STUDY ON THE FUNCTIONAL SITES OF Mg~(2+) -INDUCED Chl α FLUORESCENCE AND SURFACE CHARGE CHANGES OF THYLAKOID MEMBRANES

Thylakoid polypeptide components, Mg~(2+) -induced Chl a fluorescence at 77K and surface chargechanges were measured to investigate the functional sites of Mg~(2+) in the regulation of energy distri-bution between two photosystems in completely and incompletely developed barley chloroplasts. It wasfound that in contrast to the completely developed chloroplasts, the incompletely developed chloroplastslacked Chl b and did not contain the 23KDa and 25KDa polypeptide components of LHC-PSII. In themeantime, these membranes did nor present Mg~(2+) -induced Cbl a fluorescence and surface charge changesof thylakoids. These results provided strong evidence that the 23KDa and 25KDa polypeptides ofLHC-PSII are the specific acting sites of the cation that induced these two phenomena. It is suggested in this paper that Mg~(2+) -induced change of excitation energy distribution betweentwo photosystems is produced by the mechanism due to electrostatic neutralization of LHC-PSII bythe cation to cause a structural or con     

假根羽藻LHCⅡ的同质和异质三聚体的能量传递动力学研究

采用飞秒时间分辨荧光光谱技术在室温下用波长为495nm的光激发,研究了假根羽藻LHCⅡ的同质三聚体和异质三聚体这两种不同聚集形式蛋白复合物的光谱特性和传能特性.将实验获得的两样品的荧光光谱分别进行高斯解析,各得到5条亚谱带,并分析得出同质三聚体的四种特征Chl分子的光谱特性和异质三聚体的五种特征Chl分子光谱特性,另外还对两蛋白复合物的荧光光谱作了比较.利用Global优化处理方法,建立多指数拟合联立方程对荧光衰减曲线进行拟合处理,得到同质三聚体Chl分子传能的寿命：165±8.5 fs、2.2±0.6 ps、5.1±0.1 ns;异质三聚体Chl分子传能的寿命：255±6.3 fs、4±0.8 ps和3.8±0.1 ns;并且将这些时间常数作出归属.将其分析比较,快组分同质三聚体的传能速率较高,推断组成同质三聚体的同种脱辅基蛋白间的结合程度较组成异质三聚体的不同脱辅基蛋白间的更紧密,使其上结合的Chl分子空间位置更近,更有利于能量的迅速传递;而慢组分同质三聚体的偏大,可能是由于其经历的传能途径较长,传能机制上也和异质三聚体不同所致.     

光系统ⅡChl分子能量传递超快光谱动力学

利用ICCD飞秒扫描成象和飞秒时间分辨光谱装置实验研究了高等植物捕光天线LHCⅡ三聚体和PSⅡ颗粒复合物的超快光谱动力学,经过吸收光谱和发射光谱分析,确定在LHCⅡ三聚体中至少存在7种Chl分子光谱特性,分别是Chlb_653/656^658.7、Chla_662.0^665.2、Chla/b_670/671^677.1、Chla_675.0^677.1、Chla_680/681^682.9、Chla_685.0^689.1和Chla_695.0^695.6.采用光强1013光子/cm²/脉冲激励浓度为30μg/mL的捕光天线LHCⅡ三聚体,在650nm到705nm谱段逐点探测分析处理,产生了2组短寿命组分210fs、520fs和5.2ps、36.7ps及2个长寿命组分1.8ns、2ns.最快的3个寿命210fs、520fs和5.2ps反映了三聚体Chlb分子向Chla分子的激发能传递过程;寿命36.7ps反映了Chla分子向相邻单体Chla分子的激发能传递过程;最长的2个寿命1.8ns和2ns是在三聚体中Chla分子通过中间体Chla分子辐射荧光,分别跃迁回基态的过程.获得的6个寿命组分有把激发能传递时间与Chla/b分子发射光谱相结合的特点.经拟合处理解析PSⅡ颗粒复合物光谱,得到3个组分谱,其峰值分别为686.8nm、692.2nm和694.9nm,与LHCⅡ比较分析,说明天然构型的PSⅡ有很强的吸收光能和有效传递光能的本领.     

关于光合膜上蛋白质的功能分析

本文用胰蛋白酶作探针研究了菠菜叶绿体膜结构与功能之间的关系.在胰蛋白酶消化过的叶绿体中,观察到低浓度的CCCP(10μM)对放O₂和电子传递速度有强烈的抑制作用.胰蛋白酶诱导的这类CCCP的抑制作用,可被牛血清蛋白所“阻滞”.牛血清蛋白可使胰蛋白酶消化引起的叶绿体光还原DCIP的抑制得到恢复,但对酶消化产生的放O₂抑制无恢复效果. 根据上述结果推论:在PS-Ⅱ膜上可能存在有类似“通道”和“门”的结构;囊状体膜上的蛋白质组分有微区域的差异.     

