蓝藻NAD（P）H脱氢酶复合体的结构与生理功能研究

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英文题名：Investigation on the Structure and Physiological Function of Cyanobacterial NAD(P)H Dehydrogenase Complex 作者：邓勇 论文级别：博士 学科专业名称：植物学 中文关键词：NAD(P)H ; 脱氢酶 ; 集胞蓝藻 ; PCC6803 ; PSI ; 循环电子传递 ; CO2浓缩机制 ; 疏水亚基 ; 藻胆蛋白 英文关键词：NAD(P)H dehydrogenase ; Synechocystis PCC6803 ; PSI-cyclic electro flow ; CO2 concentrating mechanism ; hydrophobic subunit ; phycobiliproteins 学位年度：2003 导师：米华玲 ; 沈允钢 学科代码：071001 学位授予单位：中国科学院研究生院（上海生命科学研究院） 论文提交日期：2003-06-01

摘要

一般认为，蓝藻和高等植物类囊体膜上都存在 4 种膜蛋白复合体，即 PSII
    复合体，细胞色素 b6/f 复合体，PSI 复合体和 ATP 合酶复合体。近年来，在蓝
    藻、高等植物等的类囊体膜上又发现了另一类膜蛋白复合体，称为 NAD(P)H 脱
    氢酶复合体（NDH），它是一类与线粒体复合体 I 高度同源的多亚基复合体。此
    复合体在蓝藻中参与呼吸和光合循环电子传递，在高等植物中参与光合循环电
    子传递和叶绿体呼吸等过程。越来越多的证据表明，NDH 对蓝藻的生理活动甚
    至生存起着重要的作用，因而对蓝藻 NDH 的结构与生理功能的研究正日益受
    到重视。然而，目前蓝藻 NDH 复合体的结构与功能研究仍处于起步阶段。本
    文的研究工作主要集中于 NDH 在蓝藻对低 CO2浓度的响应和适应中的作用、
    NDH 复合体的分离纯化以及其与藻胆蛋白的相互作用等方面。
    1．低 CO2 浓度对集胞蓝藻 NDH 复合体的影响
     比较了高 CO2（H-cells）和低 CO2（L-cells）下培养的集胞蓝藻 PCC6803
    中 NAD(P)H 脱氢酶复合体（NDH）的活性以及亚基表达。Western 印迹分析表
    明，L-cells 所含的 NDH 亚基，NdhH、NdhI 和 NdhK 的量明显比 H-cells 高。
    非变性凝胶电泳以及活性染色分析显示，L-cells 中，NADPH 专一的 NDH 亚复
    合体具有更高的 NADPH-氮蓝四唑（NBT）氧化还原酶活性；此外，L-cells 中，
    NADPH-menadione 氧化还原酶和光系统 I（PSI）驱动的 NADPH 氧化活性也明
    显比 H-cells 高，这都表明 NDH 的活性在低 CO2下增强了。在 DCMU 和背景
    远红光存在下，测得的作用光关闭后 P700+的暗中还原初始速率在 L-cells 中明
    显提高，表明 L-cells 中围绕 PSI 的循环电子传递增强。NDH 的专一抑制剂，
    鱼藤酮（rotenone）对 L-cells 的作用光关闭后叶绿素荧光瞬时上升的抑制程度
    比 H-cells 要大得多，这说明在低 CO2浓度下 NDH 介导的循环电子传递明显加
    强。以上结果表明，NDH 及其介导的循环电子传递均为低 CO2促进，提示 NDH
     IV
    
    
    参与蓝藻对低 CO2的适应过程。
    2．集胞蓝藻中 NDH 复合体对 CO2 浓度变化的响应
     通过非变性凝胶电泳分离和活性染色，从集胞蓝藻 PCC6803 的细胞粗提
    物中检测到一个 NADPH 专一的 NDH 亚复合体。当高 CO2浓度培养的蓝藻细
    胞转为低 CO2浓度培养时，此 NDH 亚复合体的活性迅速提高，同时伴随 NdhK
    亚基蛋白质表达水平和 PSI 驱动的 NADPH 氧化活性明显增加。而当低 CO2浓
    度培养的蓝藻转为高 CO2浓度培养时，一开始，此 NDH 亚复合体和 PSI 驱动
    的 NADPH 氧化活性以及 NdhK 的蛋白质表达水平均有轻微的提高，但随着培
    养时间的增加，都有不同程度的明显下降。这些结果表明体外的 CO2浓度作为
    信号可调控 NDH 复合体的活性，同时 NDH 复合体反过来也可参与 CO2吸收的
    调节。 驱动的 NADPH 氧化活性可以反映 NDH 介导的循环电子传递的活性，
     PSI
    因此我们的结果也表明 NDH 介导的 PSI 循环电子传递活性受低 CO2诱导而为
    高 CO2抑制。综合以上结果，说明 NDH 及其介导的循环电子传递可能参与蓝
    藻对 CO2浓度变化的响应。
    3．集胞蓝藻 PCC6803 含疏水亚基的 NAD(P)H 脱氢酶亚复合体的分离
     蓝藻 NDH 复合体的分离纯化比较困难。迄今，人们只分离到几个 NDH 亲
    水亚复合体，含疏水亚基的蓝藻 NDH 亚复合体的分离还未见报道。我们利用
    离子交换与凝胶过滤层析，从 n-dodecyl β-D-maltoside (DM)处理的集胞蓝藻
    Synechocystis PCC6803 细胞粗提液中，首次分离到两个包含 NDH 疏水亚基
    NdhA 的亚复合体。酶活分析表明，分离到的 NDH 亚复合体具有 NADPH-氮蓝
    四唑(NBT)氧化还原酶活性，以 NADPH 为电子供体可以还原铁氰化钾、二溴百
    里香醌（DBMIB）、二氯酚靛酚（DCPIP）、duroquinone 以及 UQ-0 等质醌类电
    子受体。
    4．集胞蓝藻 PCC6803 中 NAD(P)H 脱氢酶与藻胆蛋白结合的初步证据
     通过非变性凝胶电泳（native-PAGE）和 NADPH-氮蓝四唑（NBT）氧化还
    原酶活性染色，从集胞蓝藻 PCC6803 的细胞粗提物中检测到一个 NADPH 专一
    的 NAD(P)H 脱氢酶（NDH）亚复合体，Western 印迹分析表明此亚复合体含
     V
    
    
    NdhA、NdhB 和 NdhH 但不含 NdhK 和 Fd-NADP+氧化还原酶（FNR）。吸收光
    谱和 77K 荧光光谱显示此亚复合体中含别藻蓝蛋白和藻蓝蛋白。进一步利用离
    子交换与凝胶过滤层析从细胞粗提物中分离 NDH 时，发现 NADPH-铁氰化钾
    氧化还原酶和NADPH-NBT氧化还原酶活性总是与藻胆蛋白共分离，无法分开。
    分离得到的NDH经 native-PAGE 分离和活性染色后，在凝胶上检测到两条 NDH
    活性条带，Western 印迹分析显示此两条带均含 NDH 的亚基，表明它们都是
    NDH 亚复合体。此两 NDH 条带在活性染色前均为蓝色，在非变性条件下将其
    蛋白质溶出后，吸收光谱以及 77K 和室温荧光光谱均显示此两条带含有藻胆蛋
    白。本文的结果表明，至少在我们的实验条件下，集胞蓝藻 PCC6803 中 NDH
    复合体与藻胆蛋白相互结合。
It had been known that there are mainly four kinds of complexes, photosystem II (PSII),
    cytochrome b6/f, photosystem I (PSI) and ATP synthase, in cyanobacterial and higher plant
    cloroplastic thylakoid membranes. Since mid-1980’s, another protein complex, NAD(P)H
    dehydrogenase (NDH) complex, with a high degree homology to mitochondrial complex I has
    been identified in thylakoid membranes of cyanobacteria and higher plants. NDH complex has
    been suggested to function as a mediator of both PSI-cyclic and respiratory electron flow to
    the intersystem chain in cyanobacteria or of PSI-cyclic and chlororespiratory electron flow in
    higher plants. Evidences show that NDH complex has very important physiological functions
    in cyanobacteria and is even indispensable to the viability of cyanobacteria. Therefore,
    presently, more and more attentions are being paid to this area. Up to date, studies on the
    structure and functions of NDH complex are still in primary stage. This work mainly focuses
    on the roles of NDH complex in the responses and acclimation of cyanobacteria to low CO2
    concentration, the separation and purification of NDH complex and its interaction with
    phycobiliproteins.
    1. Effects of low CO2 on cyanobacterial NDH complex
     The expression and activity of type-1 NAD(P)H dehydrogenase (NDH) was compared
    between cells of Synechocystis PCC6803 grown in high (H-cells) and low (L-cells) CO2 conditions.
    Western analysis indicated that L-cells contain higher amounts of the NDH subunits, NdhH, NdhI
    and NdhK. An NADPH-specific subcomplex of NDH showed higher NADPH-nitroblue tetrazolium
    oxidoreductase activity in L-cells. The activities of both NADPH-menadione oxidoreductase and
     VII
    
    
    light-dependent NADPH oxidation driven by photosystem I were much higher in L-cells than in
    H-cells. The initial rate of re-reduction of P700+ following actinic light illumination in the presence of
    DCMU under background far-red light was enhanced in L-cells. In addition, rotenone, a specific
    inhibitor of NDH, suppressed the relative rate of post-illumination increase in Chl fluorescence of
    L-cells more than that of H-cells, suggesting that the involvement of NDH in cyclic electron flow
    around photosystem I was enhanced by low CO2. Taken together, these results suggest that NDH
    complex and NDH mediated-cyclic electron transport are stimulated by low CO2 and function in the
    acclimation of cyanobacteria to low CO2.
    2. Response of NDH complex to the alteration of CO2 concentration
     An NADPH-specific NDH subcomplex was separated by native-polyacrylamide gel
    electrophoresis and detected by activity staining from the whole cell extracts of Synechocystis
    PCC6803. Low CO2 caused increase in the activity of this subcomplex quickly, accompanied by an
    evident increase in the expression of NdhK and PSI-driven NADPH oxidation activity that can
    reflect the activity of NDH-mediated cyclic electron transport. During incubation with high CO2,
    the activities of NDH subcomplex and PSI-driven NADPH oxidation as well as the protein
    level of NdhK slightly increased at the beginning, but decreased evidently in various degrees
    along with incubation time. These results suggest that CO2 concentration in vitro as a signal can
    control the activity of NDH complex and NDH complex may in turn function in the regulation of CO2
    uptake
    3. Separation of hydrophobic NDH subcomplexes from Synechocystis PCC6803
     Some efforts have been performed to separate an integrated NA(D)PH dehydrogenase
    (NDH) complex from cyanobacteria, which proved the purification of NDH complex very
    difficult due to its friability in vitro. Until now, no report has ever been showed to illustrate the
    purification of a hydrophobic NDH subcomplex from cyanobacteria yet. In this work, two NDH
    subcomplexes were separated from n-dodecyl β-D-maltoside (DM)-treated whole cell extracts
    of Synechocystis PCC6803 by anion exchange chromatography and gel filtration. Both
    subcomplexes contained the hydrophobic subu

引文

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    中国科学院上海植物生理生态研究所博士论文
    实 验 部 分
     集胞蓝藻 NDH 复合体的结构与生理功能研究
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
     （一）低 CO2浓度对集胞蓝藻 NDH 复合体的影响
     （二）集胞蓝藻 NDH 复合体对 CO2浓度变化的响应
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
     （一）集胞蓝藻含疏水亚基的 NDH 亚复合体的分离
     （二）集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
     第二章: NDH 在蓝藻对不同 CO2浓度的响应
     和适应中的作用
     （一）低 CO2浓度对集胞蓝藻 NDH 复合体的影响
     摘要
     比较了高 CO2（H-cells）和低 CO2（L-cells）下培养的集胞蓝藻 PCC6803
    中 NAD(P)H 脱氢酶复合体（NDH）的活性以及亚基表达。Western 印迹分析表
    明，L-cells 所含的 NDH 亚基，NdhH、NdhI 和 NdhK 的量明显比 H-cells 高。
    非变性凝胶电泳以及活性染色分析显示，L-cells 中，NADPH 专一的 NDH 亚复
    合体具有更高的 NADPH-氮蓝四唑（NBT）氧化还原酶活性；此外，L-cells 中，
    NADPH-menadione 氧化还原酶和光系统 I（PSI）驱动的 NADPH 氧化活性也明
    显比 H-cells 高，这都表明 NDH 的活性在低 CO2下增强了。在 DCMU 和背景
    远红光存在下，测得的作用光关闭后 P700+的暗中还原初始速率在 L-cells 中明
    显提高，表明 L-cells 中围绕 PSI 的循环电子传递增强。NDH 的专一抑制剂，
    鱼藤酮（rotenone）对 L-cells 的作用光关闭后叶绿素荧光瞬时上升的抑制程度
    比 H-cells 要大得多，这说明在低 CO2浓度下 NDH 介导的循环电子传递明显加
    强。以上结果表明，NDH 及其介导的循环电子传递均为低 CO2促进，提示 NDH
    参与蓝藻对低 CO2的适应过程。
    关键词 CO2；NAD(P)H 脱氢酶；PSI 循环电子传递；集胞蓝藻 PCC6803
     引言
     在哺乳动物、真菌和植物线粒体中，NADH-泛醌还原酶（NDH）复合体，
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    低 CO2浓度对集胞蓝藻 NDH 复合体的影响
    也称为复合体 I，催化由 NADH 到泛醌的电子传递并伴随跨膜质子梯度的形成，
    以用于 ATP 合成。集胞蓝藻 PCC6803 的基因组中也含有 12 个与线粒体复合体
    I 高度同源的 ndh 基因（ndhA~L）（Kaneko et al., 1996）。在集胞蓝藻 PCC6803
    中，NDH既介导循环电子传递也介导呼吸电子传递（Mi et al., 1992a, 1992b, 1994,
    1995），一般认为 NDH 介导的电子传递可能为其它需能的过程，如胁迫条件下
    的 CO2同化等补充 ATP。
     蓝藻和许多藻类具有在体内浓缩 CO2 的机制，称为 CO2 浓缩机制
    （CO2-concentrating mechanism），可以提高核酮糖-1,5-二磷酸羧化/加氧酶
    （Rubisco）活性位点附近的 CO2浓度，从而弥补 Rubisco 对 CO2很低的亲和性
    （Kaplan and Reinhold, 1999）。过去一直认为，蓝藻通过此机制吸收 CO2的过
    程是由 NDH 介导的围绕 PSI 的循环电子传递提供能量的（Ogawa, 1991）。然而，
    最新的研究结果表明，蓝藻中存在两种功能完全不同的 NDH 复合体：一种含
    NdhD1 或/和 NdhD2，参与呼吸和 PSI 循环电子传递；而另一种则含 NdhD3 或/
    和 NdhD4，参与 CO2吸收，但并不介导 PSI 循环电子传递（Klughammer et al.,
    1999; Ohkawa et al., 2000a; Ohkawa et al., 2000b）。这一发现大大削弱了此前人们
    对 NDH 在 CO2吸收中所起作用的设想。到目前为止，NDH 参与 CO2吸收的机
    制还不清楚，NDH 介导的 PSI 循环电子传递在 CO2吸收中的作用仍存在争论。
     以前人们对 NDH 在 CO2吸收中的功能的研究主要是通过对蓝藻单/双 ndh
    基因突变体的遗传分析进行的。然而，由于蓝藻 NDH 复合体由多亚基组成，
    一个或几个 ndh 基因的缺失往往会影响其他 ndh 基因的表达或 NDH 复合体的
    组装（Ohkawa et al., 2000; Pieulle et al., 2001），从而可能导致间接表型的产生，
    因此应用这些研究方法不能阐明不同 NDH 亚基在 CO2吸收中所可能各自具有
    的不同功能。研究显示低 CO2诱导 ndhB、ndhD2、ndhD3 和 ndhD5 的转录，而
    对ndhD1和ndhD4的转录水平则没有明显的影响（Figge et al., 2001; Marco et al.,
    1993; Ohkawa et al., 1998）。由于叶绿体基因的表达主要受转录后水平的调控
    （Danon, 1997），因此 ndh 基因的表达还可能受其他层次的调控，所以还需要
    从蛋白质水平上进行研究。此外，至今也还没有关于 NDH 酶活性在蓝藻对不
    同 CO2浓度的适应过程中变化的报道。阐明这一问题将有助于揭示 NDH 复合
    体是否参与蓝藻对不同 CO2浓度的适应过程以及其具体机制。
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    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
     为研究 NDH 复合体在蓝藻对不同 CO2浓度适应中的作用，我们检测了不
    同 CO2浓度培养的集胞蓝藻 PCC6803 中 NDH 亚基的蛋白质水平、NDH 酶活性
    及其介导的循环电子传递活性。我们的结果显示，低 CO2浓度下 NDH 亚基蛋
    白质量、NDH 酶活性及其介导的 PSI 循环电子传递均增加，表明 NDH 确实参
    与蓝藻对低 CO2浓度的适应过程。
     材料与方法
    1． 蓝藻细胞的培养
     集胞蓝藻 PCC6803 细胞用含 2 g/L HEPES-KOH（pH 7.4）的 BG-11 培养
    基（Allen, 1968），在 30 ℃、连续光照（60 μEm-2s-1），分别在空气（低 CO2浓
    度）和含 2% CO2的空气（高 CO2浓度）下震荡（100 转/分钟）培养。
    2． 细胞粗提液的提取
     对数后期生长的细胞（A730=0.4~0.6）于 10 000 g 离心5 min 收集，细胞以
    medium A（10 mmol/L HEPES-NaOH, 5 mmol/L sodium phosphate (pH 7.5), 10
    mmol/L MgCl2，10 mmol/L NaCl）洗涤一次，再次离心，将蓝藻细胞悬浮于含 20%
    (体积比) 甘油和 0.5 mmol/L phenylmethylsulfonyl fluoride（PMSF）的 mediumA 中。
    收集的细胞立即于冰浴中，以 Bead-Beater（Biopsec，日本）破碎，每次破碎
    20 s，间隔 3 min，共破碎 5 次。细胞匀浆于 4 ℃、10 000 g 离心 5 min 以去除未
    破碎的细胞和残渣。收集上清，加入0.1% (体积比)TritonX-100，冰浴中缓慢震荡1
    h。样品立即用于电泳及酶活性分析。
    3．凝胶电泳、NADPH-NBT 氧化还原酶活性染色和 Western blotting
     SDS-PAGE 按照 Laemmli（1970）的方法，在 12%的聚丙烯酰胺凝胶上进
    行。Western blotting 采用 ECL 分析试剂盒（Amersham Pharmacia），按照厂家的
    方法手册进行。文中所采用的 NdhB 亚基抗体由 Asada 教授（Department of
    Biotechnology, Faculty of Engineering, Fukuyama University）、NdhH 抗体由 Ogawa
    教授（Bioscience Center, Nagoya University）、NdhI 抗体由 Takabe 教授（Research
    Institute of Meijo University）、NdhK 抗体由 Arizmendi 博士（Department de
    Bioquimicay Biologia Molecular, Universidad del País Vasco）分别惠赠。
     50
    
