光和水分胁迫对臭柏实生幼苗光化学效率及色素组成的影响

为探明毛乌素沙地3年生臭柏(Sabina vulgaris)实生苗在不同光照和水分条件下的光抑制响应机制,研究了各处理臭柏实生苗的最大光化学效率(F_v/F_m)及叶绿素(Chla+Chlb)和叶黄素(A+V+Z)含量,分析了其叶绿素循环和叶黄素循环的变化规律。结果表明,77%透光区通过减少Chlb含量,升高Chla/Chlb,避免光能过剩;同时,增加A+V+Z及热散逸色素(A+Z)含量、提高(A+V+Z)/(Chla+Chlb)和(A+V)/(A+V+Z)值,耗散过剩光能,避免光破坏。25%透光区的叶绿素和叶黄素循环机制随着水分条件的变化迅速发生改变。10%透光区通过增加Chlb含量,降低Chla/Chlb,捕捉更多的光能,几乎不存在光抑制。毛乌素臭柏实生幼苗能够适应不同的光照和水分条件,在恶劣的沙漠中完成天然更新,形成独特的群落景观,与叶绿素循环和叶黄素循环有着密切的关系。     

福建梅花山57种常绿树叶片叶绿素特征分析

选取福建梅花山海拔1200m和455m地区57种常绿植物为研究对象,测定两海拔的植物叶片叶绿素a和叶绿素b含量,并计算叶绿素a/b、总叶绿素含量。结果表明:(1)植物叶绿素a、b,叶绿素a+b及叶绿素a/b值大多分布在一个相对集中的区域。(2)多数植物新叶叶绿素含量高于老叶,叶绿素a/b值低于老叶,但是新、老叶无显著差异。(3)不同海拔的植物叶片总叶绿素含量和叶绿素a/b值有明显差异,低海拔地区明显高于高海拔地区。植物通过总叶绿素含量和a/b值的变化以适应不同环境条件。     

On the spatial organization of hemes and chlorophyll in cytochrome b(6)f. A linear and circular dichroism study

The organization of chromophores in the cytochrome b(6) f from Chlamydomonas reinhardtii has been studied spectroscopically. Linear dichroism (LD) measurements, performed on the complex co-reconstituted into vesicles with photosynthetic reaction centers as an internal standard, allow the determination of the orientations of the chromophore with respect to the membrane plane. The orientations of the b(H)- and b(L)-hemes are comparable to those determined crystallographically on the cytochrome bc(1). The excitonic CD signal, resulting from the interaction between b-hemes, is similar to that reported for the cytochrome bc(1). LD and CD data are consistent with the differences between the b(6) f and bc(1) leaving the orientation of the b-hemes unaffected. By contrast, the LD data yield a different orientation for the heme f as compared either to the heme c(1) in the crystallographic structures or to the heme f as studied by electron paramagnetic resonance. This difference could either result from incorrect assumptions regarding the orientations of the electronic transitions of the f-heme or may point to the possibility of a redox-dependent movement of cytochrome f. The chlorophyll a was observed in a well defined orientation, further corroborating a specific binding site for it in the b(6) f complex.     

Interaction between light harvesting chlorophyll-a/b protein (LHCII) kinase and cytochrome b6/f complex. In vitro control of kinase activity

We have previously reported that the cytochrome b6/f complex may be involved in the redox activation of light harvesting chlorophyll-a/b protein complex of photosystem II (LHCII) kinase in higher plants (Gal, A., Shahak, Y., Schuster, G., and Ohad, I. (1987) FEBS Lett. 221, 205-210). The aim of this work was to establish whether a relation between the cytochrome b6/f and LHCII kinase activation can be demonstrated in vitro. Preparations enriched in cytochrome b6/f obtained from spinach thylakoids by detergent extraction and precipitation with ammonium sulfate followed by different procedures of purification, contained various amounts of LHCII kinase activity. Analysis of the cytochrome b6/f content and kinase activity of fractions obtained by histone-Sepharose and immunoaffinity columns, immunoprecipitation and sucrose density centrifugation, indicate functional association of kinase and cytochrome b6/f. Phosphorylation of LHCII by fractions containing both cytochrome b6/f and kinase was enhanced by addition of plastoquinol-1. LHCII phosphorylation and kinase activation could be obtained in fractions prepared by use of beta-D-octyl glucoside but not when 3-[(cholamidopropyl)dimethyl-ammonio]-1-propanesulfonate was used as the solubilizing detergent. Kinase activity could be inhibited by halogenated quinone analogues (2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone and 2,3-diiodo-5-t-butyl-p-benzoquinone) known to inhibit cytochrome b6/f activity. However, kinase activity was inhibited by these analogues in all preparations including those which could not phosphorylate LHCII. We thus propose that the redox activation of LHCII phosphorylation is mediated by kinase interaction with cytochrome b6/f while the deactivation may be related to a distinct quinone binding site of the enzyme molecule.     

