条斑紫菜孢子体与配子体PSⅡ的特性以及单颗粒分析
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摘要
选择条斑紫菜(Porphyra yezoensis)两个不同发育阶段(孢子体和配子体)作为研究对象,分别从孢子体与配子体的类囊体膜上分离到具有高放氧活性的PSII复合物。配子体PSII复合物的放氧活性为2269.77±152.94μmolO2/chl(mg).h ,孢子体PSII复合物的放氧活性为2256.33±141.81μmolO2/chl(mg).h。对分离到的PSII复合物的稳定性和放氧活性进行了研究,结果表明:孢子体和配子体的PSII复合物,在4℃条件下保存比-80℃下放氧活性高,稳定性高。配子体跟孢子体比较,在-80℃保存条件下第六天就已经没有放氧活性,而此时-80℃下孢子体PSII复合物仍然具有放氧活性。同时对4℃下保存的PSII复合物进行分子筛柱层析,室温吸收光谱测定以及放氧活性测定,发现随着放氧活性逐渐降低的同时,蛋白大分子有聚合现象。室温吸收光谱表明经过长期的保存,吸收峰向短波长方向偏移,同时叶绿素易降解成为脱镁叶绿素。分别测定了配子体与孢子体PSII复合物的SDS-PAGE电泳蛋白条带的氨基酸序列,配子体PSII复合物经过SDS-PAGE电泳后的蛋白条带,经过质谱测定,鉴定出CP-47、D2、D1蛋白。孢子体PSII复合物,经过SDS-PAGE电泳后的蛋白条带,经过质谱分析鉴定出CP-47、CP-43、D2、D1蛋白。CP-47, CP-43蛋为PSII反应中心叶绿素P680的脱辅基蛋白,D1与D2蛋白为PSII的反应中心蛋白。对纯化的PSII复合物,并首次采用单颗粒技术分析比较其形态结构,电镜观察证实纯化的PSII复合物为二聚体,颗粒大小约为13nm×7.8nm,经过计算总分子量约为500kDa,但是孢子体与配子体的PSII复合物在形态上有轻微差别,配子PSII复合物要略宽于孢子体复合物,孢子体PSII复合物周边棱角形较配子体明显。
The life cycle of a red alga,Porphyra yezoensis consists of two phases:sporophyte and gametophyte. PSII complexes with high oxygen evolution activiteswere purified from both sporophyte and gametophyte. The oxygen evolution activitiesof PSII complexes of gametophyte is 2269.77±152.94 μmolO2/chl(mg).h and that ofsporophyte is 2256.33±141.81 μmolO2/chl(mg).h. The isolated PSII complexes werethen stored at 4oC and -80oC.Measurement of the oxygen evolution activity werecarried out every other day. The oxygen evolution activity of PSII at 4oC is higherthan that at -80oC. But the PSII complex of gametophyte at -80oC didn't have activityafter 6 days while the PSII complex of Sporophyte at -80oC was still active .The PSIIcomplexes stored under 4oC were analysed in three ways: gel filtration;oxygenevolution activity and absorption spectrum at room tempreture.From the gel filtrationprofile, we found the evolution peaks of the protein shift to left meaning the proteinaggregated during the store. From the absorption spectrum ,it was found that the peakof chlorophyll left shifted and chlorophyll became into phaeophytin.The oxygenevolution activity of PSII from both of gametophyte and sporophyte reduced duringthe store.The subunits of PSII isolated by the SDS-PAGEwere measured and theresults showed that PSII complexes purified from gametophyte contained PSII P680chlorophyll a apoprotein CP-47 and PSII reaction center protein D2 andD1,sporophyte PSII complexes contained PSII P680 chlorophyll a apoprotein CP-47,PSII P680 chlorophyll a apoprotein CP-43 and PSII reaction center protein D2 andD1.The single particle analysis showed dimmerized structure features and the size ofthe molecule was 13nm×7.8nm.The molecular mass of the oxygen evolving PSIIcomplex was estimated to be 500kDa that corresponds to the complex of PSII fromPorphyridium cruentum and the PSII complex from gametophyte was a little differentfrom that from sporophyte.
The life cycle of a red alga,Porphyra yezoensis consists of two phases:sporophyte and gametophyte. PSII complexes with high oxygen evolution activiteswere purified from both sporophyte and gametophyte. The oxygen evolution activitiesof PSII complexes of gametophyte is 2269.77±152.94 μmolO2/chl(mg).h and that ofsporophyte is 2256.33±141.81 μmolO2/chl(mg).h. The isolated PSII complexes werethen stored at 4oC and -80oC.Measurement of the oxygen evolution activity werecarried out every other day. The oxygen evolution activity of PSII at 4oC is higherthan that at -80oC. But the PSII complex of gametophyte at -80oC didn't have activityafter 6 days while the PSII complex of Sporophyte at -80oC was still active .The PSIIcomplexes stored under 4oC were analysed in three ways: gel filtration;oxygenevolution activity and absorption spectrum at room tempreture.From the gel filtrationprofile, we found the evolution peaks of the protein shift to left meaning the proteinaggregated during the store. From the absorption spectrum ,it was found that the peakof chlorophyll left shifted and chlorophyll became into phaeophytin.The oxygenevolution activity of PSII from both of gametophyte and sporophyte reduced duringthe store.The subunits of PSII isolated by the SDS-PAGEwere measured and theresults showed that PSII complexes purified from gametophyte contained PSII P680chlorophyll a apoprotein CP-47 and PSII reaction center protein D2 andD1,sporophyte PSII complexes contained PSII P680 chlorophyll a apoprotein CP-47,PSII P680 chlorophyll a apoprotein CP-43 and PSII reaction center protein D2 andD1.The single particle analysis showed dimmerized structure features and the size ofthe molecule was 13nm×7.8nm.The molecular mass of the oxygen evolving PSIIcomplex was estimated to be 500kDa that corresponds to the complex of PSII fromPorphyridium cruentum and the PSII complex from gametophyte was a little differentfrom that from sporophyte.
引文
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