    
    低 CO2浓度对集胞蓝藻 NDH 复合体的影响
     非变性凝胶电泳（native-PAGE），按 Davis 等（1964）的方法用 7.5%聚丙
    烯酰胺凝胶于 4 ℃下进行。活性染色时将凝胶于室温、20 mmol/LTris-HCl (pH
    7.5)中缓慢摇动 5 min，然后暗中加入含 1 g/L NBT 及 1 mmol/L NADPH 的 20
    mmol/L Tris-HCl（pH 7.5），直至紫色条带呈现。
    4. PSI 驱动的 NADPH 氧化和 NADPH-menadione 氧化还原酶活性分析
     PSI 驱动的 NADPH 氧化按照 Teicher 和 Scheller（1998）的方法并稍作修改
    进行。于 3 ml 反应缓冲液[10 μmol/L DCMU, 0.25 mmol/L NaN3, 0.1 mmol/L
    KCN，0.2% (体积比) Triton X-100，medium A]中加入蓝藻细胞粗提液（5 μg 叶
    绿素）。测定前，上述反应液先以作用光（>660 nm, 200 μE·m-2·s-1）照射 2 min
    以激活 NDH，然后加入 100 μmol/L NADPH，开启作用光后立即用分光光度计
    （UV-3000, 岛津）记录 340 nm 光吸收的下降。
     NADPH-menadione 氧化还原酶活性按照 Matsuo 等（1998）的方法并稍作
    修改进行。NADPH 的氧化通过记录加入 50 μmol/L NADPH 后 340 nm 处光吸
    收的下降进行测量。反应混合液为含 10 mmol/LMgCl2, 0.15 mmol/Lmenadione 的
    10 mmol/LTris-HCl (pH 8.0)。
     以上反应均在25 ℃进行。NADPH 的吸光系数为 6.22 mmol/L-1 cm-1。
    5. PQ 和 P700 的氧化还原动力学
     对数后期的蓝藻细胞（A730 = 0.4~0.6）于10 000 g 离心5 min 收集，重新悬浮
    于新鲜的含2 g/L HEPES-KOH（pH 7.4）的 BG-11 培养基（Allen, 1968）中，叶
    绿素浓度调整为10 μg/mL。PQ 的氧化还原用带有激发-检测附件（ED-101US）的
    PAM 叶绿素荧光光度计（Walz, Germany）通过检测叶绿素荧光动力学来测定；P700
    的氧化还原按照 Klughammer 和 Schreiber 的方法（1998）用带有附件
    ED-P700DW-II 的 PAM 叶绿素荧光光度计通过测定 810 nm 与 830 nm 处光吸收的
    差值来检测。
     结 果
     通过 Western 印迹分析，分析了高低 CO2浓度下培养的蓝藻中不同 NDH 亚
     51
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
    基的蛋白量。如图 1 所示，L-cells 中 NdhH（46 kD）、NdhI（24 kD）和 NdhK
    （27 kD）的量分别为 H-cells 的 3，2 和 7 倍，表明这些亚基为低 CO2诱导表达。
    相反，在相同的条件下，NdhB 的量却下降了 40%。与他人以前对 ndh 基因转
    录水平的分析结果相同，我们的结果也表明，一些 NDH 亚基可为低 CO2诱导，
    但并不是所有的 NDH 亚基都受低 CO2诱导。
     H-cells L-cells
     NdhB 55 kD
     NdhH 46 kD
     A
     NdhI 24 kD
     NdhK 27 kD
     900
     800
     H-cells
     (%) 700 L-cells
     nt 600
     B ou
     ma 500
     evitlaeR400
     300
     200
     100
     0
     NdhB NdhH NdhI NdhK
     Fig. 1 Western analysis (A) of the amount of NDH subunits in Synechocystis
     PCC6803 cells cultured in air (low CO2) and 2% CO2 (high CO2). Whole cell
     extracts were separated on 12% SDS-PAGE gels and immunoblotted with
     antibodies against NdhB, NdhH, NdhI and NdhK, respectively. Fifteen
     micrograms of protein were loaded in each lane. (B) Band intensities were
     quantified using a software Gel-Pro analyzer 3.0 with the levels in the H-cells
     being set as 100%. Standard errors were calculated from three separate experiments.
     我们进一步分析了 H-和 L-cells 细胞粗提液中 NDH 的活性。经非变性凝胶
    电泳分离和 NADPH-NBT 氧化还原酶活性染色分析后，在 300kD 处检测到一条
    NADPH 专一的活性条带（Fig. 2A）。免疫印迹分析显示此活性条带中含 NdhH
    亚基（数据未列出），表明其是 NDH 亚复合体。在 L-cells 中，此 NDH 活性条
     52
    
    
    低 CO2浓度对集胞蓝藻 NDH 复合体的影响
    带的 NADPH-NBT 氧化还原酶活性约为 H-cells 的两倍（Fig. 2）。Fig. 3 的结果
    也表明，L-cells 中 NADPH-menadione 氧化还原酶活性明显高于 H-cells。为避
    免来自铁氧还蛋白-NADP+氧化还原酶（FNR）的黄递酶活性的干扰，我们还分
    析了依赖光的 PSI 驱动的 NADPH 氧化活性，它可以反映 PSI 驱动的 NDH 活性
    （Mi et al., 1995; Teicher and Scheller, 1998）。结果也显示 PSI 驱动的 NADPH 氧
     kD H-cells L-cells Fig. 2. Activity staining of NADPH-NBT
     669
     440 oxidoreductase (A) in the whole cell
     232 NDH extracts in H- and L-cells of Synechocystis
     140 PCC6803. After solubilization with 0.1%
    A
     (v/v) Triton X-100, the crude extract (25
     6 μg protein per lane) was separated on 7.5%
     native PAGE gels. NADPH oxidoreductase
     activity was detected using NBT reduction
     in the presence of 1 mmol/L NADPH as an
     electron donor. (B) Band intensities were
     estimated as in Fig.1. Each experiment was
     300
     repeated three times and the standard errors
     250
     )
     (% were calculated and represented as the
     200
    B tyivi lengths of the vertical bars.
     150
     act
     e
     100
     Relativ
     50
     0
     H-cells L-cells
    化活性在低 CO2 下明显增加（Fig. 3A）。以上结果均表明 NDH 活性为低 CO2
    培养条件促进。
     作用光关闭后叶绿素荧光的瞬时上升现象是由于光下积累的还原产物在
    暗中还原 PQ 所致，在高等植物和蓝藻中可反映围绕 PSI 的循环电子传递（Asada
    et al.，1993；Mano et al., 1995; Mi et al., 1995）。因此，我们可利用此现象检测
    低 CO2对 NDH 介导的 PSI 循环电子传递的影响。集胞蓝藻 PCC6803 典型的叶
    绿素荧光动力学见 Fig. 4A，方框圈起来的部分为作用光关闭后叶绿素荧光的瞬
     53
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
    时上升。L-cells 中，作用光关闭后叶绿素荧光的瞬时上升现象比 H-cells 更加明
    显（Fig. 4B），这意味着 PSI 循环电子传递在低 CO2下可能加强了。为证实此结
    果，我们比较了 H-和 L-cells 的 P700 氧化还原动力学。在 DCMU 和背景远红
    光存在下，作用光关闭后 P700+暗中还原初始速率可反映 PSI 循环电子传递的速
    率（Klughammer and Schreiber, 1998）。 Fig. 5 的结果表明，L-cells 的 P700+暗
    中还原初始速率明显高于 H-cells，这进一步证实 PSI 确实在 L-cells 中加强了。
    鉴于 NDH 在蓝藻中既介导循环电子传递又介导呼吸电子传递，我们分析了 H-
     ) A
     chl
     -1 2
     mg
     -1
     min
     μM
     -1 01(
     no 1
     idati
     ox
     NADPH
     0
     ein)
     otrp B
     3
     -1
     mg
     -1 ni
     m
     μM 2
     -1
     (10
     iontad 1
     oxi
     NADPH
     0
     H-cells L-cells
     Fig. 3 Activity of light-dependent NADPH oxidation (A) and
     NADPH-menadione oxidoreductase (B) in H- and L-cells. The
     standard errors calculated from 5 replicates of each measurement are
     represented as the lengths of the vertical bars.
    和 L-cells 的暗中呼吸活性，没有发现明显的差异（数据未列出），这表明低 CO2
    下 NDH 亚基表达和酶活性的增加并没有促进细胞的呼吸活性，因此在此条件
     54
    
    
    低 CO2浓度对集胞蓝藻 NDH 复合体的影响
    下增加的 NDH 可能主要参与循环电子传递。
     AL off
     AL on
     L-cells H-cells
     RFp Control
    A scence
     re B
     uofllhC 20 μM rotenone
     Fo 30s 6s
     140 control
     20 μM rotenone
     120
     100
     (%)
     p
     80
     RF
     C vetia 60
     Rel 40
     20
     0
     H-cells L-cells
     Fig. 4 Post illumination increase in Chl fluorescence in H- and L-cells. The Chl
     concentration was adjusted to 10 μg/mL before measurement. Actinic light (AL,
     >645nm, 200 μEm-2s-1) was applied with a cut-off light filter. A shows the typical
     Chl fluorescence kinetics of Synechocystis PCC6803: the area surrounded by a
     square shows post illumination increases in Chl fluorescence and the slope of the
     indicated line given the relative rate of post illumination increase in Chl
     fluorescence (RFp). B and C show the effects of 20 μmol/L rotenone on the kinetics
     of post illumination increase in Chl fluorescence and RFp in H- and L-cells. RFp
     before addition of inhibitor was regarded as 100%. Where indicated, rotenone was
     added in the dark for 5 min before measurement. Each experiment was repeated
     three times and standard errors were calculated.
     55
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
     前面的结果已经显示 NDH 的亚基表达和酶活性均可为低 CO2诱导，而且
    PSI 循环电子传递在低 CO2 下增强。然而，鉴于蓝藻中存在多条不同的 PSI 循
    环电子传递途径，各条途径所占的比例仍不清楚，因此我们利用抑制剂进一步
    分析了低 CO2对 NDH 介导的 PSI 循环电子传递途径的影响。NDH 的专一抑制
    剂鱼藤酮（Yagi, 1991）可以专一抑制集胞蓝藻 PCC6803 的循环电子传递（Mi et
    al., 1995），此外作用光关闭后叶绿素荧光瞬时上升的初始速率（RFp）可反映
    PSI 循环电子传递的速率（Asada et al.，1993；Mano et al., 1995; Mi et al., 1995），
    我们以此为指标检测了鱼藤酮对 H-和 L-cells 循环电子传递的影响。结果显示，
    在 20 μmol/L 鱼藤酮存在下， L-cells 的 RFp只有处理前的 30%，而 H-cells 仍
    具有高于 60%的活力（Fig. 4B, C），这表明 L-cells 中 NDH 介导的 PSI 循环电
    子传递途径在所有途径中的比例明显高于 H-cells，推测其在低 CO2下可能起主
    要作用。
     250
     ative,%)l
     (re
     200
     ionct
     redu 150
     +
     00
     P7 100
     of
     te
     ra 50
     Initial
     0
     H-cells L-cells
     Fig. 5 Initial rates of P700+ re-reduction after turning off saturating actinic
     light (600~620 nm, 1000 μE m-2 s-1) in the presence of DCMU (10 μmol/L
     final) under background far-red light (>705 nm, 5.2 μE m-2 s-1) in H- and
     L-cells. The Chl concentration was adjusted to 10 μg/mL. The cells were
     dark-adapted sufficiently before measurement. Each experiment was repeated
     four times and the standard errors were calculated.
     56
    