Energy Transfer among Chlorophylls in Trimeric Light-harvesting ComplexⅡ of Bryopsis corticulans

A study on energy transfer among chlorophylls(Chls)in the trimeric unit of the major light-harvesting complex Ⅱ(LHC Ⅱ)from Bryopsis corriculan,was carried out using time-correlated singlephoton counting.In the chlorophyll Q region of LHC Ⅱ,six molecules characterized as Chlb_(628),Chlb_(646),Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683) were discriminated according to their absorption spectrumand fluorescence emission spectrum.Then,excited by pulsed light of 628 nm,fluorescence kinetics spectrain the chlorophyll Q region were measured.In accordance with the principles of fluorescence kinetics,thesekinetics data were analyzed with a multi-exponential model.Time constants on energy transfer were obtained.An overwhelming percentage of energy transfer among chlorophylls undergoes a process longer than 97picoseconds(ps),which shows that,before transferring energy to another Chl,the excited Chl might convertenergy to vibrations of a lower state with different multiplicity(intersystem crossing).Energy transfer at thelevel of approximately 10 ps was also obtained,which was interpreted as the excited Chls may go throughinternal conversion before transferring energy to another Chl.Although with a higher standard deviation,timeconstants at the femtosecond level can not be entirely excluded,which can be attributed to the ultrafastprocess of direct energy transfer.Owing to the arrangement and direction of the dipole moment of Chls inLHC Ⅱ,the probability of these processes is different.The fluorescence lifetimes of Chlb_(652)~(654,657),Chla_(664)~(666),Chla_(674)~(677.680)and Chla_(682)~(683)were determined to be 1.44ns,1.43 ns,636 ps and 713 ps,respectively.Thepercentages of energy dissipation in the pathway of fluorescence emission were no more than 40% in thetrimeric unit of LHC Ⅱ.These results are important for a better understanding of the relationship between thestructure and function of LHC Ⅱ.     

Functional implications of pigments bound to a cyanobacterial cytochrome b6f complex

A highly purified cytochrome b(6)f complex from the cyanobacterium Synechocystis sp. PCC 6803 selectively binds one chlorophyll a and one carotenoid in analogy to the recent published structure from two other b(6)f complexes. The unknown function of these pigments was elucidated by spectroscopy and site-directed mutagenesis. Low-temperature redox difference spectroscopy showed red shifts in the chlorophyll and carotenoid spectra upon reduction of cytochrome b(6), which indicates coupling of these pigments with the heme groups and thereby with the electron transport. This is supported by the correlated kinetics of these redox reactions and also by the distinct orientation of the chlorophyll molecule with respect to the heme cofactors as shown by linear dichroism spectroscopy. The specific role of the carotenoid echinenone for the cytochrome b(6)f complex of Synechocystis 6803 was elucidated by a mutant lacking the last step of echinenone biosynthesis. The isolated mutant complex preferentially contained a carotenoid with 0, 1 or 2 hydroxyl groups (most likely 9-cis isomers of beta-carotene, a monohydroxy carotenoid and zeaxanthin, respectively) instead. This indicates a substantial role of the carotenoid - possibly for strucure and assembly - and a specificity of its binding site which is different from those in most other oxygenic photosynthetic organisms. In summary, both pigments are probably involved in the structure, but may also contribute to the dynamics of the cytochrome b(6)f complex.     

