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
邓勇,叶济宇,米华玲 (2003)集胞蓝藻 PCC6803 含疏水亚基的 NAD(P)H 脱氢酶亚复合体的分离. 生物
化学与生物物理学报,印刷中
Alpes, I., Scherer, S. and B?ger, P. (1989) The respiratory NADH dehydrogenase of the cyanobacterium Anabaena
variabilis: purification and characterization. Biochim. Biophys. Acta 973, 41-46
Anderson, S.L. and McIntosh, L. (1991) Partial conservation of the 5' ndhE-psaC-ndhD 3' gene arrangement of
chloroplasts in the cyanobacterium Synechocystis sp. PCC6803: implications for NDH-D function in
cyanobacteria and chloroplasts. Plant Mol. Biol. 16, 487-499
Aoki, M. and Katoh, S. (1983) Size of the plastoquinone pool functioning in photosynthetic and respiratory
electron transport of Synechococcus sp. Plant Cell Physiol. 24, 1379-1386
Asada, K., Heber, U. and Schreiber, U. (1993) Electron flow to the intersystem chain from stromal components and cyclic
electron flow in maize chloroplasts, as detected in intact leaves by monitoring redox change of P700 and chlorophyll
fluorescence. Plant Cell Physiol. 34, 39-50
Badger MR and Price GD. (1992) The carbon dioxide concentrating mechanism in cyanobacteria and microalgae.
Physiol. Plantrum. 84, 606-615
Bendall, D.S. and Manasse, R.S. (1995) Cyclic photophosphorylation and electron transport. Biochim. Biophys.
Acta. 1229, 23-38
Bennoun, P. (1982) Evidence for a respiratory chain in the chloroplast. Proc. Natl. Acad. Sci. USA 79, 4352-4356.
Bennoun, P. (1983) Effects of mutations and of ionophore on chlororespiration in Chlamydomonas reinhardtii.
FEBS Lett. 165, 363-365
Berger, S., Ellersiek, U. and Steinmuller, K. (1991) Cyanobacteria contain a mitochondrial complex I-Homologoues
NADH-dehydrogenase. FEBS Lett. 286, 129-132
Berger, S., Ellersiek, U., Kinzelt, D. and Steinmuller, K. (1993a) Immunopurification of a subcomplex of the
NAD(P)H-plastoquinone-oxidoreductase from the cyanobacterium Synechocystis sp. PCC6803. FEBS Lett. 326,
246-250
Berger, S., Ellersiek, U., Westhoff, P. and Steinmuller, K. (1993b) Studies on the expression of NDH-H, a subunit
of the NAD(P)H-plastoquinone-oxidoreductase of higher-plant chloroplasts. Planta 190, 25-31
Biggins, J. (1969) Respiration and photosynthesis in energy transducing membranes of cyanobacteria. J. Bacteriol.
99, 570-575
36
中国科学院上海植物生理生态研究所博士学位论文
Bonfil, D.J., Ronen-Tarazi, M., Sültemeyer, D., Lieman-Hurwitz, J., Schatz, D. and Kaplan, A. 1998. A putative
HCO3 transporter in the cyanobacterium Synechococcus PCC7942. FEBS Lett. 430, 236-240
-
Burrows, P.A., Sazanov, L.A., Svab, Z., Maliga, P. and Nixon, P.J. (1998) Identification of a functional respiratory
complex in chloroplasts through analysis of tobacco mutants containing disrupted plastid ndh genes. EMBO J.
17, 868-876
Canaani, O., Schuster, G., Ohad, I. (1989) Photoinhibition in Chlamydomonas reinhardtii: Effect of state transition,
intersystem energy distribution and photosystem cyclic electron flow. Photosynth. Res. 20, 129-146
Canaani, O. (1990) The role of cyclic electron flow around photosystem I and excitation energy distribution
between the photosystems upon acclimation to high ionic strength in Dunaliela salina. Photochem. Photobiol.
52, 591-599
Carol, P., Stevenson, D., Bisanz, C., Breitenbach, J., Sandmann, G., Mache, R., Coupland, G. and Kuntz, M. (1999)
Mutations in the Arabidopsis gene IMMUTANS cause a variegated phenotype by inactivating a chloroplast
terminal oxidase associated with phytoene desaturation. Plant Cell 11, 57-68
Carol, P. and Kuntz, M. (2001) A plastid terminal oxidase comes to light: implications for carotenoid biosynthesis
and chlororespiration. Trends Plant Sci. 6, 31-36
Casano, L.M., Martín, M., Zapata, J.M. and Sabater, B. (1999) Leaf age- and paraquat concentration-dependent
effects on the levels of enzymes protecting against photooxidative stress. Plant Science, 149, 13-22
Casano, L.M., Zapata, J.M., Martin, M., and Sabater, B. (2000) Chlororespiration and poising of cyclic electron
transport. Plastoquinone as electron transporter between thylakoid NADH dehydrogenase and peroxidase. J.
Biol. Chem. 275, 942-948
Corneille, S., Cournac, L., Guedeney, G., Havaux, M. and Peltier, G. (1998) Reduction of the plastoquinone pool
by exogenous NADH and NADPH in higher plant chloroplasts. Characterization of a
NAD(P)H-plastoquinone oxidoreductase activity. Biochim. Biophys. Acta 1363, 59-69
Cournac, L., Redding, K., Ravenel, J., Rumeau, D., Josse, E. M., Kuntz, M., and Peltier, G. (2000) Flexibility in
photosynthetic electron transport: a newly identified chloroplast oxidase involved in chlororespiration. J. Biol.
Chem. 275, 17256-17262
Cuello J., Quiles, M.J., Albacete, M.E. and Sabarer, B. (1995) Properties of a large complex with NADH
dehydrogenase activity from barley thylakoids. Plant Cell Physiol. 36, 265-271
Darrouzet, E., Issartel, J.P., Lunardi, J. and Dupuis, A. (1998) The 49-kDa subunit of NADH-ubiquinone
oxidoreductase (Complex I) is involved in the binding of piericidin and rotenone, two quinone-related
inhibitors. FEBS Lett. 431, 34-38
37
第一章 蓝藻 NAD(P)H 脱氢酶的结构与功能研究进展
Deng, Y., Ye, J. and Mi, H. (2003a) Low CO2 stimulates the expression of NAD(P)H degydrogenase which
mediates cyclic electron transport around PSI to the intersystem in the cyanobacterium Synechocystis
PCC6803. Plant Cell Physiol. 44, 534-540
Deng, Y., Ye, J., Mi, H. and Shen, Y. (2003b) Response of NAD(P)H dehydrogenase complex to the alteration of
CO2 concentration in the cyanobacterium Synechocystis PCC6803. J. Plant Physiol. (in press)
Dzelzkalns, V.A., Obinger, C., Regelsberger, G., Niederhauser, H., Kamensek, M., Peschek, G.A. and Bogorad, L.
