradientandADPphosphorlation.

Therefore,heobtainedNobelPrizesinChemistryin1978.

◆Oxidativephosphorylation(氧化磷酸化):AprocessofthesynthesisofATPcoupledwithrespiratorychainoxidationinMi.底物水平的磷酸化(A)Inhibiterofelectrontransport(B)Inhibiterofprotontransport(C)Uncouplingagent(解偶联剂):acompoundthatdisruptstheusualtightcouplingbetweenelectrontransportandADPphosphorylation.

UncouplersblockoxidativephosphorylationbydissipatingtheH+electrochemicalgradientInhibiterofoxidativephosphorylation（寡霉素）鱼藤酮粉蝶毒素阿米妥汞度冷丁噻吩甲酰三氟丙酮奎諾酮（未加工的蜂蜡）抗霉素A氰化物叠氮化物二环己基碳二亚胺2，4-二硝基苯酚（质子载体）血液凝固防止剂三氟甲氧基苯腙羰基氰化物萎锈灵：杂环类杀菌剂4.ProliferationofMitochondria◆Thenewmitochondria

derivesfrom

thepreviousmitochondriadivision.4)MitochondrialDiseases◆Mitochondria

abnormityleadstodiseases.◆Keshandisease

(克山病:1935在黑龙江克山县发现)：lackofSeleadstofewer

cristaorswellingofcardiacmuscle(心肌)mitochondria

duetoSe

stabilizingmitochondrialmembrane.◆mtDNAabnormity

（mutation,deletion,rearrangement）leadstotheabnormityofenzymesystemsofelectrontransportandoxidativephosphorylation.2.Peroxisome（过氧化物酶体）◆peroxisomes(ormicrobody-微体)arewidelydistributedoverallanimalcellsandmanyplantcells.◆Adiameterof0.1to1.0μmvesiclesthatoftencontainadense,crystallinecoreofoxidativeenzymes--urateoxidase（nourateoxidasesfromhuman,birdandtetrahymena(四膜虫)）尿酸氧化酶1)Structureofperoxisomes

尿酸氧化酶：痛风？◆Peroxisomesaresmallvesicularcompartmentspackedby

singlelayermembraneand

containmanytypesofoxidativeenzymes.Peroxisomes

usuallyhavetwotypesofenzymes:

●Fla