Studies of cGMP analog specificity and function of the two intrasubunit binding sites of cGMP-dependent protein kinase

The specificity of the two intrasubunit cGMP binding sites of cGMP-dependent protein kinase was determined by measuring the ability of 46 cGMP analogs to compete with [3H]cGMP. Both sites of the enzyme exhibited high specificity for the ribose cyclic phosphate moiety, and lower specificity for the guanine moiety. Effects of modifications in the ribose cyclic phosphate moiety suggested that cGMP is bound at both sites by three hydrogen bonds at 2'-OH, 3'-O, and 5'-O. A negative charge in the cyclic phosphate is apparently required. Modifications of the pyrimidine part of guanine, particularly at C-1, generally caused selectivity for the rapidly exchanging site while modifications of the imidazole part of guanine at C-7 and C-8 caused selectivity for the slowly exchanging site. These increases in selectivity for a site were mainly due to losses in affinity of the other site. There was an apparent requirement of the intact amino group at C-2, particularly for the slowly exchanging site. Comparison of the molecular interactions of cAMP and cGMP with their specific protein kinases showed that both nucleotides are bound by similar forces in the 2', 3' and 5' region, both bases may be bound in syn conformation, but that each base moiety is bound by different molecular interaction, thus leading to the selectivity of the two enzymes. cGMP analogs which possessed strong selectivity for the rapidly exchanging site, but not those selective for the slowly exchanging site, stimulated the binding of [3H]cGMP. Only a few cGMP analogs were more potent than cGMP in stimulating protein kinase activity. The potency of cGMP analogs as stimulators of kinase activity correlated better with the mean binding affinity for both binding sites than with the affinity for either site alone. Two analogs added in combination were synergistic in kinase activation, particularly if one analog was selective for the slowly exchanging site and the other for the rapidly exchanging site. These observations are suggestive that cGMP binding at the rapidly exchanging site stimulates cGMP binding at the slowly exchanging site and that both sites are involved in the activation process.     

金叶银杏半同胞子代无性系的叶色和色素含量变化及呈色机制分析

对4月份至11月份金叶银杏‘万年金'( Ginkgo biloba ‘Wannianjin')32个半同胞子代无性系与亲本的叶色差异进行比较;比较了不同色系叶片的色素含量和比值及叶色参数(L*、a*和b*)的变化,分析了叶色参数与叶片色素含量的相关性;并观察了不同色系的叶绿体超微结构。结果表明：32个半同胞子代无性系可被分为金黄、浅黄、草绿和蓝绿4个色系。随时间推移,草绿和蓝绿色系叶片的总叶绿素( Chl)、叶绿素a( Chla)、叶绿素b( Chlb)和类胡萝卜素( Car)含量均呈“双峰型”变化趋势,Car/Chl和Car/Chla比值的变幅均较小;而金黄和浅黄色系叶片的上述色素含量呈“升高—降低—升高”变化趋势,Car/Chl和Car/Chla比值总体呈“迅速下降—相对稳定—缓慢升高”的变化趋势。各色系叶片的上述色素含量在夏季均不同程度下降,Car/Chlb比值变化差异较大,且金黄和浅黄色系的各色素含量均低于草绿和蓝绿色系。随时间推移,金黄和浅黄色系叶片的L*、a*和b*值以及草绿和蓝绿色系叶片的L*和b*值均先降低后升高,后2个色系的a*值则先升高后下降;并且,前2个色系的L*和b*值总体上显著高于后2个色系,而a*值则总体上低于后2个色系。金黄色系的Chla和Chl含量与L*和a*值显著负相关,而其Car/Chl和Car/Chla比值则与L*、a*和b*值显著或极显著正相关;浅黄色系的Chlb含量与a*值显著负相关,其Car/Chla比值与L*和b*值以及Car/Chlb比值与a*值均显著正相关;草绿色系的Chla含量与L*值显著负相关,其Car/Chla比值与L*和b*值以及Car/Chlb比值与a*值均显著正相关;这3个色系叶片的其余指标间以及蓝绿色系叶片的各指标间均无显著相关性。观察结果显示：金黄和浅黄色系的叶绿体基粒片层发育不健全,基粒片层可见但排列较疏松,且无明显垛叠,分布范围小而稀疏;蓝绿和草绿色系叶绿体的基粒类囊体垛叠层数均较多,基粒片层发达且排列紧致、整齐,分布范围大而稠密。综合分析结果表明：‘万年金'4个色系半同胞子代无性系叶片的呈色差异和叶色变化由多种因素控制,其中,Car/Chl和Car/Chla比值高且叶绿体基粒片层发育不健全是叶片呈黄色的主要原因。     

