Effect of Sodium Dodecyl Sulfate on Structure and Spectroscopic Characteristics of Water-Soluble Chlorophyll Protein Complex Isolated from Stems of Lepidium virginicum

The relationship between structure and spectroscopic characteristicsof the watersoluble chlorophyll protein complex isolated fromstems of Lepidium virginicum (CP663S) was studied. Additionof 0.08% SDS induced a red shift of the 663 nm absorption maximum.At the same time, under excitation at 435 nm, the maximum offluorescence emission shifted from 672 nm to 675 nm and thefluorescence yield increased. When CP663S was excited at 480nm, the 660 nm emission band of chlorophyll b became more prominent.Fluorescence lifetime of emission from chlorophyll a increasedon addition of SDS. The energy transfer from chlorophyll b tochlorophyll a was decreased by the SDS addition, as judged bythe fluorescence spectra and lifetime measurement. Symmetricalpositive and negative peaks of the circular dichroism (CD) spectrumaround 669 nm, which indicate the interaction between chlorophylla molecules at short distances, disappeared after addition ofSDS. These SDS-induced changes of spectroscopic characteristicsoccurred in similar SDS concentration ranges and were reversible.SDS polyacrylamide gel electrophoresis cleaved CP663S into subunits.Chlorophyll molecules moved with protein moieties. Glutaraldehydetreatment suppressed the effects of SDS on absorption, fluorescenceand CD characteristics. We conclude that chlorophyll moleculesin CP663S are in the hydrophobic region of the protein and theinteraction between chlorophyll a molecules occurs at shortdistances. Changes of spectroscopic characteristics are a resultof cleavage of CP663S. ¹Present address: National Institute for Basic Biology, Okazaki444, Japan. (Received November 22, 1982; Accepted May 31, 1983)     

Micropropagation of Lepidium virginicum (Brassicaceae), a plant with antiprotozoal activity

Summary Micropropagation is a technique to ensure a constant and uniform source of medicinal plants. In this report, we describe the micropropagation of Lepidium virginicum L. (Brassicaceae), a wild plant used as an antiamoebic in traditional Mexican medicine. In vitro-germinated seeds were cultured in Murashige and Skoog (MS) medium to obtain pathogen-free cotyledons, hypocotyls, and apical bud (AB) explants. For induction of morphogenesis, the effect of cytokinins, benzyladenine (BA) and kinetin (KN), combined with auxin, indole-3-acetic acid (IAA) was evaluated. The best rate of shoot proliferation was induced 15 d after culture on MS mineral medium supplemented with IAA∶KN (0.57∶13.94 μM) from AB explants. Maximum shoot elongation was achieved without plant growth regulators. The effect of indole-3-butyric acid (IBA) (14.76 μM) was evaluated for in vitro root induction; 60 d after culture all the shoots had developed roots. All rooted plants were successfully transferred to pots and 100% acclimatized in ex vitro conditions. The methanol extracts from the micropropagated active explants of L. virginicum showed and IC₅₀ antiprotozoal value between 141.90 and 268.53 μg ml⁻¹.     

芸苔（菜心）茎叶中的几丁质结合蛋白的分离纯化和某些特性分析

用脱氨再生几丁质亲和层析和羧甲基-纤维素CM52离子交换柱层析从芸苔（菜心）的茎叶中纯化了4种几丁质结合蛋白：CBPa,CBPb、CBPc和CBPd,SDS-PAGE显示CBPa和CBPb的分子量分别为29．04kDa和30．68kDa。凝胶过滤方法测定CBPa和CBPb的分子量分别为29．04kDa和31．2kDa,表明两者都是单体酶。4种CBP蛋白均有几丁质酶活性,其中CBPa和CBPb还有溶菌酶活性。EDTA对CBPa和CBPb溶菌酶的活性均无影响,各种类型的几丁质对CBPa和CBPb都有较强的吸附作用,PAS染色法分析表明CBPa和CBPb均为糖蛋白。     

Chemical, physicochemical and spectrophotometric properties of crystalline chlorophyll-protein complexes from Lepidium virginicum L