(1994) Deletion of the structural gene for the NADH-dehydrogenase subunit 4 of Synechocystis 6803 alters
respiratory properties. Plant Physiol. 106, 1435-1442
Ellersiek, U. and Steinmuller, K. (1992) Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and
the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803. Plant Mol. Biol. 20, 1097-1110
Elortza, F., Asturias, J.A. and Arizmendi, J.M. (1999) Chloroplast NADH dehydrogenase from Pisum sativum:
characterization of its activity and cloning of ndhK gene. Plant Cell Physiol. 40, 149-154
Endo, T., Mi, H., Shikanai, T. and Asada, K. (1997) Donation of electrons to plastoquinone by NAD(P)H
dehydrogenase and by ferredoxin-quinone reductase in spinach chloroplasts. Plant Cell Physiol. 38,
1272-1277
Endo, T., Shikanai, T., Takabayashi, A., Asada, K. and Sato, F. (1999) The role of chloroplastic NAD(P)H
dehydrogenase in photoprotection. FEBS Lett. 457, 5-8
Espie, G.S. and Kanasamy, R.A. (1992) Na+-independent HCO3 - transport and accumulation in the
cyanobacteriuim Synechococcus UTEX 625. Plant Physiol. 98, 560-568
Espie, G.S. and Kanasamy, R.A. (1994) Monensin inhibition of Na+-dependent HCO3 transport distinguishes it
-
from Na+-independent HCO3 transport and provides evidence for Na+/HCO3 symport in the cyanobacterium
- -
Synechococcus UTEX 625. Plant Physiol. 104, 1419-1428
Figge, R.M., Cassier-Chauvat, C., Chauvat, F. and Cerff, R. (2001) Characterization and analysis 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
Fridlyand, L., Kaplan, A. and Reinhold, L. (1996) Quantitative evaluation of the role of a putative CO2-scavenging
entity in the cyanobacterial CO2-concentrating mechanism. Biosystems 37, 229-238
Friedrich, T. (1998) The NADH:ubiquinone oxidoreductase (complex I) from Escherichia coli. Biochim. Biophys.
Acta. 1364, 134-146
Friedrich, T., Strohdeicher, M., Hofhaus, G., Preis, D., Sahm, H. and Weiss, H. (1990) The same domain motif for
ubiquinone reduction in mitochondrial or chloroplast NADH dehydrogenase and bacterial glucose
38
中国科学院上海植物生理生态研究所博士学位论文
dehydrogenase. FEBS Lett. 265, 37-40
Friedrich, T., Abelmann, A., Brors, B., Guenebaut, V., Kintscher, L., Leonard, K., Rasmussen, T., Scheide, D.,
Schlitt, A., Schulte, U. and Weiss, H. (1998) Redox components and structure of the respiratory
NADH:ubiquinone oxidoreductase (complex I). Biochim. Biophys. Acta 1365, 215-219
Friedrich, F., Steinmüller, K. and Weiss, H. (1995) The proton-pumping respiratory complex I of bacteria and
mitochondrial and its homologue in chloroplast. FEBS Lett. 367, 107-111
Fulda, S. and Hagemann, M. (1995) Salt treatment induces accumulation of flavodoxin in the cyanobacterium
Synechocystis sp. PCC6803. J. Plant Physiol. 146, 520-526
Grohmann, L., Herz, U., Thieck, O., Heiser, V., Schmidt-Bleek, K., Lin T. and Brennicke, A. (1995) In: F. Palmieri,
S. Papa, C. Saccone and M.M. Gadaleta, Editors, Progress in Cell Research - Symposium on `Thirty Years of
Progress in Mitochondrial Bio-energetics and Molecular Biology', Elsevier Science B.V., Amsterdam
Guedeney, G., Corneille, S., Cuine, S and Peltier, G. (1996) Evidence for an association of ndhB, ndhJ gene
products and ferredoxin-NADP-reductase as components of a chloroplastic NAD(P)H dehydrogenase
complex. FEBS Lett. 378, 277-280
Guenebaut, V., Vincentelli, R., Mills, D., Weiss, H. and Leonard, K.R. (1997) Three-dimensional structure of
NADH-dehydrogenase from Neurospora crassa by electron microscopy and conical tilt reconstruction. J.
Mol. Biol. 265, 409-418
Guéra, A. and Sabater, B. (2002) Changes in the protein and activity levels of the plastid
NADH-plastoquinone-oxidoreductase complex during fruit development. Plant Physiol. Biochem. 40,
423-429
Hagemann, M., Jeanjean, R., Fulda, S., Havaux M., Joset, F. and Erdmann, N. (1999) Flavodoxin accumulation contributes
to enhanced cyclic electron flow around photosystem I in salt-stressed cells of Synechocystis sp. strain PCC6803.
Physiol. Plant. 105, 670-678
Hibino, T., Lee, B.H., Rai, A.K., Ishikawa, H., Kojima, H., Tawada, M., Shimoyama, H. and Takabe, T. (1996) Salt
enhances photosystem I content and cyclic electron flow via NAD(P)H dehydrogenase in the halotolerant
cyanobacterium Aphanothece halophytica. Aust. J. Plant Physiol. 23, 321-330
Hihara, Y., Kamei, A., Kanehisa, M., Kaplan, A. and Ikeuchi, M. (2001) DNA microarray analysis of cyanobacterial gene
expression during acclimation to high light. Plant Cell 13, 793-806
Horvath, E. M., Peter, S. O., Jo?t, T., Rumeau, D., Cournac, L., Horvath, G. V., Kavanagh, T. A., Schafer, C.,
Peltier, G., and Medgyesy, P. (2000) Targeted inactivation of the plastid ndhB gene in tobacco results in an
enhanced sensitivity of photosynthesis to moderate stomatal closure. Plant Physiol. 123, 1337-1349
39
第一章 蓝藻 NAD(P)H 脱氢酶的结构与功能研究进展
Howitt, C.A., Whelan, J., Price, G.D. and Day, D.A. (1996) Cloning, analysis and inactivation of the ndhK gene
encoding a subunit of NADH quinone oxidoreductase from Anabaena PCC 7120. Eur. J. Biochem. 240,
173-180
Howitt, C.A., Udall, P.K. and Vermaas, W.F. (1999) Type 2 NADH dehydrogenases in the cyanobacterium
Synechocystis sp. strain PCC6803 are involved in regulation rather than respiration. J. Bacteriol. 181,
3994-4003
Howitt, C.A., Mith, G.D. and Day, D.A. (1993) Cyanide-insensitive oxygen uptake and pyridine nucleotide
dehydrogenase in the cyanobacterium Anabaena PCC7120. Biochim. Biophys. Acta 1141, 313-320
Jeanjean, R., Bedu, S., Havaux, M., Matthijs, H.C.P. and Joset, F. (1998) Salt-induced photosystem I cyclic electron
transfer restores growth on low inorganic carbon in a type 1 NAD(P)H dehydrogenase deficient mutant of
Synechocystis PCC6803. FEMS Microbiol. Lett. 167, 131-137
Jeanjean, R., van Thor, J.J., Havaux, M., Joset, F. and Matthijs, H.C.P. (1999) Identification of
plastoquinone-cytochrome/b6f reductase pathways in direct or indirect phtosystem 1 driven cyclic electron
flow in Synechocystis PCC6803. In The Phototrophic Prokaryotes, edited by Peschek et al. pp: 251-258.
