类囊体膜的提取

浓度名称分子量捣碎液:PH7.82500ml5000ml1000ml20mMTris-HCl[121.14](6.057g)（12.114）2.42280.4MSucrose[342.3](342.3g)(684.6)136.9215mMNaCl[58.44](2.192g)(4.383)0.87662mMEDTA二钠[372.2](1.861g)(3.722)0.7444胀破液：PH7.81000ml2000ml20mMTris-HCl[121.14](2.423g)(4.846)5mMMgCl2[203.3](1.017g)(2.033)15mMNaCl[58.44](0.8766g)(1.753)保存液：PH7.01000ml2000ml20mMTris-HCl[121.14](2.423g)(4.846)35mMNaCl[58.44](2.045)(4.09)0.4MSucrose[342.3](136.92g)(273.84)Activitiesofelectrontransport:ThylakoidmembraneswereisolatedfromtheleavesasdescribedbyBertholdetal.(1981).Wholechainelectrontransport(H2O→methylviologen,MV)andpartialreactionsofphotosyntheticelectrontransportmediatedbyPS2(H2O→2,6-dichloro-p-benzoquinone,DCBQ;H2O→silicomolybdate,SiMo)andPS1(DCPIPH2→MV)weremeasuredasdescribedbyNedunchezhianetal.(1997).Thylakoidsweresuspendedat10mg(Chl)m-3intheassaymediumcontaining20mMTris-HCl,pH7.5,10mMNaCl,5mMMgCl2,5mMNH4Cl,and100mMsucrosesupplementedwith500μMDCBQand200μMSiMo.类囊体的提取步骤：Highlyactiveintactthylakoidmembraneswerepreparedasin[6].Subchloroplastmembranescapableofoxygenevolutionwerepreparedbysuspensionoftheintactthylakoids(2mgchl/ml)inMgC12(5mM),NaCl(15mM)andHepesbuffer(20mM,pH7.5)andincubationwithTritonX-100(25mg/mgchl)at4°Cfor30min.Thefractionofchl-containingmaterialsedimentingat40000Xg(30min)wasresuspendedinincubatingbuffer(2mgchl/ml)withTritonX-100(5mg/mgchl),recentrifugedimmediately(40000Xg,30min)andstoredinsucrose(0.4M),MgClz(5mM),NaCl(15mM)andHepes(20mM,pH7.5)at-35°Cforsubsequentanalyses.Inthisprocedure,whichissuperficiallysimilartothatin[3],wehaveproducedasetofconditionswithregardtosaltconcentrationswherebyO2evolutionactivityisresistanttodenaturationbyexposuretoTritonX-100.Proceduresforassayofoxygenevolution,forTris-inhibition,andEPRdetectionofsignalsII,andIIfhavebeenreportedin[7,8].CytochromecontentwasassayedopticallybyusinganAmincoDW-2spectrophotometer.AssayofpigmentscontentLeaftissueswerehomogenizedinchilledN,N-dimethylformamideusingamortarandpestleindarkat4°C,andthehomogenateswerecentrifugedat8,800×gfor10minutes.Thesupernatantswerecollectedandtheabsorptionspectraat663and646nmwererecordedfortheestimationofchlorophylla(Chla)andchlorophyllb(Chlb)followingtheproceduredescribedbyInskeepandBloom(1985).Fortheestimationoftotalcarotenoids(Car),leaftissues(300mg)werehomogenizedinchilled80%(v/v)acetone,andthencentrifugedat8,800×gfor10minutesindarkat4°C.Theabsorbanceoftheacetoneextractswasmeasuredat663,646and470nm.TotalcarotenoidscontentwascalculatedasdescribedbyWellburn(1983).