模拟酸雨对龙眼叶绿体活性的影响

研究了模拟酸雨对龙眼叶绿体活性的影响,结果表明,pH≤3．5的酸雨胁迫使用叶绿素（Chl),类胡萝卜素（Car)含量下降,Chl含量的下降是Chla含量下降的结果,Chlb对酸雨的反应不敏感,因而Chla/Chlb下降,Chl和Car含量,Chla/Chlb随胁迫时间的延长而下降,光照加剧了酸雨对光合色素的伤害,酸雨胁迫使叶绿体光还原活性,光合磷酸化活性,H^ -ATPase活性降低,电子传递链的破坏与磷酸化解偶联,从而使叶绿体不能有效吸收,传递,转换光能,光合磷酸化过程对酸雨胁迫的反应比光还原过程敏感。     

低浓度NaCl对玉米幼苗光合作用的影响

以Hoagland溶液为基础培养液,采用沙培法,研究了0,5,10,20,30mmol／LNaCl对玉米（Zeamays）幼苗光合作用的影响。结果表明,低浓度NaCl（5,10mmol／LNaCl）处理使其叶绿素含量增加,Chla／Chlb增大,PEPCase活性提高,光合速率增强。说明低盐促进玉米的光合作用可能与叶绿素含量增加,Chla／Chlb增大以及PEPCase活性提高有关。     

美丽箬竹对模拟大气O3浓度倍增胁迫的生理响应

运用开顶式气室(OTCs)模拟当前环境大气O3浓度(对照,40～45 nL·L-1)、O3浓度倍增4倍(TR-1,92～106 nL·L-1)和O3浓度倍增2倍(TR-2,142～160 nL·L-1)胁迫,以叶片光合色素、MDA、可溶性蛋白质和可溶性糖含量及SOD和POD活性和相对电导率为指标,分析了美丽箬竹(Indocalamus decorus Q.H.Dai)对O3胁迫的生理响应规律.结果显示:随O3浓度的提高,叶片叶绿素a(Chla)、叶绿素b(Chlb)、总叶绿素和类胡萝卜素含量,SOD和POD活性,可溶性蛋白质和可溶性糖含量及二者的比值(SPC/SSC)均呈下降趋势;叶绿素a口与b的比值(Chla/Chlb)、MDA含量和相对电导率均呈增加趋势.TR-1条件下叶片POD活性和可溶性糖含量分别比对照下降38.86％和10.37％,差异显著;Chla/Chlb比值显著高于对照,其余指标均与对照无显著差异.而在TR-2条件下叶片Chla、Chlb、总叶绿素和类胡萝卜素含量显著低于对照,降幅分别为26.89％、40.91％、30.47％和20.37％;SOD和POD活性、可溶性蛋白质和可溶性糖含量以及PC/SSC比值也均显著低于对照,降幅分别为42.03％、46.62％、45.09％、11.52％和40.15％;Chla/Chlb比值、MDA含量和相对电导率均显著高于对照.另外,TR-1与TR-2处理组间光合色素含量和POD活性差异不显著,TR-2处理组MDA含量和相对电导率显著高于TR-1处理组,SOD活性显著低于后者.研究结果表明:美丽箬竹对O3胁迫具有强耐受性,可种植于O3浓度较高的环境中.     