Kluwer Academic/Plenum Publishers, New York
Joet, T., Cournac, L., Peltier, G. and Havaux, M. (2002) Cyclic electron flow around photosystem I in C3 plants. In
vivo control by the redox state of chloroplasts and involvement of the NADH-dehydrogenase complex. Plant
Physiol. 128, 760-769
Josse, E. M., Simkin, A. J., Gaffe, J., Laboure, A. M., Kuntz, M., and Carol, P. (2000) A plastid terminal oxidase
associated with carotenoid desaturation during chromoplast differentiation. Plant Physiol. 123, 1427-1436
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., Badger, M.R. and Berry, J.A. (1980) Photosynthesis and intracellular inorganic carbon pool in the
blue-green algae Anabaena variabilis: responses to external CO2 concentration. Planta 149, 219-226
Kaplan, A. and Reinhold, L. (1999) CO2 concentrating mechanism in photosynthetic microorganisms. Annu. Rev.
Plant Physiol. Plant Mol. Biol. 50, 539-570
Kaplan, A., Schwarz, R., Ariel, R. and Reinhold, L. (1990) The CO2-concentrating system in cyanobacteria:
perspective and prospects. In Regulation of Photpsynthetic Processes, ed. by Kanai, R., Katoh, S., Miyachi,
40
中国科学院上海植物生理生态研究所博士学位论文
S., pp. 53-72. Bot. Mag., spec. issue
Kaplan, A., Zenvirth, D., Marcus, Y., Omata, T. and Ogawa, T. (1987) Energization and activation of inorganic
carbon uptake by light in cyanobacteria. Plant Physiol. 84, 210-213
Klughammer, B., Sultemeyer, D., Badger, M.R., Price, G.D. (1999) The involvement of NAD(P)H dehydrogenase
subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in Synechococcus sp. PCC7002 gives evidence for
multiple NDH-1 complexes with specific roles in cyanobacteria. Mol. Microbiol. 32, 1305-1315
Kofer, W., Koop, H.U., Wanner, G. and Steinmüller, K. (1998) Mutagenesis of the genes encoding subunits A, C, H,
I, J and K of the plastid NAD(P)H-plastoquinone- oxidoreductase in tobacco by polyethylene
glycol-mediated plastome transformation. Mol. Gen. Genet. 258, 168-173
Leif, H., Sled, V.D., Ohnishi, T., Weiss, H. and Friedrich, T. (1995) Isolation and characterization of the
proton-translocating NADH:ubiquinone oxidoreductase from Escherichia coli. Eur. J. Biochem. 230,
538-548
Lelong, C., Boekema, E.J., Kruip, J., Bottin, H., R?gner, M. and Sétif, P. (1996) Characterization of a redox active
cross-linked complex between cyanobacterial photosystem I and soluble ferredoxin. EMBO J. 15, 2160-2168
Li, Q. and Canvin, D.T. (1998) Energy sources for HCO3 and CO2 transport in air-grown cells of Synechococcus
-
UTEX 625. Plant Physiol. 116, 1125-1132
Maeda, S., Badger, M.R., Price, G.D. (2002) Novel gene products associated with NdhD3/D4-containing NDH-1
complexes are involved in photosynthetic CO2 hydration in the cyanobacterium, Synechococcus sp.
PCC7942. Mol. Microbiol. 43, 425-35
Maeda, S., Price, G.D., Badger, M.R., Enomoto, C. and Omata, T. (2000) Bicarbonate-binding protein of the
cyanobacterium Synechococcus sp. PCC7942. Plant Cell Physiol. 41: supplement, s89
Malkin, S. and Canaani, O. (1994) The use and characteristics of the photoacoustic method in the study of photosynthesis.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 45, 493-526
Mano, J., Miyake, C., Schreiber, U. and Asada, K. (1995) Photoactivation of the electron flow from NADPH to
plastoquinone in spinach chloroplasts. Plant Cell Physiol. 36, 1589-1598
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
Martín, M., Casano, L.M. and Sabater, B. (1996) Identification of the product of ndhA gene as a thylakoid protein
synthesized in response to photooxidative treatment. Plant Cell Physiol. 37, 293-298
Matthijs, H.C.P., Ludérus, E.M.E., L?ffler, H.J.M. Scholts, M.J.C. and Kraayenhof, R. (1984) Energy metabolism in the
41
第一章 蓝藻 NAD(P)H 脱氢酶的结构与功能研究进展
cyanobacterium Plectonema boryanum. Participation of the thylakoid photosynthetic electron transfer chain in the dark
repiration of NADPH and NADH. Biochim. Biophys. Acta 766, 29-37
Matsuo, M., Endo, T. and Asada, K. (1998a) Properties of the respiratory NAD(P)H dehydrogenase isolated from
the cyanobacterium Synechocystis PCC6803. Plant Cell Physiol. 39, 263-267
Matsuo, M., Endo, T. and Asada, K. (1998b) Isolation of a novel NAD(P)H-quinone oxidoreductase from the
cyanobacterium Synechocystis PCC6803. Plant Cell Physiol. 39, 751-755
Mayes, S.R., Cook, K.M. and Barber, J. (1990) Nucleotide sequence of the second psbG gene in Synechocystis 6803.
Possible implications for psbG function as a NAD(P)H dehydrogenase subunit gene. FEBS Lett. 262, 49-54
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
Mi, H., Endo, T., Ogawa, T., and Asada, K. (1995) Thylakoid membrane-bound, NADPH-specific pyridine
nucleotide dehydrogenase comples mediates cyclic electron transport in the cyanobacterium Synechocystis
PCC6803. Plant Cell Physiol. 36, 661-668
Mi, H., Endo, T., Schreiber, U., Ogawa, T. and Asada, K. (1992) 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. and Asada, K. (1992) Donation of electrons to the intersystem chain in the
cyanobacterium Synechcoccus sp. PCC7002. Plant Cell Physiol. 33, 1099-1105
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
Mi, H., Klughammer, C. and Schreiber, U. (2000) Light-induced dynamic changes of NADPH fluorescence in
Synechocystis PCC6803 and its ndhB-defective mutant M55. Plant Cell Physiol. 41, 1129-1135
Miller, A.G., Espie, G.E. and Canvin, D.T. (1991) Active CO2 transport in cyanobacteria. Can. J. Bot. 69, 925-935.