    
    低 CO2浓度对集胞蓝藻 NDH 复合体的影响
     讨 论
     为了研究 NDH 在蓝藻对低 CO2条件适应中的作用，本文比较了高低 CO2
    培养的集胞蓝藻 PCC6803 中四个 NDH 亚基（NdhB, NdhH, NdhI 和 NdhK）蛋
    白质量以及 NDH 复合体酶活性的差异。我们发现，低 CO2条件下，NdhH, NdhI
    和 NdhK 的蛋白质水平以及 NDH 酶活性均显著提高。有研究表明，NdhH 亚基
    对集胞蓝藻 PCC6803 的生存是必需的（Pieulle et al., 2000）；在同样的蓝藻中，
    缺失 NdhK 会使 CO2吸收活性降至几乎为 0，并且 NdhK 突变体在光异养条件
    不能生长（Ogawa, 1992），因此推测它们可能参与 CO2吸收。本文中观察到的
    低 CO2条件下 NdhH 和 NdhK 蛋白质量的增加进一步证明这些亚基在蓝藻对低
    CO2 条件的适应中具有重要的作用，此外 NdhI 蛋白质量的增加也表明此亚基可
    能也参与这一过程。
     有意思的是，我们发现 NdhB 的蛋白质量在低 CO2条件下并没有增加（Fig.
    1）。有研究表明，ndhB 基因编码的膜蛋白是 NDH 复合体的一个核心组分，它
    对蓝藻的呼吸和循环电子传递以及无机碳吸收都是必需的（Klughammer et al.,
    1999; Marco et al., 1993; Ogawa, 1992）。此结果看起来与 NdhB 参与蓝藻无机碳
    吸收和低 CO2条件下 NDH 酶活性的增加的事实相矛盾。然而，也有证据显示，
    在 NdhB 缺失的集胞蓝藻突变体 M55 中，在类囊体膜和质膜上几乎检测不到
    NdhH、NdhJ 和 NdhK 的存在（Pieulle et al., 2000），表明 NdhB 的缺失使得这些
    亚基不能组装到类囊体膜上。这说明 ndhB 基因的缺失可能导致 NDH 复合体解
    体，从而产生其它间接表型（Ohkawa et al., 2000a）。Matsuo 等（1998）从集胞
    蓝藻 PCC6803 中分离到一个含 NADPH 结合位点和具有 NADPH 脱氢酶活性的
    NDH 亲水亚复合体，此亚复合体含有 NdhH，但是不含 NdhA 和 NdhB，这表
    明至少在体外 NdhB 对 NDH 的 NADPH 脱氢酶活性并不是必需的。因此，NdhB
    对体内 NDH 复合体的结构和功能上的完整性可能是必需的，但对其体外的脱
    氢酶活性则不是必需的。本文所示的低 CO2对 NdhB 和 NDH 活性的相反影响
    也支持此观点。
     我们的结果还表明，在低 CO2下各种 NDH 亚基蛋白质表达水平之间存在
    差异，这与他人以前在低 CO2下用 Northern 印迹分析（Ohkawa et al., 1998）和
    在强光下用 DNA 微阵列（Hihara et al., 2001）得出的结论一致。然而这种不同
     57
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
    亚基差异表达的机制还不清楚。蓝藻中参与 CO2吸收的 NDH 可以分为两种类
    型：NdhD3/NdhF3 和 NdhD4/NdhF4 类型的 NDH 分别参与低 CO2诱导的、具有
    较高亲和性的 CO2 吸收和组成性的、亲和性较低的 CO2 吸收（Shibata et al.,
    2001）。各种 NDH 亚基在低 CO2 下表达水平的差异也提示蓝藻中可能确实存在
    功能上完全不同的 NDH 复合体（Ohkawa et al., 2000a），并为阐明各种类型的
    NDH 复合体在两种 CO2 吸收类型（Shibata et al., 2001; Maeda et al., 2002）中的
    作用具有指导意义。然而，NDH 复合体在呼吸、循环电子传递以及 CO2吸收中
    的功能和机制的多样性仍需要进一步研究。
     蓝藻中具体的循环电子传递途径以及各条途径所占的比例至今仍存在争论
    （Golbeck, 1994）。研究表明，NADPH 向电子传递链传递电子的过程既可由
    NDH 介导（Mi et al., 1992a, 1992b, 1994, 1995），也可通过其他不依赖 NDH 的
    循环电子传递途径（Bendall and Manasse, 1995; Jeanjean et al., 1998; van Thor et
    al., 2000）。本文的结果显示，L-cells 中 NDH 途径所占的比例明显高于 H-cells，
    这可能是 NDH 活性增加的结果。低 CO2浓度对其它循环电子传递途径的影响
    还不清楚。
     过去的研究表明，PSI 循环电子传递可能是蓝藻 CO2吸收必需的。Kaplan
    和 Reinhold（1999）提出了 CO2吸收的碱性口袋模型，指出在类囊体膜或质膜
    的某些区域，光合电子传递可以向类囊体腔内泵出电子，从而导致这些区域的
    pH 升高形成“碱性口袋”。“碱性口袋”中比较高的 pH 有利于 CO2 在碳酸苷
    酶催化下转变为 HCO3 ，从而可以在体内累计无机碳。在此模型中，NDH 介导
     -
    的 PSI 循环电子传递在“碱性口袋”的形成中起着重要的作用。然而最近的结
    果却与此模型相矛盾。Shibata 等（2001）等发现，NdhD1/NdhD2 类型的 NDH
    复合体是 PSI 循环电子传递必需的但不参与 CO2吸收，而 ?ndhD3/?ndhD4 双突
    变体中，尽管 PSI 循环电子传递几乎没有受到影响（Ohkawa et al., 2000b），仍
    然不能吸收 CO2，这表明 PSI 循环电子传递似乎并不是 CO2吸收必需的。最近，
    关于 PSI 循环电子传递在 CO2吸收中的作用又有新的发现。Shibata 等（2001）
    从蓝藻中鉴定出两种新的 CO2吸收系统：CupA/NdhD3 和 CupB/NdhD4。研究
    表明，其中 CupB/NdhD4 系统依赖循环电子传递，不过其对 CO2的亲和性比较
    低，可能起次要作用；而 CupA/NdhD3 系统依赖光合线性电子传递，对 CO2的
    亲和性很高，可能起主要作用（Maeda et al., 2002）。然而由于蓝藻中存在多条
     58
    
    
    低 CO2浓度对集胞蓝藻 NDH 复合体的影响
    PSI 循环电子传递途径，因而各条途径在 CO2 吸收中的作用和地位仍不确定。
    Jeanjean 等（1998）报道在集胞蓝藻 ndhB 基因缺失的突变体 M55 中，盐胁迫
    可诱导PSI循环电子传递活性增加，从而使得M55恢复在低CO2下生长的能力。
    他们的结果支持 PSI 循环电子传递为 CO2吸收所必需并为其提供能量的观点。
    本文的结果表明 NDH 介导的循环电子传递为低 CO2促进，这也为此观点提供
    了进一步的证据。
     除为 CO2吸收提供能量外，还有人猜测包含 NdhD3 的 NDH 复合体也可能
    通过基因表达或蛋白质磷酸化的氧化还原控制从而参与高亲和性 CO2吸收的诱
    导等过程（Ohkawa et al., 1998）。我们的结果也提示 NDH 依赖的循环电子传递
    可能确实参与 CO2吸收的调控。当依赖或其他不依赖 NDH 的循环电子传递途
    径受到抑制或缺失后，其他非循环途径可能会作为补偿而受到诱导，这种设想
    值得进一步研究证实。
     综上所述，本文在蛋白质水平上证明某些 NDH 亚基的表达水平受低 CO2
    浓度的诱导，并首次表明 NDH 的活性也受低 CO2的正调控。我们的结果将对
    揭示 NDH 复合体的功能及其在蓝藻对低 CO2胁迫的适应中的作用具有重要意
    义。
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    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
    Ohkawa, H., Pakrasi, H.B., and Ogawa, T. (2000a) Two types of functionally distinct NAD(P)H
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     62
    
    
    低 CO2浓度对集胞蓝藻 NDH 复合体的影响
    Effects of Low CO2 on NAD(P)H Dehydrogenase, a Mediator
    of Cyclic Electron Transport Around Photosystem I in the
    Cyanobacterium Synechocystis PCC6803
     Abstract
     The expression and activity of type-1 NAD(P)H dehydrogenase (NDH) was compared
    between cells of Synechocystis PCC6803 grown in high (H-cells) and low (L-cells) CO2
    conditions. Western analysis indicated that L-cells contain higher amounts of the NDH
    subunits, NdhH, NdhI and NdhK. An NADPH-specific subcomplex of NDH showed higher
    NADPH-nitroblue tetrazolium oxidoreductase activity in L-cells. The activities of both
    NADPH-menadione oxidoreductase and light-dependent NADPH oxidation driven by
    photosystem I were much higher in L-cells than in H-cells. The initial rate of re-reduction of
    P700+ following actinic light illumination in the presence of DCMU under background far-red
    light was enhanced in L-cells. In addition, rotenone, a specific inhibitor of NDH, suppressed
    the relative rate of post-illumination increase in Chl fluorescence of L-cells more than that of
    H-cells, suggesting that the involvement of NDH in cyclic electron flow around photosystem I
    was enhanced by low CO2. Taken together, these results suggest that NDH complex and
    NDH mediated-cyclic electron transport are stimulated by low CO2 and function in the
    acclimation of cyanobacteria to low CO2.
    Key words: carbon dioxide; PSI-cyclic electron transport; NAD(P)H dehydrogenase;
     Synechocystis PCC6803
     63
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
     （二）集胞蓝藻 NDH 复合体对 CO2浓度变化的响应
     摘 要
     通过非变性凝胶电泳分离和活性染色，从集胞蓝藻 PCC6803 的细胞粗提物
    中检测到一个 NADPH 专一的 NDH 亚复合体。当高 CO2浓度培养的蓝藻细胞转
    为低 CO2浓度培养时，此 NDH 亚复合体的活性迅速提高，同时伴随 NdhK 亚基
    蛋白质表达水平和 PSI 驱动的 NADPH 氧化活性明显增加。而当低 CO2浓度培养
    的蓝藻转为高 CO2浓度培养时，一开始，此 NDH 亚复合体和 PSI 驱动的 NADPH
    氧化活性以及 NdhK 的蛋白质表达水平均有轻微的提高，但随着培养时间的增加，
    都有不同程度的明显下降。这些结果表明体外的 CO2 浓度作为信号可调控 NDH
    复合体的活性，推测 NDH 复合体反过来也可参与 CO2吸收的调节。PSI 驱动的
    NADPH 氧化活性可以反映 NDH 介导的循环电子传递的活性，因此我们的结果也
    表明 NDH 介导的 PSI 循环电子传递活性受低 CO2诱导而为高 CO2抑制。综合以
    上结果，说明 NDH 及其介导的循环电子传递可能参与蓝藻对 CO 浓度变化的响
     2
    应。
    关键词 CO2浓度；CO2吸收；NAD(P)H 脱氢酶；集胞蓝藻 PCC6803
     引 言
     蓝藻具有迅速适应 CO2 水平限制的能力，称为 CO2 浓缩机制（CO2-
    concentrating mechanism，简称 CCM），这对蓝藻在低 CO2 浓度下的生存是至关重
    要的。蓝藻 NAD(P)H 脱氢酶（NDH）是与线粒体复合体 I 同源的多亚基复合体，
    由 12 个亚基组成，其序列分别与线粒体复合体 I 的相应亚基高度同源（Kaneko et
    al., 1996）。研究表明，NDH 复合体参与蓝藻的 CO2 吸收（Ogawa, 1991a, 1991b）。
    在集胞蓝藻 PCC6803 种，NDH 复合体既位于类囊体膜也位于质膜上（Berger et al.,
    1991）。在类囊体膜上，它介导呼吸和循环电子传递（Mi et al., 1992a, 1992b, 1994,
     64
    
    
    集胞蓝藻中 NDH 复合体对 CO2浓度变化的响应
    1995）。
     过去很多研究认为，蓝藻的 CO2吸收是由 NDH 介导的电子传递提供能量或
    控制的（Ogawa, 1991a; Ohkawa et al., 1998）。最近，Maeda 等（2002）从聚球藻
    （Synechococcus）PCC7942 中鉴定出两种类型的 CO2吸收系统：ChpX（CupA）
    /NdhD3 和 ChpY（CupB）/NdhD4，分别依赖光合线性和循环电子传递。然而，
    其具体机制仍不清楚。
     以前对NDH在CO2吸收中功能的研究主要是通过对蓝藻单/双ndh基因突变
    体的遗传分析进行的。然而，蓝藻的 NDH 是一个多亚基复合体，其作为整个复
    合体在 CO2吸收及其调节机制中的作用仍不清楚。有研究表明，低 CO2条件在转
    录水平影响几个 ndh 基因的表达（Figge et al., 2001; Marco et al., 1993; Ohkawa et
    al., 1998），然而至今还没有关于 NDH 参与蓝藻对 CO2 浓度变化的响应的报道。
     因此，本文对集胞蓝藻 PCC6803 中，外界 CO2浓度变化对 NDH 的活性以及
    NDH 亚基表达水平的影响进行了研究，并对 NDH 复合体在蓝藻对不同 CO2 水平
    响应中的作用进行了初步探讨。
     材料和方法
     集胞蓝藻 PCC6803 细胞用含 2 g/L HEPES-KOH, pH 7.4 的 BG-11 培养基
    （Allen, 1968），在 30 ℃，连续光照（60 μEm-2s-1），分别在空气（低 CO2浓度）
    和含 2% CO2的空气（高 CO2浓度）下通气培养。细胞的收集以及细胞粗提物的
    制备采用 Mi 等（2001）的方法。制备的样品立即用于进一步实验分析。
     SDS-PAGE 按照 Laemmli（1970）的方法，在 12%的聚丙烯酰胺凝胶上进行。
    蛋白质经 SDS-PAGE 分离后，立即电转移至硝酸纤维素膜（Bio-Rad）上用于
    Western 印迹分析，杂交信号用碱性磷酸酶底物检测。Native-PAGE 和 NADPH-
    氮蓝四唑（NBT）氧化还原酶活性染色按照 Mi 等（2001）的方法进行，活性染
    色后的凝胶经扫描后进行定量分析。
     PSI 驱动的 NADPH 氧化按照 Teicher 和 Scheller（1998）的方法并稍作修改
    进行。于3 mL反应缓冲液[10 μmol/L DCMU, 0.25 mmol/L NaN3, 0.1 mmol/L KCN，
     65
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
    0.2% (体积比) Triton X-100，medium A] 中加入蓝藻细胞粗提液至 5 μg 叶绿素。
    在测定前，上述反应液先以作用光（>660 nm, 200 μE·m-2·s-1）照射 2 min 以激
    活 NDH，然后加入 100 μmol/L NADPH，开启作用光后立即用分光光度计
    （UV-3000, 岛津）记录 340 nm 处光吸收的下降。
     结果和讨论
     蓝藻中，NDH 对 CO2吸收是必需的（Ogawa, 1991a, b; Marco et al., 1993）。
    尽管已有报道指出一些 ndh 基因的转录受低 CO2条件诱导，NDH 复合体对 CO2
    浓度变化的响应仍不清楚。本文首次研究了外界 CO2浓度改变时，蓝藻 NDH 复
    合体活性的变化。通过非变性凝胶电泳分离和活性染色，我们从集胞蓝藻
    PCC6803 的细胞粗提物中检测到一个 NADPH 专一的 NDH 亚复合体。此亚复合
    体含 NdhH（数据未列出）和 NdhB（Mi et al., 2001），但没有检测到 NdhK（数据
    未列出）。当高 CO2浓度生长的细胞（H-cells）转到低 CO2浓度培养时，NDH 复
    合体的活性在 2 个小时内迅速增加到原来的两倍，随后其活性有所下降。低 CO2
    浓度培养 4 小时后，NDH 的活性仍约为 H-cells 的 1.4 倍（Fig. 1A）。鉴于 NDH
    在蓝藻中介导循环电子传递，我们分析了可反映 NDH 介导的循环电子传递的 PSI
    驱动的 NADPH 氧化活性。与 NDH 活性相似，当 H-cells 转为低 CO2培养 2 小时
    后，PSI 驱动的 NADPH 氧化活性增加了大约 40%（Fig. 2B）。然而，此活性在培
    养 3 小时后才达到最高值，比 NDH 活性迟了一个小时，这表明 NDH 复合体可能
    比循环电子传递途径中的其它组分对低 CO2更加敏感。同时，伴随 NDH 复合体
    活性的增加， NdhK 亚基的蛋白质水平在 H-cells 转为低 CO2培养后 2 小时内升
    高了 6 倍（Fig. 1C），此后，又缓慢下降至 H-cells 的 3 倍，这表明 NdhK 的表达
    为低 CO2诱导。推测其他 NDH 亚基也为低 CO2诱导。尽管本文中检测到的 NDH
    亚复合体不含 NdhK，我们结果仍表明低 CO2可能通过诱导 NDH 亚基的表达从
    而促进 NDH 复合体的活性。
     当低 CO2培养的蓝藻细胞（L-cells）转为高 CO2培养 1 小时后，NDH 亚复
    合体的活性先是迅速小幅度地增加，继而在随后的 6 小时内缓慢下降至大约
    L-cells 的 60%（Fig. 2A）。而 PSI 驱动的 NADPH 氧化活性在开始的 3 小时内没
    有明显的变化，而在随后的 6 小时内却迅速下降了大约 50%（Fig. 2B）。同样，
     66
    