移植和钾肥对降香黄檀光合特性与叶绿素含量的影响

以降香黄檀(Dalbergia odorifera T. Chen)为材料,分析不同移植方式和外施钾肥等培育措施对其光合参数和叶绿素(Chl)含量的影响。结果显示,断根处理后植物的最大净光合速率(Pnmax)比去冠、全冠移、对照(CK)和去冠移4种处理方式分别提高了19.25%、34.79%、40.88%和219.86%。光合参数分析结果显示,光饱和点(LSP)最高的为去冠处理,光补偿点(LCP)最高的为断根处理。断根处理后植物的净光合速率(Pn)和蒸腾速率(Tr)最大。断根、去冠、全冠移3种处理的Chla、Chlb、Chl(a+b)含量及胡萝卜素均高于CK,且断根处理后上述各指标达到最高值。外施钾肥处理中,降香黄檀最大净光合速率随钾肥用量的增加而增加,K2处理下的LSP值最大。随着钾肥施用量的增加,植物叶片的Pn、气孔导度呈上升的趋势,且植株的Chla、Chlb和Chl(a+b)含量均随钾肥用量的增加而增加,CK处理下Chla/Chlb比值显著高于K1、K2处理。研究结果表明断根、去冠、施用钾肥等处理均可显著提升降香黄檀叶片的叶绿素含量,有利于植株进行光合作用,促进生长发育。     

The quaternary structure of the plasma membrane b-type cytochrome of human granulocytes

Hydrodynamic, crosslinking and immunoprecipitation studies were performed on detergent solubilized cytochrome b to demonstrate that the two copurifying polypeptides of molecular weight 91,000 (glycosylated) and 22,000 [1,2] formed a molecular complex. The hydrodynamic studies indicated that the cytochrome b/detergent complex had a sedimentation coefficient, partial specific volume and Stokes radius of 5.25 S, 0.82 cm3/g and 6.2 nm in Triton X-100 and 6.05 S, 0.80 cm3/g and 5.6 nm in octylglucoside, respectively. These studies also indicated that the detergent-protein complex has a molecular mass of 202 and 188 kDa in Triton X-100 and octylglucoside, respectively, is asymmetric in shape with a frictional coefficient of 1.3-1.4 and binds significant amounts of detergent. The molecular mass of the protein portion of the detergent-cytochrome complex was estimated to be between 100 and 127 kDa. Crosslinking studies with disuccinimidyl suberate and alkaline cleavable bis[2-(succinimidooxy-carbonyloxy)ethyl]sulfone revealed that the Mr = 91,000 and Mr = 22,000 components of purified cytochrome b are closely associated and can be covalently bound to form a polypeptide which, by SDS-polyacrylamide gel electrophoresis, has Mr values of 110,000-120,000 and 120,000-135,000 on 8% and 11% (w/v) SDS-polyacrylamide gels, respectively. Cleavage of the crosslinked species resulted in the reappearance of the Mr = 91,000 and Mr = 22,000 species. Sedimentation profiles of crosslinked cytochrome b in linear sucrose density gradients made up in H2O were identical to those of non-crosslinked controls. A close association of the two protein species was further confirmed by the ability of antibody specific for the smaller subunit to immunoprecipitate the larger one also. Experiments aimed at identifying the heme-carrying subunit(s) were inconclusive, since dissociation of the complex resulted in loss of cytochrome b spectrum. These results, in combination with our SDS-polyacrylamide gel electrophoresis molecular-weight estimates, provide strong evidence for the cytochrome b being an alpha-beta-type heterodimer composed of a glycosylated Mr = 91,000 and non-glycosylated Mr = 22,000 polypeptide.     

On the structural role of the aromatic residue environment of the chlorophyll a in the cytochrome b6f complex

Because light is not required for catalytic turnover of the cytochrome b 6 f complex, the role of the single chlorophyll a in the structure and function of the complex is enigmatic. Photodamage from this pigment is minimized by its short singlet excited-state lifetime ( approximately 200 ps), which has been attributed to quenching by nearby aromatic residues ( Dashdorj et al., 2005). The crystal structure of the complex shows that the fifth ligand of the chlorophyll a contains two water molecules. On the basis of this structure, the properties of the bound chlorophyll and the complex were studied in the cyanobacterium, Synechococcus sp. PCC 7002, through site-directed mutagenesis of aromatic amino acids in the binding niche of the chlorophyll. The b 6 f complex was purified from three mutant strains, a double mutant Phe133Leu/Phe135Leu in subunit IV and single mutants Tyr112Phe and Trp125Leu in the cytochrome b 6 subunit. The purified b 6 f complex from Tyr112Phe or Phe133Leu/Phe135Leu mutants was characterized by (i) a loss of bound Chl and b heme, (ii) a shift in the absorbance peak and increase in bandwidth, (iii) multiple lifetime components, including one of 1.35 ns, and (iv) relatively small time-resolved absorbance anisotropy values of the Chl Q y band. A change in these properties was minimal in the Trp125Leu mutant. In vivo, no decrease in electron-transport efficiency was detected in any of the mutants. It was concluded that (a) perturbation of its aromatic residue niche influences the stability of the Chl a and one or both b hemes in the monomer of the b 6 f complex, and (b) Phe residues (Phe133/Phe135) of subunit IV are important in maintaining the short lifetime of the Chl a singlet excited state, thereby decreasing the probability of singlet oxygen formation.     