Miller, A.G., Espie, G.S. and Canvin, D.T. (1990) Physiological aspects of CO2 and HCO3 transport by -
cyanobacteria: a review. Can. J. Bot. 68, 1291-1302
Miller, A.G., Salon, C., Espie, G.S. and Canvin, D.T. (1997) Measurement of the amount and isotopic composition
of the CO2 released from the cyanobacterium Synechococcus UTEX 625 after rapid quenching of the active
CO2 transport system. Can. J. Bot. 75, 981-997
Mühlenhoff, U., Kruip, J., Bryant, D.A., R?gner, M., Sétif, P. and Boekema, E. (1996) Characterization of a
42
中国科学院上海植物生理生态研究所博士学位论文
redox-active cross-linked complex between cyanobacterial photosystem I and its physiological acceptor
flavodoxin. EMBO J. 15, 488-97
Munekage, Y., Hojo, M., Meurer, J., Endo, T., Tasaka, M. and Shikanai, T. (2002) PGR5 is involved in cyclic
electron flow around photosystem I and is essential for photoprotection in Arabidopsis. Cell 110, 361-71
Nixon, P.J., Gounaris, K., Coomber, S.A., Hunter, C.N., Dyer, T.A. and Barber, J. (1989) psbG is not a
photosystem two gene but may be an ndh gene. J. Biol. Chem. 264, 14129-14135
Ogawa, T. (1991) Cloning and inactivation of a gene essential to inorganic carbon transport of Synechocystis
PCC6803. Plant Physiol. 96, 280-284
Ogawa, T. (1991) 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
Ogawa, T., Miyano, A. and Inoue, Y. (1985) Photosystem-I-driven inorganic carbon transport in the
cyanobaterium, Anacystis nidulans. Biochim. Biophys. Acta. 808, 77-84
Ogawa, T. and Ogren, W.L. (1985) Action Spectra for accumulation of inorganic carbon in the cyanobaterium,
Anabaena variabilis. Photochem. Photobiol. 41, 583-587
Ogawa, T. (1993) Molecular analysis of the CO2-concentrating mechanism in cyanobacteria. In: Photosynthetic
Responses to the Environment, pp.113-125 Rockville, MD: Am. Soc. Plant Physiol
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
Ohkawa, H., Pakrasi, H.B. and Ogawa, T. (2000) Two types of functionally distinct NAD(P)H dehydrogenases in
Synechocystis sp. strain PCC6803. J. Biol. Chem. 275, 31630-31634
Ohkawa, H., Price, G.D., Badger, M.R. and Ogawa, T. (2000) Mutation of ndh genes leads to inhibition of CO2
uptake rather than HCO3 uptake in Synechocystis sp. Strain PCC6803. J. Bacteriol. 182, 2591-2596
-
Ohnishi, T. (1993) NADH-quinone oxidoreductase, the most complex complex. J. Bioenerg. Biomembr. 25,
325-329
Ohyama, K., Fukuzawa, H., Kohchi, T., Shirai, H., Sano, T., Sano, S., Umesono, K., Inokuchi, H. and Ozeki, H.
(1986) Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha
chloroplast DNA. Nature 322, 572-574
Palmqvist, K., Sültemeyer, D., Baldet, P., Andrews, T.J. and Badger, M.R. (1995) Characterization of inorganic
carbon fluxes, carbon anhydrase(s) and ribulose-1,5-biphosphate carboxylase-oxygenase in the green
43
第一章 蓝藻 NAD(P)H 脱氢酶的结构与功能研究进展
unicellular alga Coccomyxa. Planta. 197, 352-361
Peltier, G. and Cournac, L. (2002) Chlororespiration. Annu. Rev. Plant Biol. 53, 523-550
Peschek, G.A. (1981a) Occurrence of cytochrome aa3 in Anacystis nidulans. Biochim. Biophys. Acta 635, 470-475
Peschek, G.A. (1981b) Spectral properties of a cyanobacterial cytochrome c oxidase: Evidence for cytochrome aa3.
Biochem. Biophys. Res. Commun. 98, 72-79
Peschek, G.A. (1982) The cytochrome oxidase-hydrogenase relationship in cyanobacteria. Naturwiss 69, 599-600
Peschek, G.A. and Schmetterer, G. (1982) Evidence for plastoquinol-cytochrome f/b-563 reductase as a common
electron donor to P700 and cytochrome oxidase in cyanobacteria. Biochem. Biophys. Res. Commun. 108,
1188-1195
Pieulle, L., Guedeney, G., Cassier-Chauvat, C., Jeanjean, R., Chauvat, F. and Peltier, G. (2000) The gene encoding
the NdhH subunit of type 1 NAD(P)H dehydrogenase is essential to survival of Synechocystis PCC6803.
FEBS Lett. 487, 272-276
Price, G.D., Sültemeyer, D., Klughammer, B., Ludwig, M. and Badger, M.R. (1998) The functioning of the CO2
concentrating mechanism in several cyanobacterial strains: a review of general physiological characteristics,
genes, proteins and recent advances. Can. J. Bot. 76, 973-1002
Quiles, M.J. and Cuello, J. (1998) Association of ferredoxin-NADPoxidoreductase with the chloroplastic pyridine nucleotide
dehydrogenase complex in barley leaves. Plant Physiol. 117, 235-244
Quiles, M.J., Garc?a, A. and Cuello, J. (2000) Separation by blue-native PAGE and identification of the whole NAD(P)H
dehydrogenase complex from barley stroma thylakoids. Plant Physiol. Biochem. 38, 225-232
Reiskind, J.B., Madsen, T.V., Van Ginkel, L.C. and Bowes, G. (1997) Evidence that inducible C4-type
photosynthesis is a chloroplastic CO2-concentrating mechanism in Hydrilla, a submersed monocot. Plant
Cell Environ. 20, 211-220
Ronen-Tarazi, M., Bonfil, D.J., Schatz, D. and Kaplan, A. (1998) Cyanobacterial mutants impaired in bicarbonate
isolated with the aid of an inactivation library. Can. J. Bot. 76, 942-948
Salon, C., Mir, N.A. and Canvin, D.T. (1996) Influx and efflux of inorganic carbon in Synechococcus UTEX 625.
Plant Cell Environ. 19, 247-259
Sandmann, G. and Malkin, R. (1983) NADH and NADPH as electron donors to respiratory and photosynthetic
electron transport in the blue-green alga, Aphanocaspsa. Biochim. Biophys. Acta 234, 105-111
Sazanov, L.A., Burrows, P.A., Nixon, P.J. (1998) The plastid ndh genes code for an NADH-specific dehydrogenase:
isolation of a complex I analogue from pea thylakoid membranes. Proc. Natl. Acad. Sci. U S A. 95,1319-1324
Sazanov, L.A., Burrows, P., Nixon, P.J. (1996) Detection and characterization of a complex I-like NADH-specific
44
中国科学院上海植物生理生态研究所博士学位论文
dehydrogenase from pea thylakoids. Biochem. Soc. Trans. 24, 739-743
Scheller, H.V. (1996) In vitro cyclic electron transport in barley thylakoids follows two independent pathways.