    
    集胞蓝藻中 NDH 复合体对 CO2浓度变化的响应
     Time 0 1 2 3 4 (h)
     NDH 300 kDa
     A
     ) 2.2
     chl
     -1 g
     m 2.0
     -1
     min
     B Mμ 1.8
     -1
     10(
     niot 1.6
     idaxo
     H 1.4
     NADP
     0.0
     0 1 2 3 4 5
     Time (h)
     Time 0 1 2 3 4 (h)
     C NdhK 27 kDa
    Fig. 1 Response of NDH to low CO2 concentration. The whole H-cell extracts of
    Synechocystis PCC6803 after incubation in low CO2 for different time were
    applied for measurement. A, proteins were separated by a non-denaturing gel and
    stained by NADPH-NBT oxidoreductase activity as described in Materials and
    Methods; B, PSI-driven NADPH oxidation. Each experiment was independently
    repeated three times and the standard errors were calculated and represented as the
    lengths of the vertical bars; C, Western blotting with an antibody raised against
    NdhK.
     67
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
    `
     Time 0 1 3 5 7 h
     NDH 300 kDa
     A
     ) 5
     chl
     -1
     mg 4
     -1
     min
     mM 3
     B -1 01(
     niot 2
     idaxo 1
     H
     NADP0
     0 2 4 6 8 10 12
     Time (h)
     Time 0 1 3 5 7 h
     C Ndh K 27 kDa
     Fig. 2 Response of NDH to high CO2 concentration. The whole L-cell extracts of
     Synechocystis PCC6803 after incubation in high CO2 for different time were
     applied for measurement. A, proteins were separated by a non-denaturing gel and
     stained by NADPH-NBT oxidoreductase activity as described in Materials and
     Methods; B, PSI-driven NADPH oxidation. Each experiment was independently
     repeated three times and the standard errors were calculated and represented as the
     lengths of the vertical bars; C, Western blotting with an antibody raised against
     NdhK.
     68
    
    
    集胞蓝藻中 NDH 复合体对 CO2浓度变化的响应
    NdhK 的蛋白质水平在一开始的 1 小时内上升了 20%，随后即迅速下降，至 7 小
    时时降低了大约 60%（Fig. 2C）。Fig. 2 的结果表明，作为对 CO2浓度升高的响应，
    NDH 复合体的活性开始轻微上升，随后，随着蓝藻对高 CO2 浓度的适应，NDH
    亚基的表达收到明显抑制，从而导致 NDH 复合体及其介导的 PSI 循环电子传递
    活性降低。
     鉴于 NDH 参与蓝藻的 CO2吸收（Ogawa 1991a,b; Shitbata et al. 2001; Maeda et
    al. 2002），可以推测 NDH 复合体可能也参与蓝藻对低 CO2 浓度的响应和适应过
    程。本文的结果表明 NDH 复合体的活性在蓝藻对低 CO2浓度响应过程中受诱导
    增加，提示 NDH 复合体在此过程中可能具有重要作用。此外，高 CO2浓度下 NDH
    复合体活性以及NdhK蛋白水平的降低也说明NDH可能受高CO2浓度的负调控。
    有证据表明集胞蓝藻 PCC6803 中存在低 CO2诱导的 CO2吸收系统，NDH 对此系
    统是必需的（Shibata et al., 2001; Maeda et al., 2002）。NDH 复合体及其介导的循
    环电子传递分别为低 CO2 浓度诱导和高 CO2 浓度抑制，这表明它们可能在此低
    CO2 诱导的 CO2 吸收系统中起着重要的作用。同时，我们的结果也支持 PSI 循环
    电子传递参与 CO2吸收的观点。
     在蓝藻细胞由高到低和由低到高 CO2 浓度的转换过程中，NDH 活性以及
    NdhK 蛋白水平一开始迅速上升，这表明 NDH 可能参与蓝藻对 CO2 水平迅速改
    变的响应。由此推测，NDH 复合体可能也参与蓝藻对其它迅速改变的外界环境条
    件的响应过程。
     综上所述，我们的结果表明体外的 CO2 浓度作为信号可调控 NDH 复合体的
    活性，同时推测 NDH 复合体反过来也可能参与 CO2吸收的调节。
     参考文献
    Allen, M.M. (1968) Simple conditions for growth of unicellular blue-green algae on plates.
     J .Phycol. 4: 1-4
    Berger, S., Ellersiek, U. and Steinmüller, K. (1991) Cyanobacteria contain a mitochondrial
     complex I-homologoues NADH-dehydrogenase. FEBS Lett. 286: 129-132
    Figge, R.M., Cassier-Chauvat, C., Chauvat, F. and Cerff, R. (2001) Characterization and analysis
     69
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
     of an NAD(P)H dehydrogenase transcriptional regulator critical for the survival of
     cyanobacteria facing inorganic carbon starvation and osmotic stress. Mol. Microbiol. 39:
     455-468
    Kaneko, T., Sato, S., Kotani, H., Tanaka, A., Asamizu, E., Nakamura, Y., Miyajima, N., Hirosama,
     M., Sugiura, M., Sasamoto, S., Kimura, T., Hosouchi, T., Matsuno, A., Muraki, A., Nakazaki,
     N., Naruo, K., Okumura, S., Shimpo, S., Takeuchi, C., Wada, T., Watanabe, A., Yamada, M.,
     Yasuda, M. and Tabata, S. (1996) Sequence analysis of the genome of the unicellular
     cyanobacterium Synechocystis sp. strain PCC6803 II. Sequence determination of the entire
     genome and assignment of potential protein-coding regions. DNA Res. 3: 109-136.
    Kaplan, A. and Reinhold, L. (1999) CO2 concentrating mechanism in photosynthetic
     microorganisms. Annu. Rev. Plant Physiol. Plant Mol. Biol. 50: 539-570
    Lammli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of
     bacteriophasge T4. Nature 227: 680-685
    Maeda, S., Badger, M.R. and Price, G.D. (2002) Novel gene products associated with
     NdhD3/D4-containing NDH complexes are involved in photosynthetic CO2 hydration in the
     cyanobacterium, Synechococcus sp. PCC7942. Mol. Microbiol. 43: 425-435
    Marco, E., Ohad, N., Schwarz, R., Lieman Hurwitz, J., Gabay, C. and Kaplan, A. (1993) High CO2
     concentration alleviates the block in photosynthetic electron transport in an ndhB-inactivated
     mutant of Synechococcus sp. PCC7942. Plant Physiol. 101: 1047-1053
    Mi, H., Endo, T., Ogawa, T. and Asada, K. (1995) Thylakoid membrane-bound, NADPH-specific
     pyridine nucleotide dehydrogenase complex mediates cyclic electron transport in the
     cyanobacterium Synechocystis PCC6803. Plant Cell Physiol. 36: 661-668
    Mi, H., Endo, T., Schreiber, U. and Asada, K. (1992a) Donation of electrons to the intersystem
     chain in the cyanobacterium Synechococcus sp. PCC7002. Plant Cell Physiol.33: 1099-1105
    Mi, H., Endo, T., Schreiber, U., Ogawa, T. and Asada, K. (1992b) Electron donation from cyclic
     and respiratory flows to the photosynthetic intersystem chain is mediated by pyridine
     nucleotide dehydrogenase in the cyanobaterium Synechocystis PCC6803. Plant Cell Physiol.
     33: 1233-1237
    Mi, H., Endo, T., Schreiber, U., Ogawa, T. and Asada, K. (1994) NAD(P)H-dehydrogenase-
     dependent cyclic electron flow around photosystem I in the cyanobaterium Synechocystis
     PCC6803: a study of dark-starved cells and spheroplasts. Plant Cell Physiol. 35: 163-173
     70
    
    
    集胞蓝藻中 NDH 复合体对 CO2浓度变化的响应
    Mi, H., Deng, Y., Tanaka, Y., Hibino, T. and Takabe, T. (2001) Photo-induction of an NADPH
     dehydrogenase which functions as a mediator of electron transport to the intersystem chain in
     the cyanobacterium Synechocystis PCC6803. Photosyn. Res. 70: 167-172
    Ogawa, T. (1991a) Cloning and inactivation of a gene essential to inorganic carbon transport of
     Synechocystis PCC6803. Plant Physiol. 96: 280-284
    Ogawa, T. (1991b) A gene homologous to the subunit-2 gene of NADH dehydrogenase is essential
     to inorganic carbon transport of Synechocystis PCC6803. Proc. Natl. Acad. Sci. USA 88,
     4275-4279
    Ogawa, T. (1992) Identification and characterization of the ictA/ndhL gene product essensial to
     inorganic carbon transport of Synechocystis PCC6803. Plant Physiol. 99: 1604-1608
    Ohkawa, H., Sonoda, M., Katoh, H. and Ogawa, T. (1998) The use of mutants in the analysis of the
     CCM in cyanobacteria. Can. J. Bot. 76: 1025-1034
    Shibata, M., Ohkawa, H., Kaneko, T., Fukuzawa, H., Tabata, S., Kaplan, A. and Ogawa, T. (2001)
     Distinct constitutive and low-CO2-induced CO2 uptake systems in cyanobacteria: genes
     involved and their phylogenetic relationship with homologous genes in other organisms.
     Proc. Natl. Acad. Sci. USA 98: 11789-11794
    Teicher, H.B. and Scheller, H.V. (1998) The NAD(P)H dehydrogenase in barley thylakoids is
     photoactivatable and uses NADPH as well as NADH. Plant Physiol. 117: 525-532
     71
    
    
    第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
    Response of NAD(P)H Dehydrogenase Complex to the
    Alteration of CO2 Concentration in the Cyanobacterium
    Synechocystis PCC6803
     Abstract
     An NADPH-specific NDH subcomplex was separated by native- polyacrylamide gel
    electrophoresis and detected by activity staining from the whole cell extracts of Synechocystis
    PCC6803. Low CO2 caused increase in the activity of this subcomplex quickly, accompanied by
    an evident increase in the expression of NdhK and PSI-driven NADPH oxidation activity that
    can reflect the activity of NDH-mediated cyclic electron transport. During incubation with high
    CO2, the activities of NDH subcomplex and PSI-driven NADPH oxidation as well as the protein
    level of NdhK slightly increased at the beginning, but decreased evidently in various degrees
    along with incubation time. These results suggest that CO2 concentration in vitro as a signal
    can control the activity of NDH complex and NDH complex may in turn function in the regulation
    of CO2 uptake
    Key words: CO2 concentration; CO2 uptake; NAD(P)H dehydrogenase;
     Synechocystis PCC6803
     72
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白质的相互作用
     第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白
     的相互作用
     （一）集胞蓝藻含疏水亚基的 NDH 亚复合体的分离
     摘 要
     本文利用离子交换与凝胶过滤层析，从 n-dodecyl β-D-maltoside (DM)处理的
    集胞蓝藻 Synechocystis PCC6803 细胞粗提液中，首次分离到两个包含 NDH 疏
    水亚基NdhA的亚复合体。酶活分析表明，分离到的NDH亚复合体具有NADPH-
    氮蓝四唑(NBT)氧化还原酶活性，以 NADPH 为电子供体可以还原铁氰化钾、二
    溴百里香醌（DBMIB）、二氯酚靛酚（DCPIP）、duroquinone 以及 UQ-0 等质醌
    类电子受体。
    关键词 集胞蓝藻; NAD(P)H 脱氢酶; 疏水亚基
     引言
     集胞蓝藻 Synechocystis PCC6803 的基因组包含 12 个 ndh 基因（ndhA~L）
    （Kaneko et al., 1996），它们都编码一个与线粒体复合体 I 高度同源的 NAD(P)H
    脱氢酶(NDH,EC1.6.99.3)（Berger et al., 1991）。在蓝藻中，NDH 是一个多亚基复
    合体，已知其 ndhA~G 和 ndhH~K 基因分别编码 NDH 的疏水亚基和亲水亚基
    （Berger et al., 1991, 1993）。
     有证据表明蓝藻 NDH 既位于质膜也位于类囊体膜上（Berger et al., 1991），
    在类囊体膜上介导呼吸和光合循环电子传递（Mi et al., 1995; Sandmann et al.,
     73
    