A trimeric supercomplex of the oxygen-tolerant membrane-bound [NiFe]-hydrogenase from Ralstonia eutropha H16

The oxygen-tolerant membrane-bound [NiFe]-hydrogenase (MBH) from Ralstonia eutropha H16 consists of three subunits. The large subunit HoxG carries the [NiFe] active site, and the small subunit HoxK contains three [FeS] clusters. Both subunits form the so-called hydrogenase module, which is oriented toward the periplasm. Membrane association is established by a membrane-integral cytochrome b subunit (HoxZ) that transfers the electrons from the hydrogenase module to the respiratory chain. So far, it was not possible to isolate the MBH in its native heterotrimeric state due to the loss of HoxZ during the process of protein solubilization. By using the very mild detergent digitonin, we were successful in isolating the MBH hydrogenase module in complex with the cytochrome b. H(2)-dependent reduction of the two HoxZ-stemming heme centers demonstrated that the hydrogenase module is productively connected to the cytochrome b. Further investigation provided evidence that the MBH exists in the membrane as a high molecular mass complex consisting of three heterotrimeric units. The lipids phosphatidylethanolamine and phosphatidylglycerol were identified to play a role in the interaction of the hydrogenase module with the cytochrome b subunit.     

Consecutive binding of chlorophylls a and b during the assembly in vitro of light-harvesting chlorophyll-a/b protein (LHCIIb)

The apoprotein of the major light-harvesting chlorophyll a/b complex (LHCIIb) is post-translationally imported into the chloroplast, where membrane insertion, protein folding, and pigment binding take place. The sequence and molecular mechanism of the latter steps is largely unknown. The complex spontaneously self-organises in vitro to form structurally authentic LHCIIb upon reconstituting the unfolded recombinant protein with the pigments chlorophyll a, b, and carotenoids in detergent micelles. Former measurements of LHCIIb assembly had revealed two apparent kinetic phases, a faster one (tau1) in the range of 10 s to 1 min, and a slower one (tau2) in the range of several min. To unravel the sequence of events we analysed the binding of chlorophylls into the complex by using time-resolved fluorescence measurements of resonance energy transfer from chlorophylls to an acceptor dye attached to the apoprotein. Chlorophyll a, offered in the absence of chlorophyll b, bound with the faster kinetics (tau1) exclusively whereas chlorophyll b, in the absence of chlorophyll a, bound predominantly with the slower kinetics (tau2). In double-jump experiments, LHCIIb assembly could be dissected into a faster chlorophyll a and a subsequent, predominantly slower chlorophyll b-binding step. The assignment of the faster and the slower kinetic phase to predominantly chlorophyll a and exclusively chlorophyll b binding, respectively, was verified by analysing the assembly kinetics with a circular dichroism signal in the visible domain presumably reflecting the establishment of pigment-pigment interactions. We propose that slow chlorophyll binding is confined to the exclusively chlorophyll b binding sites whereas faster binding occurs to the chlorophyll a binding sites. The latter sites can bind both chlorophylls a and b but in a reversible fashion as long as the complex is not stabilised by proper occupation of the chlorophyll b sites. The resulting two-step model of LHCIIb assembly is able to reconcile the highly specific binding sites containing either chlorophyll a or b, as seen in the recent crystal structures of LHCIIb, with the observation of promiscuous binding sites able to bind both chlorophyll a and b in numerous reconstitution analyses of LHCIIb assembly.     