刺松藻类囊体膜23kD和24kD多肽在阳离子调节激发能分配中的作用

与高等植物不同,Mg~(2+)诱导刺松藻类囊体膜Chla荧光F_(687)增高与其诱导膜表面电荷密度减小无平行相关性,用Ca~(2+)提去膜表面的30和31kD多肽(Q_B蛋白),对Mg~(2+)诱导上述两种效应无明显的影响,但用胰酶消化进一步除去膜表面的23和24kD多肽后,Mg~(2+)诱导F_(687)增高的效应随之消失,而诱导膜表面电荷密度变化的性质不变,这些结果证明,刺松藻类囊体膜表面的23和24kD多肽是Mg~(2+)诱导荧光变化的功能部位;Mg~(2+)诱导激发能分配的改变不受膜表面静电性质控制。     

EFFECTS OF TRYPSIN ON PHOTOSYSTEM Ⅱ,ELECTRON TRANSPORT AND ENERGY DISTRIBUTION

The effects of trypsin on the light-induced variable fluorescence of both the normaland tris-washed spinach chloroplasts have been investigated and it is discovered that: (i)Trypsin clearly inhibits in both cases the light-induced variable fluorescence emissions;but when the exogenous electron donor SMC B is added, this inhibitroy effects of trypsinare greatly alleviated and the recovery efficiency is similar in both cases. (ii) In thepresence of DCMU, trypsin still inhibits the variable fluorescence intensity but theexogenous electron acceptor such as DCIP or Fecy has no influence on it. Based on the above results, the following conclusion can be drawn: trypsin can evident-ly inactivate the electron transport on the oxidizing side of PS-Ⅱ, which exists between thewater splitting enzyme system and SMCB binding site. However, the water splitting enzymeis not sensitive to trypsin. It might be assumed that there is some protein on the thylakoidmembrane that acts as regulator to the functional mechanism of     

FUNCTIONAL ANALYSIS OF PROTEINS ON THE THYLAKOID MEMBRANE——EFFECTS OF Mg~(2+) AND BSA ON THE TRYPSIN-INDUCED CHLOROPHYLL a FLUORESCENCE AND THYLAKOID MEMBRANE SURFACE CHARGE CHANGES IN SPINACH CHLOROPLASTS

Trypsin digestion of spinach chloroplasts not only inhibits light-induced variable fluorescence yield (△F/F_0) but also abolishes Mg~(2+) stimulating effect on △F/F_0. If chloroplasts are incubated by Mg~(2+) prior to trypsin digestion, Mg~(2+) stimulating ability remains unchanged whereas the enzyme-induced △F/F_0 decline itself is not affected. The protective effect of pretreatment of chloroplasts with Mg~(2+) against trypsin closely correlated with alteration of the net negative charge on the outer surface of the thylakoid membrane by Mg~(2+). In contrast to this, addition of BSA to the chloroplast digested with trypsin significantly recovers the light-induced variable fluorescence yield but it does not influence Mg~(2+)-induced variable fluorescence change. The recovery effect of BSA incubation is not dependent upon Mg~(2+) concentration. Also, BSA does not alter the net negative charge on the outer surface of the thylakoid membrane.These results demonstrate the existence of two independent prote     

关于光合膜上蛋白质的功能分析——Mg~(2 )和BSA对胰酶诱导菠菜叶绿体Chla荧光及膜表面电荷变化的影响

本文证明了胰酶消化菠菜叶绿体不仅抑制Chl的可变荧光产率(△F/F_o),而且使Mg~(2 )对△F/F_o的刺激效应消失。在酶消化前加Mg~(2 )与叶绿体进行预保温,对Mg~(2 )刺激△F/F_o的效应有保护作用,但对酶诱导△F/F_o下降本身无影响。这种保护作用与Mg~(2 )诱导类囊体膜表面电荷的改变密切相关。相反,在酶消化过的叶绿体中加入BS保温,对酶诱导的△F/F_o下降有明显的恢复作用,但对酶诱导Mg~(2 )刺激△F/F_o能力的消失无影响。这种恢复作用不因Mg~(2 )冲的存在而改变;BSA对膜表面电荷亦无任何影响。这些结果证明,光合膜表面可能存在有两类蛋白质或多肽,它们通过不同的机制调节激发能在两个光系统之间的分配与传递。     

胰蛋白酶对光系统Ⅱ电子传递和能量分配的影响

本文测定了胰蛋白酶对正常的和经Tris洗过的菠菜叶绿体光诱导可变荧光的影响,发现:(1)酶消化对这两种叶绿体的可变荧光产率有明显的抑制作用;加入PS—Ⅱ电子供体SMCB可使抑制作用显著减轻,其荧光的恢复效率在两种叶绿体中接近相等.(2)在有DCMU存在下,酶诱导的上述抑制作用仍很显著,且不受外加受体的影响. 由上述结果得出结论:胰蛋白酶对PS—Ⅱ氧化侧有明显的钝化作用,钝化的部位在水裂解酶系统与SMCB插入的位置之间,而水裂解酶系统本身对酶消化不敏感;光合膜上可能有某种控制激发能转移和分配的蛋白质存在.     

关于Mg~(2+)诱导Chla荧光及类囊体膜表面电荷变化的功能部位的探讨

本文用发育完全的和发育不完全的大麦叶绿体膜观察了它们的类囊体膜多肽成分、Mg~(2+)诱导Chla低温荧光及膜表面电荷的变化。发现发育不完全的叶绿体膜与发育完全的叶绿体膜不同,它们的类囊体膜缺少Chlb,不含LHC-PSII的23KDa和25KDa多肽成分,亦无Mg~(2+)诱导Chla荧光及膜表面电荷变化的效应。这些试验结果证明,LHC-PSII的23KDa和25KDa多肽系Mg~(2+)诱导这两种相关效应的功能部位。文中讨论了Mg~(2+)对LHC-PSII的静电中和作用而引起膜结构或构型的改变,可能是阳离子诱导激发能在两个光系统之间分配改变的重要原因。