    
    集胞蓝藻含疏水亚基的 NDH 亚复合体的分离
    1983）。NDH 介导的电子传递参与无机碳转运系统的能化/活化（Ogawa, 1991;
    Klughammer et al., 1999; Ohkawa et al., 2000）以及蓝藻对盐胁迫的适应（Hibino
    et al., 1996; Tanaka et al., 1997）等生理过程。此外，研究还表明 NdhH 亚基对于
    蓝藻的生存是必需的（Pieulle et al., 2000）。最近，我们发现蓝藻中 NADPH 专
    一的 NDH 参与光调节的围绕光系统 I 的循环电子传递及呼吸电子传递，而且
    NDH 的活性和亚基表达也受光诱导（Mi et al., 2002）。
     蓝藻 NDH 复合体的分离纯化比较困难。主要的原因是，在分离纯化过程中
    常常得到几个不同的、具有酶活性的亚复合体。此外，也有报道指出，蓝藻中
    可能存在不同功能形式的 NDH（Klughammer et al., 1999; Ohkawa et al., 1998）。
    至今为止，人们还没有从蓝藻中分离到完整的 NDH 复合体。Berger 等（1993）
    用免疫亲和层析的方法，从集胞蓝藻 PCC6803 类囊体膜的 Triton X-100 溶出物
    中分离到一个 NDH 的亚复合体，包含 7 条主要的多肽，其中有 NdhH、NdhI、
    NdhJ 和 NdhK 等亲水亚基。Matsuo 等（1998）从 3-[(3-cholamidopropyl) dimethy-
    lammonio] -1-propanesulfonate (CHAPS) 处理的集胞蓝藻 PCC6803 的细胞粗提
    物中，也分离到一个对 NADPH 专一的 NDH 亲水亚复合体，包含 NdhH, I, J 和
    K 亚基，但不含 NdhA 和 NdhB 等疏水亚基。迄今，含疏水亚基的蓝藻 NDH 亚
    复合体的分离还未见报道。有研究表明线粒体呼吸链复合体 I 的疏水亚复合体可
    能含有醌结合位点（Friedrichetal.,1995），由于蓝藻NDH 与线粒体复合体I 有很高
    的同源性，可以推测蓝藻可能也是如此，但还没有直接的实验证据。因此，从蓝藻
    中分离NDH 的疏水亚复合体对研究NDH 复合体的结构与功能具有重要意义。
     本文利用离子交换层析和凝胶过滤，首次从集胞蓝藻 PCC6803 中分离到含
    疏水亚基的 NDH 亚复合体，并对此亚复合体的性质进行了初步的研究。
     材料和方法
    1. 细胞的培养
     集胞蓝藻PCC6803细胞用BG-11培养基（Allen, 1968）（含5mmol/LTris-HCl,
    pH 8.0），在 30 ℃、连续光照（60 μEm-2s-1）下通气（含 2% CO2 的空气）培养。
    对数后期生长的细胞（A730=0.4~0.6）于 5 000 g 离心5 min 收集，细胞以10 mmol/L
     74
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白质的相互作用
    Tris-HCl (pH8.0)洗涤一次，再次离心，将蓝藻细胞悬浮于含 20%（体积比）甘油的
    10 mmol/LTris-HCl (pH8.0)中，贮存于-80℃。
    2. 蛋白质的分离
     细胞冻融后加入 0.5 mmol/L phenylmethylsulfonyl fluoride (PMSF)，用
    Bead-beater 细胞破碎器（Biospec，日本）在冰浴中破碎，每次破碎 20 s，间歇
    3 min，重复 5 个循环。细胞匀浆于 10 000 g、4 ℃离心 5 min 以除去未破碎的
    细胞，上清液（细胞粗提液，蛋白质浓度调节到 25 g/L）中加入 5 g/L n-dodecyl
    β-D-maltoside (DM) (最终浓度)，冰浴中缓慢振荡 1 h，最后以 140 000 g 离心60
    min。离心后上清液先经 DEAE-52（Whatman）离子交换柱（2.5 cm×15 cm）分离，
    用含0 ~ 0.5 mol/LNaCl 线性梯度的10 mmol/LTris-HCl (pH 8.0), 0.5 mmol/LEDTA，
    在7 ℃洗脱（流速为3 mL/min，12 mL/管）。得到的活性部分，用Amicon YM-10
    超滤膜浓缩，然后再用两支串联的Hiload 26/60 Superdex 200 prep grade凝胶过滤柱
    （2.6 cm×60 cm），于10 ℃，在高压液相层析（FPLC）系统（Amersham Pharmacia）
    上分离蛋白质，洗脱液为10 mmol/LTris-HCl (pH 8.0), 0.5 mmol/LEDTA, 150 mmol/L
    NaCl，流速为 1 mL/min。收集的活性部分以 Amicon YM-30 超滤膜浓缩后-80 ℃
    贮存。
    3. NDH 酶活性分析
     NDH 酶活性通过测量人工电子受体的还原或底物 NADPH 的氧化来分析
    （Matsuo et al., 1998）。NADPH 的氧化用分光光度计（岛津 UV-3000）记录 340
    nm {ε=6.22/ [(mmol?L)-1? cm] }光吸收的下降速率来测量，当铁氰化钾为电子受体
    时记录 420 nm {ε=1.03/ [(mmol?L)-1? cm] }处光吸收下降速率来测量。测量温度为
    室温（约25℃）。1 个酶活单位为每分钟氧化 1 μmol/L NADPH 的酶量。
    4 非变性凝胶电泳及活性染色
     凝胶过滤分离到的活性部分直接进行非变性凝胶电泳（native-PAGE）分析，
    按 Davis 等（1964）的方法用 7.5%聚丙烯酰胺凝胶于 4 ℃下进行。活性染色时
    将凝胶于室温、20 mmol/LTris-HCl (pH 7.5)中缓慢摇动 5 min，然后暗中加入含
    1 g/L 氮蓝四唑（NBT）及 1 mmol/L NADPH 的 20 mmol/L Tris-HCl（pH 7.5），
    直至紫色条带呈现。
     75
    
    
    集胞蓝藻含疏水亚基的 NDH 亚复合体的分离
     )
     -1 6 700
     mL
     -1 A280
     n 5 NDH activity 600
     mi NaCl 2
     500
     4
     NADPH 400 )L/lo
     0
     3 28
     A mm(
     mmol 300 Cl
     1
     -1 Na
     10(y 2
     200
     1
     ctivitA 100
     0 0 0
     0 20 40 60
     8
     ) 1.4
     -1 A280
     NDH activity
     mL
     -1 1.2
     n 6
     mi
     1.0
     NADPH 4 .8
     280
     .6 A
     mmol
     -1
     10(y 2 .4
     .2
     ctivitA
     0 0.0
     300 350 400 450 500 550 600 650
     Volume (mL)
    Figure 1 Separation of NDH from the whole cell extracts of
    Synechocystis PCC6803
    A，anion exchange chromatography; B，gel filtration.
     76
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白质的相互作用
    5 蛋白质的洗脱、SDS-聚丙烯酰胺凝胶电泳（PAGE）及免疫印迹分析
     将多个非变性凝胶上的活性条带切下后，置于约 1 倍凝胶体积的 10 mmol/L
    Tris-HCl (pH 8.0), 0.1% (体积比) Triton X-100 和 0.1 g/L SDS 中 30 ℃振荡过夜，
    然后 10 000 g 离心10 min，上清液以15%SDS-PAGE 分离并用于免疫印迹分析。
     结 果
     NDH 复合体的分离过程见表 1。蓝藻细胞粗提液经 5 g/L DM 增溶后，经离子
    交换层析分离，集中在 0.18 ~ 0.31 mol/L NaCl 梯度洗脱范围分离到一个主要的
    NADPH-铁氰化钾氧化还原酶活性峰 (图 1A)。将这部分活性峰处的蛋白质再以凝胶
    过滤层析分离后，得到四个不同分子量的酶活性部分（图1B）。由于我们希望得到
    分子量尽可能大的 NDH 亚复合体，因此我们主要对第一个活性峰进行了分析。第
    一个活性峰的蛋白质经非变性凝胶电泳及活性染色后，主要出现了三条活性条带，
    Table 1 Procedure of the separation of NDH
     纯化步骤 蛋白质 总活性 (μmol 比活力（μmol NADPH 纯化 得率
     （毫克） NADPH min-1ml-1) min-1 (mg protein)-1）倍数 (%)
     细胞粗提液 496 123 0.249 1 100
     (DM 处理前)
     细胞粗提液 481 120 0.250 1 97
     (DM 处理后)
     DEAE52 125 65 0.522 2.1 25
     Superdex 5.5 29 5.29 22 1.1
     200
    分子量分别为240 kD (Band I)、200kD(BandII)和100kD(Band III)，考马斯亮蓝染
    色后在相同位置有对应的三条蛋白质带。由于Band III 分子量较小，活性较低，而
    且其量随Band I 和Band II 的变化而变化 (图2)，因此我们推测它可能是从Band I
    或者Band II 降解下来的，然而也不能排除其是另一种NDH 亚复合体的可能。Band
    I, II 和 III 都不能氧化 NADH（数据未列出），表明我们分离得到的复合体是 NADPH
    专一的。
     Band I 和 II 中的蛋白质切带溶出后，经 SDS-PAGE 分析，分别含有 14 和 13
     77
    
    
    集胞蓝藻含疏水亚基的 NDH 亚复合体的分离
    条蛋白质带，其中Band I 与Band II 的多肽组成大部分相同（图3A），表明分子量
    较小的Band II 可能是从Band I 上降解来的；然而，也不排除它们是两种分子组成
    有所不同的NDH 的可能。免疫印迹分析结果显示BandI 含NdhA 和NdhH；Band II
     KDa Marker 400 406 412 418 424 432 436 440 446 Elution volume (ml)
     669
     440 A
     Band I
     232 Band II
     140
     Band III
     67
     669
     440 B
     Band I
     232 Band II
     140
     Band III
     67
    Figure 2 Native-PAGE and NADPH-NBT oxidoreductase activity
    staining of the activity peak fractions separated by gel filtration
    A: Stained with Coommassie brilliant blue; B: NADPH-NBT
    oxidoreductase activity staining.
    含NdhA，但不含NdhH（图3B）。此外，从图3A 可以看出有三条很浓的蛋白质带，
    分子量分别为48KD，21KD，18KD，分别与相应藻胆体蛋白的分子量（MacColl and
    Guard-Friar, 1987）一致。我们在另一份工作中发现蓝藻中 NDH 与藻胆蛋白结合在
    一起（未发表数据），Matsuo 等（1998）在分离的蓝藻 NDH 中也发现藻胆蛋白的
    存在，因此我们推测这三个蛋白质可能为藻胆体蛋白。在这两个NDH 亚复合体中，
    我们没有检测到NdhB、NdhK 及Fd-NADP+氧化还原酶（FNR）的存在。此外，在
     78
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白质的相互作用
    离子交换层析前，用非变性凝胶电泳及活性染色分析了细胞粗提液，我们检测到一
    个300kD 的NDH 亚复合体，含有NdhA, NdhB 和NdhH（数据未列出），然而本文
    中分离到的两个NDH 亚复合体都不含NdhB，我们推测NdhB 可能在分离过程中从
    NDH 复合体上脱离下来了。
     由Band I 和Band II 在离子交换、凝胶过滤层析及电泳中的行为可以看出它们
    在分子量及电荷等蛋白质性质上非常接近，用本实验中采用的离子交换、凝胶过滤
    层析方法我们无法把它们分开；尽管非变性电泳可以将他们分开，但在从胶上洗脱
    蛋白质时，NDH 亚复合体降解严重。此外，我们还发现，NDH 的结构很不稳定，
    细胞粗提液于30 ℃处理1 h，300 kD 的NDH 即大部分消失，同时伴随240 kD 和
    200 kD 两条 NDH 活性条带的增加，其分子量大小分别对应 Band I 和 Band II（图 5）。
     Band I Band II
     kDa I II I II
     66 kDa Band Band Band Band
     45 66
     36 45
     36
     29 29
     A 24 B 24
     20 20
     14
     14
     Ndh A Ndh H
     Figure 3 Analysis of Band I and Band II
     A, SDS-PAGE; B, Western blotting.
    因此我们推测Band I 和Band II 都是从300 kD 的NDH 上降解下来的。同时，由于
    Band I 和 Band II 在亚基组成上非常相近（图 3A），而且它们均具有氧化 NADPH 和
    还原醌类似物的活性（图2），推测在降解过程中可能都没有丢失反应中心的亚基，
    因此它们在酶活性质上可能相同，所以我们在混合条件下对这两个 NDH 亚复合体
     79
    
    
    集胞蓝藻含疏水亚基的 NDH 亚复合体的分离
    的分子性质进行了研究。当以铁氰化钾为电子受体时，分离到的含疏水亚基的NDH
    亚复合体对NADPH 的Km为6.5 μmol/L，Vmax为8 单位每毫克蛋白质，均高于他人
    用亲水亚复合体得到的结果（Matsuo et al., 1998）。
     研究了分离的 NDH 亚复合体对不同电子受体的 NADPH 氧化活性（图 4），结
     ] 10
     -1
     otein)
     prgm( 8
     -1
     min 6
     DPH
     NAlomm 4
     -3 0
     [1yitvi 2
     Act 0
     1 2 3 4 5 6 7 8 9 10
    Fig. 4 NADPH oxidation activity of NDH subcomplex with different electron
    acceptors
    1, 200 μmol/L O2; 2, 100 μmol/L duroquinone; 3, 100 μmol/L DMBQ; 4, 100
    μmol/L DBMIB; 5, 1 mmol/L Ferrecyanide; 6, 100 μmol/L DCPIP; 7, 100
    μmol/L UQ-0; 8, 100 μmol/L menadione; 9, 100 μmol/L decylubiquinone; 10,
    100 μmol/L PQ.
    果表明对铁氰化钾有最高的活力，依次为二溴百里香醌（DBMIB），二氯酚靛酚
    （DCPIP）及2,6-dimethyl-p-benzoquinone (DMBQ)等。这与 NDH 亲水亚复合体的
    情况相似（Matsuo et al., 1998）。然而，对维生素K3 (menadione) 的还原活性却远小
    于亲水亚复合体，而当质醌 (PQ)，decyllubiquinone 及O2为电子受体时，则几乎检
    测不到NADPH 氧化活性。
     80
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白质的相互作用
     a b c d e
     300 kDa NDH
     240 kDa Band I
     200 kDa Band II
     Fig. 5 Analysis of the stability of cyanobacterial NDH subcomplexs.
     After treatment with 0.5% (w/v) DM in ice for 1 h, the whole cell extract of
     Synechocystis PCC6803 was incubated in ice consecutively (a) or at 30 ℃
     for 0.5 h (b), 1 h (c), 1.5 h (d) and 2 h (e) respectively. Then, the samples
     were applied to native-PAGE and subsequent NADPH-NBT oxidoreductase
     activity staining immediately.
     讨 论
     迄今，人们只分离到几种NDH 亲水亚复合体（Berger et al., 1993; Matsuo et al.,
    1998），含疏水亚基的蓝藻 NDH 亚复合体的分离至今还未见报道。本文中，我们分
    离到两个含疏水亚基 NdhA 的 NDH 亚复合体，其中一个亚复合体还含有 NdhH 亚
    基，而另外一个则不含NdhH。尽管NdhH 对集胞蓝藻的生存是必需的（Pieulle et al.,
    2000），但我们的结果表明 NdhH 丢失后并不影响 NDH 在体外的 NADPH 氧化活性
    （图 2，3）。此外，我们未能在分离得到的两个 NDH 亚复合体中检测到 NdhB 及
     81
    