In vivo labeling of mitochondrial complex I (NADH:ubiquinone oxidoreductase) in rat brain using [(3)H]dihydrorotenone

Defects in mitochondrial energy metabolism have been implicated in several neurodegenerative disorders. Defective complex I (NADH:ubiquinone oxidoreductase) activity plays a key role in Leber's hereditary optic neuropathy and, possibly, Parkinson's disease, but there is no way to assess this enzyme in the living brain. We previously described an in vitro quantitative autoradiographic assay using [(3)H]dihydrorotenone ([(3)H]DHR) binding to complex I. We have now developed an in vivo autoradiographic assay for complex I using [(3)H]DHR binding after intravenous administration. In vivo [(3)H]DHR binding was regionally heterogeneous, and brain uptake was rapid. Binding was enriched in neurons compared with glia, and white matter had the lowest levels of binding. In vivo [(3)H]DHR binding was markedly reduced by local and systemic infusion of rotenone and was enhanced by local NADH administration. There was an excellent correlation between regional levels of in vivo [(3)H]DHR binding and the in vitro activities of complex II (succinate dehydrogenase) and complex IV (cytochrome oxidase), suggesting that the stoichiometry of these components of the electron transport chain is relatively constant across brain regions. The ability to assay complex I in vivo should provide a valuable tool to investigate the status of this mitochondrial enzyme in the living brain and suggests potential imaging techniques for complex I in humans.     

Application of photoaffinity labeling with [(3)H] all trans- and 9-cis-retinoic acids for characterization of cellular retinoic acid--binding proteins I and II

Cellular retinoic acid-binding proteins (CRABPs) are carrier proteins thought to play a crucial role in the transport and metabolism of all-trans-retinoic acid (atRA) and its derivatives within the cell. This report describes a novel photoaffinity-based binding assay involving competition between potential ligands of CRABP and [(3)H]atRA or [(3)H]-9-cis-RA for binding to the atRA-binding sites of CRABP I and II. Photoaffinity labeling of purified CRABPs with [(3)H]atRA was light- and concentration-dependent, saturable, and protected by several retinoids in a concentration-dependent manner, indicating that binding occurred in the CRABP atRA-binding site. Structure-function relationship studies demonstrated that oxidative changes to the atRA beta-ionone ring did not affect ligand potency. However, derivatives lacking a terminal carboxyl group and some cis isomers did not bind to CRABPs. These studies also identified two novel ligands for CRABPs: 5,6-epoxy-RA and retinoyl-beta-D-glucuronide (RAG). The labeling of both CRABPs with 9-cis-RA occurred with much lower affinity. Experimental evidence excluded nonspecific binding of RAG to CRABPs and UDP-glucuronosyltransferases, the enzymes responsible for RAG synthesis. These results established that RAG is an effective ligand of CRABPs. Therefore, photoaffinity labeling with [(3)H]atRA can be used to identify new ligands for CRABP and retinoid nuclear receptors and also provide information concerning the identity of amino acid(s) localized in the atRA-binding site of these proteins.     

强光胁迫对濒危植物金花茶幼苗生长和叶绿素荧光参数的影响

以当年生盆栽金花茶实生苗为材料,研究不同程度的强光胁迫（25％、50％和100％自然光强,以8%自然光强为对照）对其生长、生物量、叶片光合色素含量、叶绿素荧光参数的影响。结果表明：在不同程度的强光胁迫下,金花茶幼苗的生长均受到抑制,随着胁迫程度的增强,金花茶叶片颜色由深绿变为浅绿、黄绿色,叶片灼伤愈来愈严重;植株抽稍时间推迟,抽稍后长出的新叶长势较差;幼苗死亡率越来越高。幼苗根生物量、茎生物量、叶生物量和总生物量均随胁迫程度的升高而显著降低,强光胁迫对叶生物量的影响最大,根生物量次之,对茎生物量的影响最小。随着胁迫程度的增强,叶片叶绿素总量（Chl）、叶绿素a（Chla）、叶绿素b（Chlb）含量均显著降低,Chla/Chlb和Car/Chl显著升高。叶绿素荧光参数F_o、F_m、F_v、F_v/F_m、F_v/F_o均随胁迫程度的升高降低,强光胁迫使PSⅡ受到了伤害,光合作用原初反应过程受抑制,光合电子传递受到影响,从而抑制植株的正常生长。