Plant Physiol. 110,187-194
Schluchter, W.M. and Bryant, D.A. (1992) Molecular characterization of ferredoxin-NADP+ oxidoreductase in
cyanobacteria: cloning and sequence of the petH gene of Synechococcus sp. PCC 7002 and studies on the
gene product. Biochemistry. 31, 3092-3102
Schluchter, W.M., Zhao, J. and Bryant, D.A. (1993) Isolation and characterization of the ndhF gene of
Synechococcus sp. strain PCC 7002 and initial characterization of an interposon mutant. J Bacteriol. 175,
3343-3352
Schmetterer, G., Alge, D. and Gregor, W. (1994) Deletion of cytochrome c oxidase genes from the cyanobacterium
Synechocystis sp. PCC6803: Evidence for alternative respiratory pathways. Photosynth. Res. 42, 43-50
Schreiber, U., Endo, T., Mi, H. and Asada, K. (1995) Quenching analysis of chlorophyll fluorescence by the
saturation pulse method: particular aspects relating to the study of eukaryotic algae and cyanobacteria. Plant
Cell Physiol. 36, 873-882
Schwarz, R., Friedberg, D. and Kaplan, A. (1988) Is there a role for the 42 kD polypeptide in inorganic carbon
uptake by cyanbacteria? Plant Physiol. 88, 284-288
Shen, J. and Wang, G. (1998) State transition in blue-green alga Synechocystis PCC6803. Chinese Sci. Bull. 43,
2087-2090
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. U S A.
98,11789-94
Shikanai, T., Endo, T., Yamada, Y., Hashimoto, T., Asada, K. and Yokota, A. (1998) Directed disruption of the
tobacco ndhB gene impairs cyclic electron flow around photosystem I. Proc. Natl. Acad. Sci. USA 95,
9705-9709
Shinozaki, K., Ohme, M., Tanaka, M., Wakasugi, T., Hayashida, N., Matsubayashi, T., Zaita, N., Chunwongse, J.,
Obotaka, J., Yamaguchi-Shinozaki, K., Ohto, C., Torozawa, K., Meng, B.Y., Sugita, M., Deno, H.,
Kamogashira, T., Yamada, K., Kusuda, J., Takaiwa, F., Kato, F., Tohdo, N., Shimada, H. and Sugiura, M.
(1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and
expression. EMBO J. 5, 2043-2049
Steinmüller, K. (1992a) Nucleotide sequence and expression of the ndhH gene of the cyanobacterium Synechocystis
45
第一章 蓝藻 NAD(P)H 脱氢酶的结构与功能研究进展
sp. PCC6803. Plant Mol. Biol. 18, 135-137
Steinmüller, K. (1992b) Identification of a second psaG gene in the cyanobacterium Synechocystis sp. PCC6803.
Plant Mol. Biol. 20, 997-1001
Steinmüller, K. and Bogorad, L. (1990) In: Current Research in Photosynthesis. Baltscheffsky M (ed) Vol III,
pp12557-12560. Kluwer, Dordrecht, Netherlands
Steinmüller, K., Ley, A.C., Steinmetz, A.A., Sayre, R.T. and Bogorad, L. (1989) Characterization of the
ndhC-psbG-ORF157/159 operon of maize plastid DNA and of the cyanobacterium Synechocystis sp. PCC6803.
Mol. Gen. Genet. 216, 60-69
Steinmüller, K., Ellersiek, U. and Bogorad, L. (1991) Deletion of the psbG1 gene of the cyanobacterium
Synechocystis sp. PCC6803 leads to the activation of the cryptic psbG2 gene. Mol. Gen. Genet. 226,107-112
Sültemeyer, D., Biehler, K. and Forck, H.P. (1993) Evidence for the contribution of pseudocyclic
photophosphorylation to the energy requirement of the mechanism for concentrating inorganic carbon in
Chlamydomonas. Planta. 189, 235-242
Sültemeyer, D., Price, G.D., Bryant, D.A. and Badger, M.R. (1997). Psa-E and NdhF-mediated electron transport
affect bicarbonate transport rather than carbon dioxide uptake in the cyanibacterium Synechococcus sp.
PCC7002. Planta. 201, 36-42
Takahashi, Y., Shonai, F., Fujita, Y., Kohchi, T., Ohyama, K. and Matsubara, H. (1991) Structure of a
co-transcribed gene cluster, ndh1-frxB-ndh5-ndh4L, cloned from the filamentous cyanobacterium
Plectonema boryanum. Plant Cell Physiol. 32: 969-981
Tanaka, Y., Katada, S., Ishikawa, H., Ogawa, T. and Takabe, T. (1997) Electron flow from NAD(P)H
dehydrogenase to photosystem I is requireed for adaptation to salt shock in the cyanobacterium Synechocystis
sp. PCC 6803. Plant Cell Physiol. 38, 1311-1318
Tchernov, D., Hassidim, M., Luz, B., Sukenik, A., Reinhold, L. and Kaplan, A. (1997) Sustained net CO2 evolution
during photosynthesis by marine microorganisms. Curr. Biol. 7, 723-728
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
Thomas, D.J., Thomas, J., Youderian, P.A. and Herbert. S.K. (2001) Photoinhibition and light-induced cyclic
electron transport in ndhB– and psaE– mutants of Synechocystis sp. PCC 6803. Plant Cell Physiol. 42,
803-812
van Thor, J.J., Hellingwerf, K.J. and Matthijs, H.C. (1998) Characterization and transcriptional regulation of the
Synechocystis PCC 6803 petH gene, encoding ferredoxin-NADP+ oxidoreductase: involvement of a novel
46
中国科学院上海植物生理生态研究所博士学位论文
type of divergent operator. Plant Mol. Biol. 36, 353-363
van Thor, J.J., Geerlings, T.H., Matthijs, H.C. and Hellingwerf, K.J. (1999) Kinetic evidence for the
PsaE-dependent transient ternary complex photosystem I/ferredoxin/ferredoxin: NADP(+) reductase in a
cyanobacterium. Biochemistry 38,12735-12746
van Thor, J.J., Jeanjean, R., Havaux, M., Sjollema, K.A., Joset, F., Hellingwerf, K.J. and Matthijs, H.C. (2000) Salt
shock-inducible photosystem I cyclic electron transfer in Synechocystis PCC6803 relies on binding of
ferredoxin:NADP(+) reductase to the thylakoid membranes via its CpcD phycobilisome-linker homologous
N-terminal domain. Biochim. Biophys. Acta 1457,129-144
Vermaas, W.F.J. (1994) Molecular-genetic approaches to study photosynthetic and respiratory electron transport in
thylakoids from cyanobacteria. Biochim. Biophys. Acta 1187, 181-186
Vermaas, W.F.J. (1996) Molecular genetics of the cyanobacterium Synechocystis sp. PCC6803: Principles and
possible biotechnology applications. J. Applied Phycology 8, 263-273
Viljoen, C.C., Cloete, F. and Scott, W.E. (1985) Isolation and characterization of an NAD(P)H dehydrogenase from
the cyanobacterium, Microcystis aeruginosa. Biochim. Biophys. Acta 827, 247-259
Volokita, M., Zenvirth, D., Kaplan, A. and Reinhold, L. (1984) Nature of the inorganic carbon species actively
taken up by the cyanobacterium Anabaena variabilis. Plant Physiol. 76, 599-602
Wu, D.Y., Wright, D.A., Wetzel, C., Voytas, D.F., and Rodermel, S. (1999) The IMMUTANS variegation locus of
Arabidopsis defines a mitochondrial alternative oxidase homolog that functions during early chloroplast
biogenesis. Plant Cell 11, 43-55
Yagi, T. (1991) Bacterial NADH-quinone oxidoreductase. J. Bioenerg. Biomembr. 23, 2008-2013
Yamane, Y., shikanai, T., Kashino, Y., Koike, H. and Satoh K (2000) Reduction of QA in the dark: Another cause
of fluorescence Fo increases by high temperatures in higher plants. Photosyn. Res. 63, 23-34
Yao, Z. J., Ye, J-Y, Mi, H. (2001) In: PS2001 Proceedings, 12th International congress on Photosynthesis (Online
and CD-ISBN 0643 067116), pp1-6, CSIRO Publishers, Australia
Yu, L., Zhao, J., Muhlenhoff, U., Bryant, D.A. and Golbeck, J.H. (1993) PsaE is required for in vivo cyclic electron
flow around photosystem I in the cyanobacterium Synechococcus sp. PCC7002. Plant Physiol. 103, 171-180
47
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,
48
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。
49
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。
参考文献
Allen, M.M. (1968) Simple conditions for growth of unicellular blue-green algae on plates. J.
