Inourteachinglabweencouragestudentstoworkwitheachotherandtoshareinsight,experience,andevenexperimentalresults.Tofacilitatesuchcooperationwehavestudentsworkinteamsoftwo,withtwoteamsworkingtogetheronthedissectionandliverpreparationandinconductingtheexperiments.Eachpolarographicstationincludestwochambersandrecordingsystems.Weassignoneteamtoeachchamberandthetwoteamsstartbyconductingthesameexperiment.Ifoneexperimentfails,chancesaretheotherwillsucceedandbothteamscanmoveontothesecondexperiment.Ifbothexperimentssucceedtheteamsmayusethebetterofthetworecordsfordataanalysis.

Teamssharerespiratorymediumandreagentsandareexpectedtopayattentiontowhatisgoingoninbothchambers.Workingthiswayshouldincreasethechancesofworkingoutissueswiththeexperimentalmethods.Thephilosophyisthatfourheadsarebetterthantwo.Itisalsoalotmoreefficientforaninstructororteachingassistanttoadviseorassistateamoffourthanoneortwoindividualsatatime.Tobeginaseriesofexperimentswithisolatedmitochondria,theoxygraphsystemshouldbecalibratedwiththesamerespirationmediumthatwillbeusedthroughoutthestudy.Ourmediumofchoiceconsistsof70mMsucrose,220mMmannitol,2mMHEPESbuffer,5mMmagnesiumchloride,5mMpotassiumphosphate,1mMEDTA,and0.1%fattyacidfreebovineserumalbumin,pH7.4.

Unlessnoted,substratesandADPshouldbeaddedin20µlvolumesand10µlvolumesusedforpoisons.Theoptimumvolumeofmitochondriavariesfromonesubstratetothenextandwiththequalityofthepreparation.Tocompareratesamongexperimentsyouwillneedtonormalizeforthevolumeofmitochondriaadded.Thereforeyoumustbecarefultodrawup,deliver,andrecordtheintendedvolume.

Itiscriticalthatthesystemsbecalibratedandexperimentsconductedaccordingtotheprinciplesdescribedforcalibrationanduseofourpolarographicsystemfordissolvedoxygenmeasurement.Followingeachadditionofreagentorcombinationofreagents,recordforaminuteortwobeforeaddingthenextreagent,sothataslopecanbemeasuredfromthechartrecord.WhenyouproducestateIIIrespirationyoushouldobtaintwoslopes.

Experiments1-4havebeenthemostreliableofthestudiesoutlinedhere.Theyweredesignedtobeconductedinsequence.Knowledge/experiencegainedfromeachexperimentisapplicabletoconductingthenextexperiment.Resultsfromexperiments1-4willprovidethebasisfortheresearchpaper.Aftercompletingeachexperimentyouarewelcome(encouraged!)totryotherreagentsbeforecleaningoutthechamber.Ifyouplanaheadyoumaybeabletoaddressadditionalquestionsratherthansimplytryreagentsrandomly.

ThepHofallaqueousreagentswasadjustedto7.

1.Respiratorycontrolonsuccinateandinhibitionofelectrontransport

Aftercalibratingthesystemadd15-25µlmitochondriausingayellowtippipettor,glassstopperremoved,andtrituratetosUSPendmitochondriawithoutintroducingair.Replacethestopperandrecordforaminuteortwo.Starteachoxygraphrunthisway,varyingonlytheamountofmitochondriaadded.Now,therecannotberespirationwithoutasubstrate,soifthereisacontinuingdeclineinchamberoxygenthensomethingisprovidingfuel.IstherateofdeliveryofelectronsfromthatfuelefficientenoughtomaintainachemiosmoticgrADIent?Howwouldyouchecktoseeifagradientispresent?

ToproducestateIVrespiration,add20µl0.5MsuccinateusingaHamiltonsyringe,byinjectionthroughtheholeintheglassstopper.Foreachsuchinjectionmakesurethatthetipoftheneedleiswellintothechamber(usuallyasfarasitcango)andfirmlypushtheliquidintothechamber.Injectingslowlymayresultinpoormixingofreagentandyieldequivocalresults.Itshouldtakenomorethanaminuteortwotoobtainameasureableslope.