    
    集胞蓝藻含疏水亚基的 NDH 亚复合体的分离
    NdhK 亚基的存在（数据未列出），这表明 NdhB 与 NdhK 对于 NDH 的体外 NADPH
    氧化活性也不是必需的。至于我们分离的 NDH 亚复合体是否还含有其他亚基，还
    需做进一步的分析。本文中所用的分离方法与 Matsuo 等（1998）所采用方法的主
    要差别在于破碎方法和去垢剂不同，这可能是我们能够分离到含疏水亚基的 NDH
    亚复合体的主要原因。
     有文献报道，高等植物叶绿体中NDH与FNR相互结合在一起（QuilesandCuello,
    1998; Guedeney et al., 1996）。然而，至今还没有证据表明蓝藻的 NDH 与 FNR 有相
    互作用。本文中，我们分离到的NDH 亚复合体均不含FNR，这表明，至少在我们
    的实验条件下，NDH 与FNR 并不结合在一起。
     分离到的 NDH 亚复合体可以氧化 NADPH，但不能氧化 NADH，表明其是
    NADPH 专一的。对此两个 NDH 亚复合体的 NADPH 氧化活性分析表明它可以还
    原醌的类似物。然而对PQ 的还原活性却非常低，几乎不能测到（图 4），这可能是
    由于实验系统中 PQ 的水不溶性引起的，但也可能其反应另需其它亚基的存在。
    Matsuo 等（1998）分离到的 NDH 亲水亚复合体对 PQ 的还原活性也非常低，他们
    将其原因归咎于疏水亚复合体的解离。
     线粒体呼吸链复合体I主要由带有NADH结合位点的亲水亚复合体和带有醌结
    合位点的疏水亚复合体组成（Friedrich et al., 1995），推测集胞蓝藻可能也是如此，
    但还没有直接的实验证据。有报道指出，NdhA 可能是醌的结合位点（Friedrich et al.,
    2000）。与 Matsuo 等（1998）分离到的亲水亚复合体相比较，本文中分离的 NDH
    疏水亚复合体对除PQ、维生素K3外的醌类似物都具有较高的还原活性，这可能与
    NdhA 等疏水亚基的存在有关。令人奇怪的是，前人分离的亲水亚复合体尽管自称
    不含NdhA，但仍具有还原醌的能力（Matsuoetal.,1998），这也许表明蓝藻中NdhA
    可能并不是醌的结合位点或者还存在其他的醌结合位点，然而也不排除他们分离的
    亲水亚复合体含有痕量的NdhA 或其他未检测的亚基的可能。大肠杆菌 (E. coli) 中
    含有FMN 和铁硫簇的NuoF 亚基是可能的NADH 结合位点，然而在蓝藻和高等植
    物的线粒体及叶绿体中还未发现同源的亚基（Friedrich et al., 2000）。从蓝藻中分离
    纯化NDH疏水亚复合体以及进而分离纯化完整的NDH复合体对阐明NDH复合体
    的结构与催化机理将具有重要意义。
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    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白质的相互作用
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    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白质的相互作用
    Separation of Hydrophobic NAD(P)H Dehydrogenase
    Subcomplexes from the Cyanobacterium Synechocystis
    PCC6803
     Abstract
     Some efforts have been performed to separate an integrated NA(D)PH dehydrogenase
    (NDH) complex from cyanobacteria. Several hydrophilic subcomplexes of NDH have been
    purified from the cyanobacterium Synechocystis PCC6803. However, no hydrophobic NDH
    subcomplex has ever been separated from cyanobacteria yet. In this paper, two NDH
    subcomplexes were separated from n-dodecyl β-D-maltoside (DM)-treated whole cell
    extracts of Synechocystis PCC6803 by anion exchange chromatography and gel filtration.
    Both subcomplexes contained the hydrophobic subunit NdhA, suggesting that they are
    hydrophobic NDH subcomplexes. One subcomplex contained NdhH but the other one did not.
    The subcomplexes showed NADPH-nitroblue tetrazolium (NBT) oxidoreductase activity and
    could specially oxidize NADPH when several quinone analogues such as ferricyanide,
    2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB), 2,6-dichlorophenol indophenol
    (DCPIP), duroquinone, ubiquinone-0 (UQ-0), etc. were used as electron acceptors.
    Key words: Synechocystis; NAD(P)H dehydrogenase; hydrophobic subunit
     85
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
     （二）集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
     摘 要
     通过非变性凝胶电泳（native-PAGE）和 NADPH-氮蓝四唑（NBT）氧化还原
    酶活性染色，从集胞蓝藻 PCC6803 的细胞粗提物中检测到一个 NADPH 专一的
    NAD(P)H 脱氢酶（NDH）亚复合体，Western 印迹分析表明此亚复合体含 NdhA、
    NdhB 和 NdhH 但不含 NdhK 和 Fd-NADP+氧化还原酶（FNR）。吸收光谱和 77K
    荧光光谱显示此亚复合体中含别藻蓝蛋白和藻蓝蛋白。进一步利用离子交换与凝
    胶过滤层析从细胞粗提物中分离 NDH 时，发现 NADPH-铁氰化钾氧化还原酶和
    NADPH-NBT 氧化还原酶活性总是与藻胆蛋白共分离，无法分开。分离得到的
    NDH 经 native-PAGE 分离和活性染色后，在凝胶上检测到两条 NDH 活性条带，
    Western 印迹分析显示此两条带均含 NDH 的亚基，表明它们都是 NDH 亚复合体。
    此两 NDH 条带在活性染色前均为蓝色，在非变性条件下将其蛋白质溶出后，吸
    收光谱以及 77K 和室温荧光光谱均显示此两条带含有藻胆蛋白。本文的结果表
    明，至少在我们的实验条件下，集胞蓝藻 PCC6803 中 NDH 复合体与藻胆蛋白相
    互结合。
    关键词 集胞蓝藻 PCC6803; NAD(P)H 脱氢酶; 藻胆蛋白; 藻胆体
     引 言
     集胞蓝藻 PCC6803 的基因组中含 12 个 ndh 基因（ndhA~L）（Ellerisiek and
    Steinmüller, 1992; Kaneko et al., 1996; Steinmüller et al., 1992），分别与叶绿体中的
    相应基因同源，编码一个 NAD(P)H 脱氢酶（NDH）复合体（Berger et al., 1991）。
    蓝藻 NDH 也是一个与线粒体复合体同源的多亚基复合体（Berger et al., 1991；
    Berger et al., 1993），既位于类囊体膜也位于质膜上（Berger et al., 1991），在类囊
    体膜上参与呼吸和光合循环电子传递（Mi et al., 1992a, 1992b, 1994, 1995;
     86
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    Sandmann and Malkin, 1983; Yu et al., 1993）。
     有研究表明，NDH 参与蓝藻无机碳转运的能化/活化（Ogawa, 1991a, 1991b,
    1992; Klughammer et al., 1999; Ohkawa et al., 2000）和对盐震激的响应和适应
    （Hibino et al., 1996; Tanaka et al., 1997）。此外，NDH 对蓝藻的生存也是必需的
    （Pieulle et al., 2000）。集胞蓝藻 PCC6803 中，一个 NADPH 专一的 NDH 受光诱
    导并参与光调节的围绕光系统 I（PSI）的循环电子传递和呼吸电子传递（Mi et al.,
    2002）。也有报道指出，集胞蓝藻 PCC6803 的 ndhB-突变体 M55 被“锁定”在状
    态 I，因此推测 NDH 可能还参与蓝藻的状态转换（Schreiber et al., 1995; van Thor et
    al., 2000）。
     NDH 复合体在体外很不稳定，而且蓝藻中还可能存在多种不同功能的 NDH
    复合体（Klughammer et al., 1999; Ohkawa et al., 2000），因此 NDH 复合体的分离、
    纯化以及鉴定一直比较困难。有人已经从集胞蓝藻 PCC6803 分离到一个 376 kD
    的 NDH 亚复合体，此亚复合体含有 NdhH、NdhI、NdhJ 和 NdhK 等亲水亚基，
    但不含 NdhA 和 NdhB（Matsuo et al., 1998）。值得注意的是，此亚复合体中含有
    一个 70 kD的多肽，推测可能是污染的藻胆蛋白。此外，在从 Microcystis aeruginosa
    中分离 NAD(P)H 脱氢酶复合体的过程中，也发现蓝色的藻胆蛋白总是与
    NAD(P)H 脱氢酶共纯化，只有经热丙酮处理后才能除去藻胆素（Viljoen et al.,
    1985）。因此，可以设想 NDH 可能与藻胆蛋白相互作用或者结合。然而至今还没
    有这方面的报道。
     藻胆体是蓝藻的捕光天线复合体，主要由亲水性的藻胆蛋白组成，含有共价
    结合的藻胆素。此外，藻胆体中还有少量不含色素的“连接肽”。藻胆体既可以
    与 PSII 作用也可以与 PSI 作用（Ashby and Mullineaux, 1999; Mullineaux, 1992,
    1994），并将吸收的光能向反应中心 PSII 和 PSI 传递（Mullineaux, 1992），还参与
    激发能在两个光系统间的再分配，即状态转换（Biggins and Bruce, 1989）。然而这
    些光能传递途径的结构基础还不清楚。有研究表明，集胞蓝藻 PCC6803 中，
    Fd-NADP+氧化还原酶（FNR）通过 N 端 9 kD 区域与藻胆体的核心蛋白别藻蓝蛋
    白结合，推测藻胆体可能通过与其结合的 FNR 与 PSI 相互作用，从而形成藻胆体
    -FNR-PSI 超复合体（Schluchter and Bryant, 1992; van Thor et al., 1999, 2000）。然
    而，当在低离子条件下制备集胞蓝藻 PCC6803 细胞粗提物时，发现藻胆体结合的
     87
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
    FNR 脱离（van Thor et al., 2000）。此外，研究还显示 FNR 的 N 端 9 kD 区域缺失
    后并不影响光能向两个光系统的传递，也不影响状态转换（van Thor et al., 1999）。
     本文中，我们从集胞蓝藻 PCC6803 细胞粗提物中通过 native-PAGE 分离到一
    个 NADPH 专一的 NDH 亚复合体，并注意到此亚复合体含藻胆蛋白。为排除蛋
    白质之间相互污染的可能，又用离子交换和凝胶过滤层析进一步分离了细胞粗提
    物，得到两个与藻胆蛋白共分离的 NADPH 专一的 NDH 亚复合体，研究发现此
    两个亚复合体也均含藻胆蛋白。我们的结果表明蓝藻中 NDH 与藻胆蛋白相互作
    用或结合，推测 NDH 可能通过与藻胆体的相互作用参与状态转换。
     材料与方法
    1. 蓝藻的培养
     集胞蓝藻 PCC6803 细胞用 BG-11 培养基（Allen, 1968）（含 5 mmol/LTris-HCl,
    pH 8.0），在 30 ℃，连续光照（60 μEm-2s-1）下通气（含 2% CO2 的空气）培养。
    收集对数后期生长的细胞（A730=0.4~0.6）用于进一步分析。
    2．细胞粗提液的提取
     对数后期生长的细胞（A730=0.4~0.6）于 10 000 g 离心 5 min 收集，细胞以
    10mmol/LTris-HCl（pH8.0）洗涤一次，再次离心，将蓝藻细胞以含 20% (体积比) 甘
    油和0.5 mmol/Lphenylmethylsulfonylfluoride （PMSF）的10mmol/LTris-HCl（pH8.0）
    悬浮，贮存于-80 ℃。细胞悬浮液于冰上融解后，以 Bead-Beater（Biopsec，日本）
    破碎，每次破碎 15 s，间隔 3 min，共破碎 6 次。细胞匀浆于 4 ℃、10 000 g 离心
    10 min 以去除未破碎细胞和残渣。收集上清，将蛋白浓度调整至 25 mg/mL，加入 0.5%
    (w/v) n-dodecyl β-D-maltoside (DM)，冰浴中缓慢震荡 3 h， ℃、140 000 g 离心 60 min。
     4
    离心后收集上清立即用于进一步分析。
    3. NAD(P)H 脱氢酶的纯化
     蓝藻细胞粗提液先经DEAE-52（Whatman）离子交换柱（2.5 cm×15 cm）分离，
    用含0 ~ 0.5 mol/LNaCl 线性梯度的10 mmol/LTris-HCl (pH 8.0), 0.5 mmol/LEDTA，在
    7 ℃洗脱（流速为 3 mL/min，12 mL/管）。得到的活性部分，用 Amicon YM-10 超滤
     88
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    膜浓缩，然后再用两支串联的Hiload 26/60 Superdex 200 prep grade 凝胶过滤柱（2.6 cm
    ×60 cm），于10 ℃，在高压液相层析（FPLC）系统（AmershamPharmacia）上分离
    蛋白质，洗脱液为10 mmol/LTris-HCl (pH 8.0), 0.5 mmol/LEDTA, 150 mmol/LNaCl，
    流速为 1 毫升/min。收集的活性部分以AmiconYM-30 超滤膜浓缩后-80 ℃贮存。
    4. 非变性凝胶电泳及活性染色
     非变性凝胶电泳（native-PAGE）按 Davis 等（1964）的方法用 7.5%聚丙烯酰
    胺凝胶于 4 ℃下进行。活性染色时将凝胶于室温、20 mmol/L Tris-HCl (pH 7.5)
    中缓慢摇动 5 min，然后暗中依次加入含 1 g/L 氮蓝四唑（NBT）及 1 mmol/L
    NADPH 的 20 mmol/L Tris-HCl（pH 7.5），直至紫色条带呈现。
    5. 蛋白洗脱
     将多个非变性凝胶上的活性条带切下后，置于约 1 倍凝胶体积的 10 mmol/L
    Tris-HCl (pH 8.0), 0.1% (体积比) Triton X-100 和 0.1 g/L SDS 中 30 ℃振荡过夜，
    然后 10 000 g 离心10 min，上清液用于SDS-PAGE 以及免疫印迹分析。
     活性染色前非变性凝胶上的蓝色条带切下后，于4 ℃、10 mmol/LTris-HCl (pH
    8.0), 0.5 mmol/L EDTA, 150 mmol/L NaCl 中震荡过夜，然后 10 000 g 离心 10 min，
    上清用于吸收和荧光光谱分析。
    6. SDS-PAGE 和 Western blotting
     SDS-PAGE 按照 Laemmli（1970）的方法，在 12%的聚丙烯酰胺凝胶上进行。
    蛋白经 SDS-PAGE 分离后，立即电转移至硝酸纤维素膜（Bio-Rad）上用于 Western
    印迹分析，杂交信号用碱性磷酸酶底物检测。文中所采用的 NdhA 和 NdhB 亚基
    抗体由 Asada 教授（Department of Biotechnology, Faculty of Engineering, Fukuyama
    University）、NdhH 抗体由 Ogawa 教授（Bioscience Center, Nagoya University）、FNR
    抗体由 Lda 博士（Research Institute for Food Science, Kyoto University）分别惠赠。
    7. 其它蛋白分析方法
     参见《分子克隆实验指南》（第二版，1989）。蛋白浓度测定按 Bradford（1976）
    的方法进行。
     89
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
    8. NADPH 氧化活性分析
     NADPH 氧化活性通过测量人工电子受体的还原或底物 NADPH 的氧化来分
    析（Matsuo et al., 1998），采用的反应缓冲液为 10 mmol/L Tris-HCl (pH 8.0), 0.5
    mmol/L EDTA。NADPH 的氧化用分光光度计（岛津 UV-3000）记录 340 nm {ε
    =6.22/ [(mmol?L)-1? cm] }光吸收的下降速率来测量，当铁氰化钾为电子受体时记录
    420 nm {ε=1.03/ [(mmol?L)-1? cm] }处光吸收下降速率来测量。测量温度为室温（约
     kD A B C D
     669
     440
     232 a-Band
     140
     b-Band
     67 c-Band
     Fig.1 Native-PAGE of crude extract of Synechocystis PCC6803 and comparison
     of the gels before (A) and after staining. After solubilization with 0.5% (w/v)
     DM at 25 mg protein mL-1, native-PAGE was carried out with 7.5%
     polyacrylamide gel according to Davis [1964] at 4°C and then the gel was
     subjected coommassie bright blue staining or activity staining. The enzymatic
     bands were detected by incubating the gel in 20mmol/L Tris-HCl (pH 7.5) for
     five minutes and then in 20 mmol/L Tris-HCl (pH 7.5) supplement with 0.1%
     (w/v) NBT and 1 mmol/L NADPH (B) or NADH (C) successively at room
     temperature in darkness until formazan bands emerged. Proteins in the gel were
     stained with coommassie bight blue (D) as described in Molecular Cloning (A
     laboratory manual, 2nd ed) (Sambrook et al., 1989).
     90
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    25℃）。1 个酶活单位为每分钟氧化 1 μmol/L NADPH 的酶量。
    9. 吸收和荧光光谱
     吸收光谱用岛津分光光度计 UV-3000（日本）于室温测定。低温荧光以一 44W
    的荧光光度计（实验室自制）于 77K 测定，于 580 nm 激发。室温荧光光谱用荧
    光分光光度计（970 CRT, Ling guang, 上海）于室温检测，激发光为 580nm。
     A B C
     kD
     66
     NdhB
     45 NdhA NdhH
     36
     29
     24
     20
     14
     Fig.2 Western blotting analysis of the blue NADPH-NBT
     oxidoreductase band (a-Band) in native-gel. a-Band in native-gel was
     excised from the gel, and then incubated in 10 mmol/L Tris-HCl (pH
     8.0), 0.1% Triton X-100 and 1% SDS at 30 ℃ overnight following with
     centrifugation at 10 000 g for 10 min. Then the proteins extracted were
     subjected to gel electrophoresis and western blotting with antibodies
     against NdhA, NdhB and NdhH as described in EXPERIMENTAL
     PROCEDURES.
     91
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
     结 果
     为分析 DM 增溶的集胞蓝藻 PCC6803 细胞粗提液中 NDH 的分子性质，我们
    对其进行了 native-PAGE 分离以及活性染色分析。native-PAGE 分离后，在凝胶上
    观察到两条主要的蓝色条带，分子量分别为 300 kD（a-Band）和 100 kD（Fig. 1A）。
    活性染色后，发现 a-Band 具有很高的 NADPH-NBT 氧化还原酶活性（Fig. 1A），
    此外还检测到另外两条活性条带，分子量分别为 90 kD（b-Band）和 70 kD
    （c-Band），其中 70 kD 的蛋白带既可以氧化 NADPH 也可以氧化 NADH，而另外
    两条活性带均对 NADPH 专一（Fig. 1）。此非变性凝胶经考马斯亮蓝染色后，在
    a-Band 出检测到一条单一的条带。以上结果显示，a-Band 既含有蓝色的组分也具
    有 NDH 的活性。蓝藻中主要的蓝色色素蛋白质为藻胆蛋白，因此推测此带中含
    藻胆蛋白并可能与 NDH 相互结合。因而我们对 a-Band 进行了进一步的分析。
     a-Band 中的蛋白质经割带溶出后，SDS-PAGE 和 Western 印迹分析结果表明，
    a-Band 含 NdhA、NdhB 和 NdhH（Fig. 2），但没有检测到 NdhK 和 FNR（数据未
    列出）。结合前面活性染色的结果，可以得出结论，即此带为 NDH 亚复合体。
     此外，为确定 a-Band 中到底存在何种色素蛋白质，将 a-Band 中的蛋白质在
    非变性条件下经割带溶出后，测定了其吸收和 77K 荧光光谱。Fig. 3A 为 a-Band
    蛋白的室温吸收光谱，可以发现在大约 620 nm 处有吸收峰，这与提纯的藻蓝蛋
    白（PC）或藻胆体的吸收峰（MacColl and Guard-Friar, 1987）一致，提示 a-Band
    中可能确实存在藻胆蛋白。为进一步证实这种可能性，我们测定了其 77K 荧光光
    谱。当以 580 nm 的激发光激发时，发现在 660 nm 处有一主要的荧光发射峰，这
    与别藻蓝蛋白的荧光峰是一致的（MacColl and Guard-Friar, 1987）；此外在 650 nm
    处还有一比较小的荧光发射峰，与藻蓝蛋白的荧光峰（MacColl and Guard-Friar,
    1987）一致（Fig. 3B），这表明 a-Band 中确实含有别藻蓝蛋白和藻蓝蛋白。研究
    表明，PC/APC 异六聚体的室温吸收光谱与 PC 一致，而在 77K 下，由于 PC 的激
    发能向 APC 传递从而使得 PC 的荧光减弱，其荧光光谱表现为典型的 APC 荧光
    光谱（Hu et al., 1999），这些结果与本文中观察到的一致，因此推测 a-Band 中的
    PC 和 APC 可能以异六聚体的形式存在。
     然而，上述结果并不能排除在 native-PAGE 过程中，a-Band 受到其它蛋白质
     92
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    污染的可能，因此，我们对蓝藻的 NDH 进行了分离纯化，以期尽量减少蛋白质
    间相互污染的可能。
     0.4
     A
     0.3
     ce
     an
     rb 0.2
     so
     Ab
     0.1
     0
     400 450 500 550 600 650 700
     Wavelength (nm)
     B
     eldyi
     escence
     uorfl
     veti
     Rela
     600 620 640 660 680 700 720 740
     Wavelength (nm)
    Fig.3 Absorption spectra (A) and 77K fluorescence emission spectra (B) of the
    blue NADPH-NBT oxidoreductase band (a-Band) before staining. The proteins
    in a-Band were eluted from the non-denaturing gel before staining and soaked in
    10 mmol/L Tris-HCl (pH 8.0), 0.5 mmol/L EDTA, 150 mmol/L NaCl overnight
    at 4 ℃. Then the suspension was centrifuged at 10 000 g for 5 min and the
    supernatant was subjected to absorption and fluorescence emission spectra
    analysis. Excitation was at 580 nm.
     93
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
     2.5 1.0 4
     1.2
     A )
     -1
     2.0 0.8 mL
     1.0 ) 3 -1
     /Llo min
     1.5 .8 0.6 DPH
     0 0 (mniot NA
     62 A28 .6 2
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     1.0 0.4 ncentra olmm
     co -1
     .4 01(
     NaCl 1
     0.5 0.2
     .2 tyviticA
     0 0.0 0 0
     0 20 40 60 80 100 120
     Fractionmumber
     3 1.5
     B )
     -1
     mL
     -1
     2 1.0 min
     620
     A
     or
     280 NADPHl
     A
     1 0.5 mo
     μ(
     ity
     Activ
     0 0
     50 60 70 80 90 100
     Fraction number
     94
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    Fraction number 63 64 65 66 67 68 69 70 71
     KD
     669
     440 C
     232
     140
     67
     669
     440 D
     Band 1
     232 Band 2
     140
     Band 3
     67
    Fig. 4 Purification of NAD(P)H dehydrogenase. After solubilization with 0.5%
    (w/v) DM at 25 mg protein mL-1, the crude extract of Synechocystis PCC6803 was
    applied to a DEAE-52 ion exchange column (A) The enzymatic fractions obtained
    by NaCl gradient (0.14-0.27 mol/L) elution were concentrated and applied to two
    cascaded attached Hiload 26/60 Superdex 200 prep grade column equilibrated with
    10mmol/L Tris-HCl (pH 8.0), 0.5 mmol/L EDTA, 150 mmol/L NaCl (B). Proteins
    were collected as 3 mL/fraction. During the purification processes,
    NADPH-ferrecyanide oxidoreductase activity (■)absorbance at 280 nm (○) and
    620 nm (△) were inspected. After separation by Superdex 200, the enzymatic
    63-71 fractions were analyzed by native-PAGE with 15μL per lane followed with
    coommassie bight blue (C) or NADPH-NBT oxidoreductase activity (D) staining.
     95
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
     A
     sorbanceba
     vetila
     Re
     400 450 500 550 600 650 700
     wavelength (nm)
     B
     yield
     fluorescence
     tive
     Rala
     600 620 640 660 680 700 720 740
     wavelength (nm)
     Fig.5 Absorption spectra (A) at room temperature and 77K fluorescence
     emission spectra (B) of fraction 67 (─) and 78 (…) separated by Superdex
     200. Absorption spectra were normalized at 620 nm. Emission spectra were
     recorded with excitation at 580 nm and normalized at 660 nm.
     集胞蓝藻 PCC6803 的细胞粗提液经 0.5%（w/v）DM 增溶后，先通过离子交
    换层析进行分离。离子交换层析后，在 0.14~0.27 mol/L NaCl（32~60 管，在 0.20
    mol/L NaCl 处活性最高）分离到一个 NADPH-铁氰化钾氧化还原酶活性峰（Fig.
    4A）。同时为了监测藻胆蛋白的分离，测定了各部分 620 nm 处的光吸收（Fig. 4A），
    结果显示分离到两个距离很近的藻胆蛋白峰，其中第一个藻胆蛋白峰与 NADPH-
    铁氰化钾氧化还原酶活性峰重合（Fig. 4A）。
     96
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
     kD BS AS Band1 Band2
     669 kD
     440
     66
     232 Band 1 45
     Band 2
     36
     140 C 29
     A
     Band 4 24
     Band 3 20
     67
     14
     Fig. 6 Analysis of the purified blue enzymatic
     1 2 1 2 peak. Proteins in fraction 66~69 were collected
     kD Band Band Band Band and concentrated with an Amicon YM-30
     66 membrane and then were subjected to
     45
     36 native-PAGE and NADPH-NBT oxidoreductase
     29 staining (A). A gives the native gel before (BS)
    B 24 and after (AS) activity staining. The proteins in
     20 blue enzymatic band 1 and band 2 were eluted
     from the native-gel by incubated in 10 mmol/L
     14 Tris-HCl (pH8.0), 0.1% (v/v) Triton X-100, 1%
     (w/v) SDS at 30 ℃ overnight and analyzed by
     Ndh A Ndh H Western blotting with antibodies against NdhA
     and NdhH (B) after separation by SDS-PAGE
     (C). Silver staining was performed as Smabrook
     et al. (1989).
     97
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
     离子交换层析得到的 NADPH-铁氰化钾氧化还原酶活性峰（36~56 管）收集、
    浓缩后，进一步以两根串联的 Superdex 200 凝胶过滤层析柱进行分离。分别分离
    到两个主要的酶活性峰和两个藻胆蛋白峰（Fig. 4B），有趣的是发现仍有一个酶
    活性峰与一个较小的藻胆蛋白峰重合（Fig. 4B）。而另外一个主要的藻胆蛋白峰
    则不具 NADPH-铁氰化钾氧化还原酶活性。此酶活性峰经 native-PAGE 分离以及
    活性染色后，发现有三条 NDH 酶活性条带（Fig. 4B, C, D）。我们还分析比较了
    所分离的两个藻胆蛋白峰的吸收和荧光光谱，结果表明它们均含有 PC 和 APC，
    这两个藻胆蛋白峰的吸收和荧光光谱性质也基本完全一致，表明其色素蛋白质组
    成没有明显差异。
     上述具酶活性的藻胆蛋白峰经收集、浓缩并以 native-PAGE 分离后，我们观
    察到三条蓝色条带（Fig. 6A），分子量分别为 240 kD（Band 1）、200 kD（Band 2）
    和 100 kD（Band 3）。活性染色后，发现 Band 1 和 Band 2 均有很高的 NADPH-NBT
    氧化还原酶活性，但不具有 NADH-NBT 氧化还原酶活性，这表明其对 NADPH 专
    一（数据未列出）。此外还观察到一条 120 kD 的活性带（Band 4），但不含色素蛋
    白质（Fig. 6A）。由于Band 3 和Band 4 分子量较小，活性较低，而且其量随Band 1
    和Band 2 的变化而变化 (Fig. 4D)，因此我们推测它可能是从Band 1 或者Band 2 降
    解下来的，然而也不能排除其是其它蛋白质的可能。
     Band 1 和 Band 2 中蛋白质经割带溶出后，以 SDS-PAGE 分离，结果发现分别含
    14 和 13 条蛋白质带（Fig. 6C），其中 Band 2 中大部分蛋白质的分子量均与 Band I 中
    的蛋白质相同，表明这两条带相似，而且 Band 2 有可能是从 Band 1 降解下来的。
    Western 印迹分析显示，Band 1 含 NdhA 和 NdhH，Band 2 含 NdhA 但不含 NdhH，这
    表明Band1 和Band2 都是NDH 亚复合体（Fig. 6B）。然而，我们的结果也显示Band
    1 和 Band 2 都不含 NdhB 和 FNR（数据未列出）。鉴于前面我们在细胞粗提液中分离
    到的300kD 的NDH 亚复合体含NdhA、NdhH 和NdhB（Fig. 2），因此推测NdhB 和
    NdhH 可能在蛋白质分离过程中分别从 Band 1 和 Band 2 以及 Band 2 上脱离了。此外，
    在21 kD 和18kD 处还分别发现两条很浓的蛋白质带，根据其分子量推测可能为藻胆
    蛋白（PC 或APC）的不同亚基（Ajlani and Vernotte, 1998; MacColl and Guard-Friar,
    1987）。
     98
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
     0.4
     A Fig.7 A: Absorption spectra at
    )A( 0.3
     room temperature (A) and
     oni fluorescence emission spectra at
     sorptbA 0.2 room temperature (B) and 77K
     (C) of Band 1 (─) and Band 2
     0.1
     (…). The proteins in Band 1 and
     Band 2 were eluted from the
     0
     400 450 500 550 600 650 700
     Wavelength (nm) non-denaturing gel before
     staining and soaked in 10 mmol/L
     B Tris-HCl (pH 8.0), 0.5 mmol/L
     e)iv
     atle(r EDTA, 150 mmol/L NaCl
     overnight at 4 ℃ . Emission
     nce
     spectra were recorded with
     resceo
     Flu excitation at 580 nm and
     normalized at 660 nm (B) or 650
     nm (C).
     600 610 620 630 640 650 660 670 680 690
     Wavelength (nm)
     C
     d
     yiel
     e
     ncecs
     fluore
     ve
     latieR
     600 620 640 660 680 700 720 740
     Wavelength (nm)
     99
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
     活性染色前，将非变性凝胶上的Band 1 和Band 2 在非变性条件下割带溶出后，
    对其吸收和室温及77K 荧光光谱进行了分析（Fig. 7），结果显示这两条带均具有典型
    的藻胆蛋白吸收和荧光光谱（Hu et al., 1999; MacColl and Guard-Friar, 1987），这表
    明这两条带中均含有藻胆蛋白。我们的结果同时也显示这两条带中既存在藻蓝蛋白也
    存在别藻蓝蛋白（Fig. 7）。
     综上所述，我们的结果表明集胞蓝藻PCC6803 中，NDH 亚复合体与藻胆蛋白（PC
    和 APC）共分离、共纯化，因此可以预计 NDH 结合藻胆蛋白，推测 NDH 可能也与
    藻胆体相互作用。
     讨 论
     我们的结果首次证明集胞蓝藻PCC6803中NADPH专一的NDH亚复合体与藻胆
    蛋白结合，而且这种结合并不依赖FNR。在与藻胆蛋白结合的NDH 亚复合体中，发
    现了疏水亚基 NdhA 和 NdhB，因此此亚复合体可能为疏水亚复合体。本文的结果也
    表明，当不存在 NdhB 和 NdhH 时，NDH 亚复合体仍可与藻胆蛋白结合，因此推测
    这两个亚基对此结合作用不是必需的。此外，NdhA 在所有分离的复合体中都存在，
    因此推测它可能参与 NDH 与藻胆蛋白的结合，但还不确定。在线粒体复合体 I 和细
    菌NADH 脱氢酶中，NdhA 是泛醌（UQ）的结合位点（Friedrich et al., 1995），推测
    在蓝藻中可能也是如此。因此，我们猜想蓝藻中 NDH 与藻胆蛋白的结合可能也与质
    醌（PQ）的结合或还原有关，然而还需要进一步证实。不过，他人在分离的NDH 亲
    水亚复合体中并没有检测到 NdhA，但仍具有还原醌类似物的活性（Matsuo et al.,
    1998），这表明 NDH 可能还有其它的醌结合位点。至于 NDH 与藻胆蛋白结合的具体
    机制以及哪些NDH 亚基参与了这种结合作用仍需进一步实验阐明。
     有报道指出，FNR 通过其 N 末端与藻胆体的核心蛋白质别藻蓝蛋白结合
    （SchluchterandBryant,1992; vanThor et al., 1999, 2000）。在高等植物叶绿体中，也有
    报道指出FNR 与NDH 结合（Quiles and Cuello, 1998; Guedeney et al., 1996）。然而，
    至今还没有关于蓝藻NDH 与FNR 结合的报道。本文中分离的NDH 亚复合体中并没
    有 FNR，这表明至少在我们的实验条件下，蓝藻 NDH 并不与 FNR 结合，而且 FNR
    也不参与NDH与藻胆蛋白的结合。但是也不排除本文采用的处理条件对FNR与NDH
     100
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    或藻胆蛋白的结合有不利影响的可能。此外在高等植物中FNR 主要与NDH 亲水亚基
    结合（QuilesandCuello,1998;Guedeneyetal.,1996），而本文中分离的NDH 亚复合体
    主要包含疏水亚基，这也可能是检测不到FNR 的原因。
     蓝藻 NDH 复合体的分离纯化一直比较困难。主要的原因是，在分离纯化过程
    中常常得到几个不同的、具有酶活性的亚复合体。此外，也有报道指出，蓝藻中
    可能存在不同功能形式的 NDH（Klughammer et al., 1999; Ohkawa et al., 1998，
    2000）。至今为止，人们还没有从蓝藻中分离到完整的 NDH 复合体。本文分离到
    三个结合藻胆蛋白的 NDH 亚复合体，其分子量和亚基组成各不相同，这也再次
    表明蓝藻的 NDH 复合体在体外确实很不稳定。
     PC 和 APC 是组成藻胆体的主要的藻胆蛋白（MacColl and Guard-Friar, 1987）。
    本文中分离的三个 NDH 亚复合体均结合 PC 和 APC，因此可以推测 NDH 在集胞
    蓝藻 PCC6803 体内与藻胆体结合或相互作用。至今，藻胆体与类囊体膜以及反应
    中心结合/相互作用的机制尚不清楚。研究表明，当缺乏反应中心时，藻胆体在体
    内仍可结合类囊体膜，这表明藻胆体与类囊体膜的结合并不依赖反应中心的存在
    （Yu et al., 1999）。有人推测 ApcE 的“PB-loop”可能对此种结合作用是必需的
    （Redlinger and Gantt, 1982; Capuano et al., 1991）。然而，当缺失“PB-loop”后，
    藻胆体的功能并没有收到明显的影响（Ajlani and Vernotte, 1998）。此外，FNR 也
    不参与藻胆体与类囊体膜或反应中心的相互作用（van Thor et al., 1999，2000）。
    根据本文的结果，如果 NDH 确实与藻胆体有相互作用的话，那么 NDH 很有可能
    参与藻胆体与类囊体膜或反应中心的相互作用。就此我们设想NDH 可能作为“锚”
    将藻胆体固定在类囊体膜或反应中心上。鉴于类囊体膜上的NDH 复合体介导PSI 循
    环电子传递，在空间上比较靠近PSI（Albertsson,2001;Kubickietal.,1996），因此NDH
    可能只参与藻胆体与PSI 的相互作用。
     激发能在两个光系统之间的再分配称为状态转换。蓝藻具有明显的状态转换现
    象。有研究表明，集胞蓝藻PCC6803 中，NDH 介导的呼吸和PSI 循环电子传递所导
    致的PQ 的暗还原会诱导蓝藻在暗中向状态2 转换（Schreiber et al., 1995）。缺失ndhB
    的集胞蓝藻 PCC6803 突变体 M55 的状态转换受到完全抑制，并被锁定在状态 1
    （Schreiber et al., 1995），有人认为这是由于NDH 介导的循环电子传递缺失后PQ 库
    暗中过度氧化导致的（Schreiber et al., 1995; van Thor et al., 2000）。然而，蓝藻中还存
     101
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
    在其它多条不依赖NDH 的循环电子传递途径，如依赖FNR（Jeanjean et al., 1999; van
    Thor et al., 2000）、PsaE（Yu et al., 1993）和 flavindoxin（Hagemann et al.，1999）等的
    途径。在光抑制（Thomasetal.,2001）和盐胁迫（Jeanjean et al., 1998）等条件下，M55
    中其它不依赖 NDH 的循环电子传递可被诱导从而使得蓝藻可以适应这些环境胁迫。
    此外，适应盐胁迫的 M55 中，其循环和呼吸电子传递均增强，M55 也恢复了在大气
    CO2下光合自养生长的能力（Jeanjean et al., 1998）。因此，M55 中仅 NDH 介导的循环
    电子传递受到抑制应该不会对状态转换产生这么大的影响，可能还存在其它的机制。
    有报道指出，集胞蓝藻PCC6803 中围绕PSI 的循环电子传递受盐胁迫诱导与FNR 通
    过其N 末端与类囊体膜的结合有关（van Thor et al., 2000），然而，将FNR 的N 末端
    缺失后对蓝藻的状态转换没有明显的影响（vanThor et al., 1999）。这也提示，ndhB 基
    因缺失对蓝藻状态转换的严重影响可能并不完全是 NDH 介导的循环电子传递受抑制
    的结果。因此我们设想除了影响PQ 库的氧化还原状态外，NDH 还可能通过与藻胆体
    相互作用而参与状态转换。有人认为，M55 中NdhB 的缺失可能会导致所有NDH 复
    合体解体（Ohkawaetal.,2000），那么NDH 的缺失有可能导致藻胆体不能与PSI 作用，
    从而使得M55 被锁定在状态1。
     然而，由于本文中分离的所有与藻胆蛋白结合的NDH 都含醌结合亚基NdhA，因
    而还存在这样一种可能，即NDH 介导的电子传递影响PQ 的氧化还原状态，而PQ 的
    氧化还原状态反过来影响 NDH 或藻胆体的构象，从而调节藻胆体与反应中心的相互
    作用。
     NDH 和藻胆体广泛分布于各种蓝藻中，在其它蓝藻中 NDH 是否也与藻胆蛋白结
    合还需要进一步研究。阐明 NDH 与藻胆蛋白结合的机制及其功能对研究蓝藻光合机
    构运转机理将具有重要意义。
     附：应用酵母双杂交系统检测NDH 亚基与藻胆蛋白的相互作用
     -----用于酵母双杂交检测的质粒的构建
     前面的结果已经表明，集胞蓝藻PCC6803 中NDH 在体外与藻胆蛋白结合。此结
    论主要是建立在对 NDH 复合体的体外生化分析基础上的，仍不能完全排除蛋白质之
     102
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    间相互污染的可能，因此还需要进一步实验证实。此外，NDH 是否在体内也与藻胆
    蛋白结合、参与这种结合作用的蛋白质有哪些？这些问题也都需要进一步实验分析。
    酵母双杂交系统是近些年建立起来的在体内研究蛋白质之间相互作用的一种常用
    的遗传学方法（Fields and Song, 1989）。双杂交系统灵敏度高，也可用来检测蛋白质
    之间的弱相互作用和瞬时相互作用。此外，与体外方法相比，双杂交系统是在体内进
    行的，蛋白质不易受外界条件的影响，仍保持其天然构象，因而更能反映蛋白质在体
     (A) (B)
    Fig. 8 MATCHMAKER LexA Two-Hybrid System cloning vectors. Panel A. pLexA
    (pEG202 in reference 1) is used to generate fusions of the DNA-BD (the 202-residue LexA
    protein) with a target (or bait) protein. In pLexA, fusion protein expression is controlled by
    the strong yeast ADH1 promoter. (Restriction sites shown in bold are unique.) Panel B.
    pB42AD (pJG4-5 in reference 1) expresses cDNAs or other coding sequences inserted into
    the unique EcoR I and Xho I sites as translational fusions to a cassette consisting of the
    SV40 nuclear localization sequence, the 88-residue acidic activator B42 (the AD), and the
    hemagglutinin (HA) epitope tag. In pB42AD, fusion protein expression is under the control
    of the GAL1 inducible promoter, so transcription levels are very low in the presence of
    glucose and high in the presence of galactose (raffinose is added as an aid to growth). Both
    plasmids may be propagated and selected for in E. coli and yeast. For selection in yeast,
    pLexA contains the HIS3 marker and pB42AD contains the TRP1 marker.
     103
    