Phycol. 4: 1-4.
Asada, K., Heber, U. and Schreiber, U. (1993) Electron flow to the intersystem chain from
stromal components and cyclic electron flow in maize chloroplasts, as detected in intact
leaves by monitoring redox change of P700 and chlorophyll fluorescence. Plant Cell Physiol.
34: 39-50.
Bendall, D.S. and Manasse, R.S. (1995) Cyclic photophosphorylation and electron transport.
Biochim. Biophys. Acta. 1229: 23-38.
Danon, A. (1997) Translational regulation in the chloroplast. Plant Physiol. 115: 1293-1298.
Davis, B.J. (1964) Method and application to human serum protein. Ann. N. Y. Acad. Sci. 121:
404-427.
59
第二章 NDH 在蓝藻对不同 CO2浓度的响应和适应中的作用
Figge, R.M., Cassier-Chauvat, C., Chauvat, F. and Cerff, R. (2001) Characterization and analysis
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.
Golbeck, J.H. (1994) Photosystem I in cyanobacteria. In: Molecular Biology of Cyanobacteria
(Bryant D.A. eds), pp. 319-360. Kluwer Academic Publishers, Dordrecht.
Hihara, Y., Kamei, A., Kanehisa, M., Kaplan, A. and Ikeuchi, M. (2001) DNA microarray
analysis of cyanobacterial gene expression during acclimation to high light. Plant Cell 13:
793-806.
Jeanjean, R., Bedu, S., Havaux, M., Matthijs, H.C.P. and Joset, F. (1998) Salt-induced
photosystem I cyclic electron transfer restores growth on low inorganic carbon in a type 1
NAD(P)H dehydrogenase deficient mutant of Synechocystis PCC6803. FEMS Microbiol.
Lett. 167: 131-137.
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.
Klughammer, C. and Schreiber, U. (1998) Measuring P700 absorbance changes in the near
infrared spectral region with a dual wavelength pulse modulation system. In: Photosynthesis:
Mechanism and Effects (G. Grab ed.) Vol. V, pp. 4657-4660. Kluwer Academic Publishers,
Dordrecht.
Klughammer, B., Sultemeyer, D., Badger, M.R. and Price, G.D. (1999) The involvement of
NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in
Synechococcus sp. PCC7002 gives evidence for multiple NDH complexes with specific
roles in cyanobacteria. Mol. Microbiol. 32: 1305-1315.
Laemmli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophasge T4. Nature 227: 680-685.
60
低 CO2浓度对集胞蓝藻 NDH 复合体的影响
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.
Mano, J., Miyake, C., Schreiber, U. and Asada, K. (1995) Photoactivation of the electron flow
from NADPH to plastoquinone in spinach chloroplasts. Plant Cell Physiol. 36: 1589-1598.
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.
Matsuo, M., Endo, T. and Asada, K. (1998) Properties of the respiratory NAD(P)H
dehydrogenase isolated from the cyanobacterium Synechocystis PCC6803. Plant Cell
Physiol. 39: 263-267.
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.
Mi, H., Ye, J., Wang, Y., Li, D. and Shen, Y. (1997) Mechanism of temperature dependence of
post-illumination transient increase in Chl fluorescence in cyanobacterium Synechocystis
PCC6803. Chin Sci. Bul. 42: 1106-1109.
Ogawa, T. (1991) Cloning and inactivation of a gene essential to inorganic carbon transport of
Synechocystis PCC6803. Plant Physiol. 96: 280-284.
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.
61
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
dehydrogenase in Synechocystis PCC6803. J. Biol. Chem. 275: 31630-31634.
Ohkawa, H., Price, G.D., Badger, M.R. and Ogawa, T. (2000b) Mutation of ndh genes leads to
inhibition of CO2 uptake rather than HCO3 uptake in Synechocystis sp. strain PCC6803. J.
-
Bacteriol. 182: 2591-2596.
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.
Pieulle, L., Guedeney, G., Cassier-Chauvat, C., Jeanjean, R., Chauvat, F. and Peltier, G. (2000)
The gene encoding the NdhH subunit of type I NAD(P)H dehydrogenase is essential to
survival of Synechocystis PCC6803. FEBS Lett. 487: 272-276.
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.
van Thor, J.J., Jeanjean, R., Havaux, M., Sjollema, K.A., Joset, F., Hellingwerf, K.J. and Matthijs,
H.C.P. (2000) Salt shock-inducible photosystem I cyclic electron transfer in Synechocystis
PCC6803 relies on binding of ferredoxin:NADP+ reductase to the thylakoid membranes via its
CpcD phycobilisome-linker homologous N-terminal domain. Biochim. Biophys. Acta 1457:
129-144.
Yagi, T. (1991) Bacterial NADH-quinone oxidoreductase. J. Bioenerg. Biomembr. 23: 2008-2013.
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。
82
第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白质的相互作用
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-Homologous NADH-dehydrogenase. FEBS Lett., 286: 129-132
Berger, S., Ellersiek, U., Kinzelt, D. and Steinmüller, K. (1993) Immunopurification of a
subcomplex of the NAD(P)H-plastoquinone-oxidoreductase from the cyanobacterium
Synechocystis sp. PCC6803. FEBS Lett., 326: 246-250
Davis, B.J. (1964) Disk electrophoresis. II. Method and application to human serum proteins. Ann.
