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1、植物体内的亚细胞组分分离步骤:准确称取植株鲜样0.5000g,加入20mL提取液(0.25mmol/L蔗糖+50mmol/LTrisHCl缓冲液(pH7.5)),研磨匀浆,用尼龙纱布过滤,滤渣为细胞壁部分;滤液在600r/min下离心10min,沉淀为细胞核部分;上清液在2000r/min下离心15min,沉淀为叶绿体部分;上清液在10000r/min下离心20min,沉淀为线粒体部分;上清液为含核糖体的可溶部分,每组两次离心,全部操作在4下进行。植物亚细胞组分的分离步骤:准确称取鲜样0.5000g,加入20mL提取液[0.25mol·L-1蔗糖+50mmol·L-1
2、Tris-HCl缓冲液(pH7.5)+1mmol·L-1二硫赤鲜糖醇,研磨匀浆,匀浆液在冷冻离心机300×g下离心30s,沉淀为细胞壁组分;上清液在2000×g下离心15min,沉淀为细胞核和叶绿体组分;上清液在10000×g下离心20min,沉淀为线粒体组分;上清液为含核糖体的可溶组分。全部操作在4℃下进行。FractionationofLeavesandRoots.Leavesandrootswerehomogenizedusingamortarandpestleinamediumcontaining0.25Msucrose,50mMTris-HCl(pH7.5)
3、,and1mmdithioerythritol.Allstepswereperformedat4C.Theresultingbreiwasstrainedthrougheightlayersofcheesecloth,andliquidwasexpressedfromtheresidue.Theresiduewaswashedtwicewiththegrindingmedium.Thepooledwashes,togetherwiththefirstfiltrate,werecentrifugedat300gfor30s.Theresultingpelletcombi
4、nedwiththeresidueofthecheeseclothfiltrationcontainedmainlycellwallsandcellwalldebrisandwasdesignatedascellwallfraction(I).Thesupernatantofthefirstcentrifugationstepwasthencentrifugedat20,000gfor45mintosedimentcellorganelles.Thepelletwastakenasorganellefraction(II).Theresultantsupernatan
5、tsolutionreferredtoassolublefraction(III)wasemployedinsubsequentcharacterizationstudiesasdescribed.FractionsIandIIwereanalyzedfortheirCdcontent.Thesupernatant,fractionIII,wasfurtherfractionatedbygelfiltration，usingSephadexG-100,G-25,andG-10(PharmaciaFineChemicals,Uppsala,Sweden).Analiqu
6、otoffractionIIIwasappliedtoacolumn(100x2.6cm)ofG-100using50mmTris-HCl(pH7.5)and0.1mMdithioerythritolaselutingbuffer.FractionscontainingCdwerepooledandchromatographedoncalibratedG-25(roots)orG-10(leaves)columns(70x2.3cm).FractionswerecollectedonanLKBUltraracfractioncollector.Theeffluents
7、weremonitoredat280nmwithaGilfordspectrophotometer2400S.Theactivitiesoftheglutamatedehydrogenaseandmalatedehydrogenaseweredeterminedaccordingtoreferences9and23.