    
    第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
    内的相互作用。
     因此，拟采用酵母双杂交系统进一步检测NDH 各亚基（NdhA~K）与几种藻胆蛋
    白（ApcA，CpcA，ApcE）之间的相互作用。首先构建了用于酵母双杂交检测的 28
    个质粒。
     集胞蓝藻 PCC6803 细胞用 BG-11 培养基（Allen, 1968）（含 5mmol/LTris-HCl,
    pH 8.0），在 30 ℃，连续光照（60 μEm-2s-1）下通气（含 2% CO2 的空气）培养。
    收集对数后期生长的细胞（A730=0.4~0.6）用于进一步分析。蓝藻总 DNA 的提取按
    照 Cai 和 Wolk（1990）的方法进行。根据集胞蓝藻 PCC6803 基因组中相应各基
    因序列，设计引物并同时加入合适的酶切位点，经 PCR 扩增，相应限制性酶切后
    分别插入载体：pLexA（pEG202）和 pB42AD（pJG4-5）（Fig. 8）的相应酶切位
    点，并进行测序鉴定。构建好的质粒用 Qiagen Plasmid Midi Kit 抽提后，-20℃贮
    存以用于酵母双杂交实验。
    所用的引物如下：表 1
     基因 酶切位点 配对部分 内切酶
     ndh A 5’端：CG GAA TTC ATG ACT TCA GGC ATT GAT CTT CAG EcoR I
     3’端：CCC CTC GAG CTAACC GCC AAA GGC CAT GGG AAA Xho I
     ndh B 5’端：CCC CTC GAG ATG GAC TTT TCT AGT AAC GTT GCA Xho I
     3’端：CCC CTC GAG CTA GGG TAAATC ATG GGAAAT GGC Xho I
     ndh C 5’端：CG GAA TTC GTG TTT GTT TTAACC GGT TAC GAA EcoR I
     3’端：CCC CTC GAG CTA GGA CCA CTC CAG AGC CCC TTT Xho I
     ndh D1 5’端：CG GAA TTC ATG AAC ACT TTT CCC TGG CTG ACC EcoR I
     3’端：CCC CTC GAG TTAAAAACC AAT GGT GGG GGG CCG Xho I
     ndh E 5’端：CG GAA TTC ATG CAT TTG CAG CTT CAA TAT TGT EcoR I
     3’端：CCC CTC GAG CTA CCA CTT CAG GAG ATT GAA TTG Xho I
     ndh F1 5’端：CG GAA TTC ATG GAA TTA CTC TAT CAA TTA GCC EcoR I
     3’端：CCC CTC GAG CTA GGT GAG GCT AAAAAC AAT TAC Xho I
     ndh G 5’端：CG GAA TTC GTG AAT TTA GCT GAA GGT GTT CAG EcoR I
     3’端：CCC CTC GAG CTA TTT GGAAGC GGA GGT TAA CTC Xho I
     104
    