N. Y. Acad. Sci., 121: 404-427
Friedrich, T. and Scheide, D. (2000) The respiratory complex I of bacteria, archaea and eukarya and
its module common with membrane-bound multisubunit hydrogenases. FEBS Lett., 479:1-5
Friedrich, T., Steinmüller, K. and Weiss, H. (1995) The proron-pumping respiratory complex I of
bacteria and mitochondrial and its homologue in chloroplasts. FEBS Lett., 367: 107-111
Guedeney, G., Corneille, S., Cuine, S. and Peltier, G. (1996) Evidence for an association of ndhB,
ndhJ gene products and ferredoxin-NADP+-reductase as components of a chloroplastic
NAD(P)H dehydrogenase complex. FEBS Lett., 378: 277-280
Hibino, T., Lee, BH., Rai, A.K., Ishikawa, H., Kojima, H., Tawada, M., Shimoyama, H. and Takabe,
T. (1996) Salt enhances photosystem I content and cyclic electron flow via NAD(P)H
dehydrogenase in the halotolerant cyanobacterium Aphanothece halophytica. Aust. J. Plant.
Physiol., 23: 321-330
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
Klughammer, B., Sultemeyer, D., Badger, M.R. and Price, G.D. (1999) The involvement of
NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in
Synechcoccus sp. PCC7002 gives evidence for multiple NDH complexes with specific roles in
83
集胞蓝藻含疏水亚基的 NDH 亚复合体的分离
cyanobacteria. Mol. Microbiol., 32: 1305-1315
MacColl, R. and Guard-Friar, D. (1987) Phycobiliproteins., Boca Raton, FL: CRC Press.
Matsuo, M., Endo, T. and Asada, K. (1998) Properties of the respiratory NAD(P)H dehydrogenase
isolated from the cyanobacterium Synechocystis PCC6803. Plant Cell Physiol., 39: 263-267
Mi, H., Deng, Y., Tanaka, Y., Hibino, T., Takabe, T. (2002) 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
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
Ogawa, T. (1991) 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
Ohkawa, H., Price, G.D., Badger, M.R. and Ogawa, T. (2000) Mutation of ndh genes leads to
inhibition of CO2 uptake rather than HCO3 uptake in Synechocystis sp. strain PCC6803. J.
-
Bacteriol., 182: 2591-2596
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
Pieulle, L., Guedeney, G., Cassier-Chauvat, C., Jeanjean, R., Chauvat, F. and Peltier, G. (2000) The
gene encoding the Ndh H subunit of type I NAD(P)H dehydrogenase is essential to survival of
Synechocystis PCC6803. FEBS Lett., 487: 272-276
Quiles, M.J. and Cuello, J. (1998) Association of ferredoxin-NADP oxidoreductase with the
chloroplastic pyridine nucleotide dehydrogenase complex in barley leaves. Plant Physiol., 117:
235-244
Sandmann, G. and Malkin, R. (1983) NADH and NADPH as electron donors to respiratory and
photosynthetic electron transport in the blue-green alga Aphnocapsa. Biochim. Biophys. Acta,
1983, 234: 105-111
Tanaka, Y., Katada, S., Ishikawa, H., Ogawa, T. and Takabe, T. (1997) Electron flow from
NAD(P)H dehydrogenase to photosystem I is required for adaptation to salt shock in the
cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol., 38: 1311-1318
84
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
A
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
参考文献
Ajlani, G. and Vernotte, C. (1998) Deletion of the PB-loop in the LCM subunit does not affect
phycobilisome assembly or energy transfer functions in the cyanobacterium Synechocystis sp.
PCC6714. Eur. J. Biochem. 257, 154-159.
Albertsson, P.A. (2001) A quantitative model of the domain structure of the photosynthetic
membrane. Trends in Plant Sci. 6, 349-354.
Allen, M.M. (1968) Simple conditions for growth of unicellular blue-green algae on plates. J.
Phycol. 4, 1-4.
Ashby, M.K. and Mullineaux, C.W. (1999) The role of ApcD and ApcF in energy transfer from
phycobilisomes to PSI and PSII in a cyanobacterium. Photosyn. Res. 61, 169-179.
Berger, S., Ellersiek, U. and Steinmüller, K. (1991) Cyanobacteria contain a mitochondrial complex
I-Homologous NADH-dehydrogenase. FEBS Lett. 286, 129-132.
Berger, S., Ellersiek, U., Kinzelt, D. and Steinmüller, K. (1993) Immunopurification of a
105
第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
subcomplex of the NAD(P)H-plastoquinone-oxidoreductase from the cyanobacterium
Synechocystis sp. PCC6803. FEBS Lett. 326, 246-280.
Biggins, J. and Bruce, D. (1989) Regulation of excitation energy transfer in organisms containing
phycobilins. Photosyn. Res. 20, 1-34.
Bradford, M.M. (1976) A rapid and sensitive method for the quantitation of microgram quantities
of protein utilizing the principle of protein-dye binding. Anal. Biochem. 72, 248-254.
Cai,Y. P. and Wolk, C.P. (1990) Use of a conditionally lethal gene in Anabaena sp. strain PCC 7120
to select for double recombinants and to entrap insertion sequences. J. Bacteriol. 172(6):
3138-45.
Capuano, V., Braux, A., Tandeau de Marsac, N. And Houmard, J. (1991) The ‘anchor polypeptide’
of cyanobacterial phycobilisomes. J. Biol. Chem. 266, 7239-7247
Davis, B.J. (1964) Method and application to human serum protein. Ann. N. Y. Acad. Sci. 121,
404-427.
Ellerisiek, U. and Steinmüller, K. (1992) Cloning and transcription analysis of the ndh(A-I-G-E)
gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803. Plant Mol.
Biol. 20, 1097-1110.
Friedrich, T., Steinmüller, K. and Weiss, H. (1995) The proron-pumping respiratory complex I of
bacteria and mitochondrial and its homologue in chloroplasts. FEBS Lett. 367, 107-111.
Guedeney, G., Corneille, S., Cuine, S and Peltier, G. (1996) Evidence for an association of ndhB,
ndhJ gene products and ferredoxin-NADP+-reductase as components of a chloroplastic
NAD(P)H dehydrogenase complex. FEBS Lett. 378, 277-280.
Hagemann, M., Jeanjean, R., Fulda, S., Havaux M., Joset, F. and Erdmann, N. (1999) Flavodoxin
accumulation contributes to enhanced cyclic electron flow around photosystem I in
salt-stressed cells of Synechocystis sp. strain PCC6803. Physiol. Plant. 105, 670-678.
Hibino, T., Lee, B.H., Rai, A.K., Ishikawa, H., Kojima, H., Tawada, M., Shimoyama, H. and
Takabe, T. (1996) Salt enhances photosystem I content and cyclic electron flow via NAD(P)H
dehydrogenase in the halotolerant cyanobacterium Aphanothece halophytica. Aust. J. Plant
Physiol. 23, 321-330.
Hu, Q., Marquardt, J., Iwasaki, I., Miyashita, H., Kurano, N., M?rschel, E. and Miyachi, S. (1999)
Molecular structure, localization and function of biliproteins in the chlorophyll a/d containing
oxygenic photosynthetic prokaryote Acaryochloris marina. Biochim. Biophy. Acta 1412,
106
集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
250-261.