ProducestateIIIrespirationbyadding20µl0.01MADP.StateIIIrespirationonalimitingamountofADPistransient.Afterthefirstslopechangethereshouldbeasecondsteadystate.Thesecondslopechangemaynotbeasobviousasthefirst-viewdownthetraceoruseastraightedgetocheck.Ifyoudonotseeanincreaseinoxygenconsumptionwithin10-20secofaddingADPordonotseeadefinitereductioninoxygenconsumptionfollowingstateIIIrespiration,thensomethingisnotright.ForyourpaperyouwillneedtocalculateanADP:Oratioonsuccinate.

Add10µl30µMantimycin.Therewillbeaninitialriseinoxygencontentbecausethevehicle(ethanol)raisestheoxygencontentinthesolution.Waitforasteadystate.Add10µleach0.5Mascorbateand20µl30mMTMPDinimmediatesuccession(treatthemasasinglereagent).AscorbicacidmaintainsTMPD(anartificalelectrondonor)inareducedstate.ReducedTMPDdonateselectronstocytochromec.

Waitforasteadystate,recordlongenoughtogetameasurableslope,thenstopthechartrecord.Cleanoutthechamberandstopper,andprepareforasecondrun.

Steadystatesarereachedwithinmomentsofadditionofreagents.Whatconclusioncanyoudraw,then,aboutthetimeittakesformitochondriatoestablishachemiosmoticgradient?

2.Respiratorycontrolonglutamateandinhibitionofelectrontransport

Add30-40µlmitochondria,obtainasteadystate.Weusealargervolumethanforsuccinate-supportedrespirationbecauseoxygenconsumptionisslowerviatheNADHpathway(thinkaboutwhy).Add20µl0.5Mglutamate,obtainasteadystate.Oxygenconsumptionmaybehighenoughbeforeaddingsubstratethatyouwon"tseeameasureablechange.Addingglutamate,however,providesalimitlesssourceofsubstrate.Whateversupportsrespirationbeforeaddingglutamatedoesnotlikelyallowmaintenanceofagradient.

Add20µl0.01MADP,obtainasteadystate,waitforasecondsteadystateasyoudidforsuccinate,andrecordlongenoughtobeabletomeasuretheslope.YouwillneedanADP:Oratioonglutamateforyourpaper.Add10µl10µMrotenone–therewillbeanethanolartifact.Waitacoupleofminutesforasteadystate.

AddADPagain.Recordforaboutaminute.Youknowthatsteadystatesarereachedwithinmoments.Isthereanyreasontowaitlonger?Add20µl0.5Msuccinate–asteadystateshouldbeevidentwithinafewseconds,andwithinaminuteortwoyoushouldseeasecondslopechange.Allowenoughtimetoseeit,butdon"tletthechamberrunoutofoxygen.Whatmightbethereasonforthesecondsteadystate?Add10µl0.2MKCN.

3.Uncouplingagents

ProducestateIVrespirationonsuccinate,asyoudidforexperimentone.Add10µl1mMFCCP,apotentuncouplerofoxidativephosphorylation.Recordtheslope,forcomparisonwiththeslopethatyoupreviouslyrecordedforstateIIIrespirationonsuccinate.FortheresearchpaperyouwillneedtocomparestateIV,III,anduncoupledratesonsuccinate.Doyouexpectanychangeafterthemitochondriareachasteadystate?Isthereanyreasontolettherecordcontinueuntiltheoxygenisdepleted?

***AFTERUSINGFCCPORANYUNCOUPLINGAGENT,THOROUGHLYRINSETHESYRINGE,STOPPER,ANDCHAMBERSEVERALTIMES***Evenasmallconcentrationofuncouplingagentwillruinthenextexperiment.

4.Inhibitionofoxidativephosphorylation

Youmustthoroughlyrinseoutthechamber,glassstopper,andsyringesbeforebeginningthis(orany)experiment.Ifanyuncouplingagentremainstocontaminatethemedium,forexample,youwillnothaveasuccessfulexperiment.

ProducestateIVrespirationonsuccinate.Add10µl0.1mMoligomycinandrecordaslopeafterwitnessingtheartifact.Isthereanyindicationthatthisantibioticiseitheranuncouplingagentoranelectrontransportinhibitor?MinorchangesinslopecanbeconsideredtobenegligIBLe.Nowdesignanexperimenttodetermineifoligomycininhibitsoxidativephosphorylation(hint:itwouldinhibitATPsynthase,ifthatisthecase).