    
    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
     ndh H 5’端：CCC CTC GAG ATG CCG CCT TGC CTC CCC AAT GGC Xho I
     3’端：CCC CTC GAG CTA GCG GTC CAC CGA TCC CAT GAT Xho I
     ndh I 5’端：CCC CTC GAG ATG TTT AAC AAC ATT CTC AAA CAG Xho I
     3’端：CCC CTC GAG CTA TTC TGC TTT CAC CAAATC TTC Xho I
     ndh J 5’端：CG GAA TTC GTG GCT GAG GAA GTG AAC TCC CCC EcoR I
     3’端：CCC CTC GAG CTAATA GGC ATC CTG GAG TTC GTA Xho I
     ndh K 5’端：CG GAA TTC ATG AGT CCC AAC CCT GCT AAC CCC EcoR I
     3’端：CCC CTC GAG TCA GCC ACG GTT TAA TTG CTC CTT Xho I
     apc A 5’端：CG GAA TTC ATG AGT ATC GTC ACG AAA TCAATC EcoR I
     3’端：CCC CTC GAG CTA GCT CAT TTT TCC GAT AAC GAA Xho I
     cpc A 5’端：CG GAA TTC ATG AAAACC CCT TTAACT GAA GCC EcoR I
     3’端：CCC CTC GAG CTA GCT CAG AGC ATT GAT GGC GTA Xho I
     apc E 5’端：CCC CTC GAG ATG AGT GTT AAG GCAAGT GGT GGC Xho I
     3’端：CCC CTC GAG CTAACC GCC CAC TTT TAC TAC TGG Xho I
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    集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
    NAD(P)H Dehydrogenase Complex Associates with
    Phycobiliproteins in the Cyanobacterium Synechocystis
    PCC6803
     Abstract
     An NADPH-specific subcomplex of NAD(P)H dehydrogenase, containing NdhA, NdhB and
    NdhH but lacking NdhK in the crude extract of Synechocystis PCC6803, was separated by
    native polyacrylamide gel electrophoresis (native-PAGE) and was identified. The absorption
    spectra and 77K fluorescence emission spectra of this subcomplex suggested the presence of
    allophycocyanin and phycocyanin in this subcomplex, but ferredoxin-NADP+ oxidoreductase
    was absent. During the purification of NAD(P)H dehydrogenase by anion exchange
    chromatography and gel filtration, it was determined that NADPH-ferricyanide oxidoreductase
    and NADPH-nitroblue tetrazolium oxidoreductase activity always co-purified with
    phycobiliproteins. After separation by native-PAGE, two subcomplexes of NAD(P)H
    dehydrogenase each containing phycobiliproteins were detected in the purified enzymatic peak
    indicated by their absorption and fluorescence spectra at room temperature and 77K. The
    formation of an NAD(P)H dehydrogenase/phycobilisome supercomplex has been suggested
    and the role of NAD(P)H dehydrogenase in state transition of cyanobacteria via the interaction
    with phycobilisomes is discussed.
    Key words: Synechocystis PCC6803; NAD(P)H dehydrogenase; phycobiliprotein;
     phycobilisome