Jeanjean, R., Bedu, S., Havaux, M., Matthijs, H.C.P. and Joset, F. (1998) Salt-induced photosystem
I cyclic electron transfer restores growth on low inorganic carbon in a type 1 NAD(P)H
dehydrogenase deficient mutant of Synechocystis PCC6803. FEMS Microbiol. Lett. 167,
131-137.
Jeanjean, R., Van Thor, J.J., Havaux, M., Joset, F. and Matthijs, H.C.P. (1999) in: The Phototrophic
Prokaryotes (Peschek et al. ed.), pp. 251-258, Kluwer Academic/ Plenum Publishers, NY.
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.
Klughammer, B., Sultemeyer, D., Badger, M.R. and Price, G.D. (1999) The involvement of
NAD(P)H dehydrogenase subunits, NdhD3 and NdhF3, in high-affinity CO2 uptake in
Synechococcus sp. PCC7002 gives evidence for multiple NDH complexes with specific roles
in cyanobacteria. Mol. Microbiol. 32, 1305-1315.
Kubicki, A., Funk, E., Westhoff, P., Steinmüller, K. (1996) Differential expression of
plastome-encoded ndh genes in mesophyll and bundle-sheath chloroplasts of the C4 plant
Sorghum bicolor indicates that the complex I-homologous NAD(P)H-plastoquinone
oxidoreductase is involved in cyclic electron transport. Planta 199, 276-281.
Lammli, U.K. (1970) Cleavage of structural proteins during the assembly of the head of
bacteriophasge T4. Nature 227, 680-685.
MacColl, R. and Guard-Friar, D. (1987) Phycobiliproteins, CRC Press, Boca Raton, FL, NY.
Matsuo, M., Endo, T. and Asada, K. (1998) Properties of the respiratory NAD(P)H dehydrogenase
isolated from the cyanobacterium Synechocystis PCC 6803. Plant Cell Physiol. 39, 263-267.
Mi, H., Deng, Y., Tanaka, Y., Hibino, T., and Takabe, T. (2002) 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.
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
107
第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
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.
Mullineaux, C.W. (1992) Excitation energy transfer from phycobilisomes to PSI in a
cyanobacterium. Biochim. Biophys. Acta 1100, 282-292.
Mullineaux, C.W. (1994) Excitation energy transfer from phycobilisomes to photosystem I in a
cyanobacterial mutant lacking photosystem II. Biochim. Biophys. Acta 1184, 71-77.
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., Price, G.D., Badger, M.R. and Ogawa, T. (2000) Mutation of ndh genes leads to
inhibition of CO2 uptake rather than HCO3 uptake in Synechocystis sp. strain PCC6803. J.
-
Bacteriol. 182, 2591-2596.
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.
Pieulle, L., Guedeney, G., Cassier-Chauvat, C., Jeanjean, R., Chauvat, F. and Peltier, G. (2000) The
gene encoding the NdhH subunit of type I NAD(P)H dehydrogenase is essential to survival of
Synechocystis PCC6803. FEBS Lett. 487, 272-276.
Quiles, M.J. and Cuello, J. (1998) Association of ferredoxin-NADP oxidoreductase with the
chloroplastic pyridine nucleotide dehydrogenase complex in barley leaves. Plant Physiol. 117,
108
集胞蓝藻中 NDH 复合体与藻胆蛋白结合的初步证据
235-244.
Redlinger, T. and Gantt, E. (1982) A Mr 95 000 polypeptide in Porphyridium cruentum
phycobilisomes and thylakoids: possible function in linkage of phycobilisomes to thylakoids
and energy transfer. Proc. Natl. Acad. Sci. U S A 79, 5542-5546.
Sambrook, J., Fritsch, E.F., and Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual, 2nd
Ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.
Sandmann, G. and Malkin, R. (1983) NADH and NADPH as electron donors to respiratory and
photosynthetic electron transport in the blue-green alga Aphnocapsa. Biochim. Biophys. Acta
234, 105-111.
Schluchter, W.M. and Bryant, D.A. (1992) Molecular characterization of ferredoxin-NADP+
oxidoreductase in cyanobacteria: cloning and sequence of the petH gene of Synechococcus sp.
PCC7002 and studies on the gene product. Biochemistry 31, 3092-3102.
Schreiber, U., Endo, T., Mi, H. and Asada, K. (1995) Quenching analysis of chlorophyll
fluorescence by the saturation pulse method: particular aspects relating to the study of
eukaryotic algae and cyanobacteria. Plant Cell Physiol. 36, 873-882.
Steinmüller, K. (1992) Nucleotide sequence and expression of the ndhH gene of the
cyanobacterium Synechocystis sp. PCC6803. Plant Mol. Biol. 18, 135-137.
Tanaka, Y., Katada, S., Ishikawa, H., Ogawa, T. and Takabe, T. (1997) Electron flow from
NAD(P)H dehydrogenase to photosystem I is required for adaptation to salt shock in the
cyanobacterium Synechocystis sp. PCC 6803. Plant Cell Physiol. 38, 1311-1318.
Thomas, D.J., Thomas, J., Youderian, P.A. and Herbert, S.K. (2001) Photoinhibition and
Light-Induced Cyclic Electron Transport in ndhB– and psaE– Mutants of Synechocystis sp. PCC
6803. Plant Cell Physiol. 42, 803-812.
van Thor, J.J., Gruters, O.W.M., Matthijs, H.C.P. and Hellingwerf, K.J. (1999) Localization and
function of ferredoxin-NADP+ oxidoreductase bound to the phycobilisomes of Synechocystis.
EMBO J. 18, 4128-4136.
van Thor, J. J., Jeanjean, R., Havaux, M., Sjollema, K. A., Joset, F., Hellingwerf, K. J., Matthijs, H.
C. P. (2000) Salt shock-inducible photosystem I cyclic electron transfer in Synechocystis
PCC6803 relies on binding of ferredoxin:NADP+ reductase to the thylakoid membranes via its
CpcD phycobilisome-linker homologous N-terminal domain. Biochim. Biophys. Acta 1457,
129-144.
109
第三章 蓝藻 NDH 的分离纯化及与藻胆蛋白的相互作用
Viljoen, C.C., Cloete, F. and Scott, W.E. (1985) Isolation and characterization of an NAD(P)H
dehydrogenase from the cyanobacterium, Microcystis aeruginosa. Biochim. Biophy. Acta 827,
247-259.
Yu, J., Wu, Q., Zhao, N. And Vermaas, W.F.J. (1999) Effects of chlorophyll availability on
phycobilisomes in Synechocystis sp. PCC6803. IUBMB Life 48, 625-630.
Yu, L., Zhao, J.D., Muhlenhoff, U., Bryant, D.A. and Golbeck, J.H. (1993) PsaE is required for in
vivo cyclic electron flow around photosystem I in the cyanobacterium Synechococcus sp.
PCC7002. Plant Physiol. 103, 171-180.
110
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