Assumingthatyoudesignandperformanappropriateexperiment,whatsortofcontrolsmightbeneeded(hint:whatagentshouldyouaddtodetermineifthemitochondriawereexercisingrespiratorycontrol?Thatis,howcanyouconfirmthattherewasanintactgradient,andtheETSisrestrictedbyit?

Ifyouarequick,or(better)ifyouthoughtaboutthisproblembeforeyoucametolab,youcancompletetheexperimentwithouthavingtostartover.

Thefollowingexperimentsneednotbeconductedinsequence.Theirsuccessratedependsonthequalityofthemitochondriapreparationandtosomeextenttheskilloftheinvestigators.

5.Electrontransportinhibitioncontinued

Addavolumeofmitochondriathatproducedgoodslopeswithglutamateassubstrate.Add5µl0.5Msuccinate,confirmtheslopeincrease.Withouttoomuchdelay,add10µl0.5Mmalonate,obtainaslope.Malonateisacompetitiveinhibitor.Whatwouldyouexpecttobetheeffectofsuccessiveadditionsofsmallamountsofmalonateontheslopeofoxygenconsumption.Repeatthemalonateadditiontoensurethatyouhaveobtainednearmaximuminhibiton.Add10µl0.01MADP.

AfterdeterminingwhetherornotADPspeduprespiration,add20µl0.1MNADHor20µl0.5Mglutamate(recallthatinanearlierexperimenttheadditionofglutamatedidnotspeedupstateIVrespirationagreatdeal).

AlittlereflectionwillrevealthatthispartofthestudyisthecounterparttothesecondexperimentinwhichNADH-supportedrespirationwasblockedandinhibitionovercomebyaddingsuccinate.Itoftengivesusverygoodresults,butjustasoftenthepreparationdoesnotrespondtoaddingNADHorglutamate.

Add20µl0.01MADP,recordslopechangesifevident.IfyouobtainresponsestoglutamateandADPadditions,thenadd10µl30µMantimycin.Addascorbate+TMPD.

6.ADP:Oratioonascorbate+TMPD

Thisexperimentwillworkbestwithverywellcoupledmitochondria(RCRforNADHsupportedrespirationof4orbetter).YoumayhavenoticedthattheRCRwasloweronsuccinatethanonglutamate.ThetrendcontinuesonthecombinationofascorbateandTMPD,infactthemitochondriabehaveasiftheyarenearlyuncoupled.

KnowingthatTMPDdonateselectronstocytochromec,whatistheexpectedADP:Oratioonascorbate+TMPD?Testthisexpectationbyputting15µlmitochondriaintostateIVrespirationonascorbate+TMPD,thenadding10µlADP.Ifpossible,calculateanADP:Oratio.Youmayhavetostretchyourimaginationinordertodetectaslopechange.Useastraightedgetolineuptheslopes.

7.Siteofactionofantimycin

ThiselegantlittleexperimentshouldnarrowthepossibilitiesforthelocationatwhichantimycinbindstheETS.Startwith15-25µlmitochondriaandaddFCCP.Theuncouplingagentshouldputthemitochondriainastateofrapidrespirationonceasubstrateisadded.Addsuccinate,confirmaslope,theninhibitrespirationwithmalonate.Addglutamate,confirmaslope,theninhibitrespirationwithantimycin.Addascorbate+TMPD.

Youalreadyknewthatantimycininhibitsrespirationonsuccinate.Withthisnewinformation,canyoudeterminewithsomeprecisionwhereantimycinbinds?

8.Mixedactionsof2,4-dinitrophenol(DNP)

ThemixedactionsofDNParesometimesevidentandsometimesnot.Italldependsonthequalityofthepreparation.

Repeatthethirdexperiment,butsubstitute10µl200mMDNPforFCCP.DNPhasmixedeffectsonmitochondria.Oneactionshouldbeapparentrightaway,providedthemitochondriaarestillexercisingasignificantdegreeofrespiratorycontrol.Thesecondwillbedelayedandgraduallytakeeffect.

Still"alive?"

Feelfreetotryanythingelse,especiallyifyouhaveunansweredquestions.Atypicalmitochondriapreparationshouldgivegoodresultsfor21/2to3hours.Whentheybegintodeterioratetheyuncouple.StateIVrateswillincreaseandthepreparationbecomeslessresponsivetoADP.

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