Two kinds of water-soluble chlorophyll-protein complexes were prepared from leaves of Lepidium virginicum L., one (CP661) from the plant cultivated in a green house from seeds collected near Mono Lake, CA, and the other (CP-663) from a plant collected at Narashino, Chiba, Japan, by ammonium sulfate fractionation followed by column chromatography on DEAE-cellulose and Sephacryl S-200. The chlorophyll . proteins were further purified by crystallization. CP661 has absorption peaks at 661, 468, 439, 419, 380, 339 and 272 nm. CP663 had absorption peaks at 663, 469, 438, 419, 379, 338 and 272 nm. Estimated molecular weights were 78 000 for CP661 and 80 000 for CP663 by gel filtration chromatography and 83 000 for CP661 and 107 000 for CP663 by an equilibrium sedimentation method. 1 mol chlorophyll . protein contained 4 mol chlorophyll a and b with ratios of 1.0 in CP661 and 1.6 to 1.9 in CP663, but no carotenoids. These characters are different from those of chlorophyll-protein complexes which are prepared from the thylakoid membranes of chloroplasts with detergents.     

杨树胞外蛋白的提取分离及2-D电泳体系的建立

该研究以美洲黑杨杂种优良无性系NL895杨(Populus deltoides×Populus euramericana cv.)组培苗叶片和茎段为研究对象,对NL895杨叶片细胞壁蛋白(cell wall proteins,CWPs)和茎段质外体蛋白(apoplastic proteins,APPs)的提取、分离和双向电泳等技术进行了系统研究。结果表明:10g以上叶片、超声波破碎10min、CaCl2法提取的细胞壁蛋白效果较好,经G-6-PDH酶活性检测,提取的细胞壁蛋白胞质污染率较低;真空渗透法提取的茎段质外体蛋白胞质污染率较前者高,但在允许范围之内。TCA/丙酮沉淀法纯化提取的细胞壁蛋白、pH 4~7的24cm胶条、上样量为500μg的双向电泳体系,其蛋白电泳图谱中的斑点多而清晰,斑点数达550多个,是叶片细胞壁蛋白电泳分析较适合的体系;pH 3~10胶条对茎段质外体蛋白的电泳分离效果较好。该研究初步建立了杨树胞外蛋白的提取、分离及2-DE电泳体系,为木本植物胞外蛋白的研究奠定了基础。     

不同叶龄黄瓜叶片叶绿素蛋白质复合物组分的比较研究

用SDS — 聚丙烯酰胺凝胶电泳的方法,对比分析了老、嫩黄瓜叶片叶绿素蛋白质复合物之间的差异,发现嫩黄瓜叶片中缺少1条属光系统I的CPIb带。从低温荧光发射光谱观察到,嫩黄瓜的光系统I相对高于光系统Ⅱ,而老黄瓜则相反。指出在叶绿体发育过程中首先形成光系统I,以后是光系统Ⅱ。我们还注意到,叶片中的F685/F735比值与叶绿素蛋白质复合物中的单体／寡聚体比值之间呈正相关关系。     

光质对绿豆幼苗叶片超微弱发光及叶绿素含量的影响

以绿豆幼苗为试材,测定其叶片超弱发光(UBE)及叶绿素含量在不同光质条件下的变化,并探讨两者之间的关系.结果表明,生长在不同光质下绿豆幼苗叶片的UBE及延迟发光衰减参数1/P都随着其生长不断增强,且生长在白光下绿豆幼苗的UBE是生长在其他光质(红、黄、蓝、绿)下幼苗的2倍以上,而红光、黄光和绿光处理之间无显著差异;生长在白光下的绿豆幼苗叶片叶绿素含量显著高于红、黄、蓝、绿光处理幼苗,而红光和黄光处理又显著高于蓝光和绿光处理.研究发现,光质对绿豆幼苗叶片超弱发光和叶绿素含量影响相似,绿豆幼苗叶片超弱发光可能与叶绿体的发育和光合作用有关.     

Changes in the Levels of Chlorophyll and Light-Harvesting Chlorophyll a/b Protein of PS II in Rice Leaves Aged under Different Irradiances from Full Expansion through Senescence

Effects of irradiance on changes in the amounts of chlorophyll(Chl) and light-harvesting chlorophyll a/b protein of PS II(LHCII) were examined in senescing leaves of rice (Oryza sativaL.). Results of treatments at two irradiances (100% and 20%natural sunlight) were examined after the full expansion ofthe 13th leaf throughout the course of senescence. With 20%sunlight, the Chl content decreased only a little during leafsenescence, while with 100% sunlight it decreased appreciably.Similarly, the amount of LHCII protein during treatment with20% sunlight remained almost constant. However, the ratio ofChl a/b during the shade treatment decreased significantly andthe rate of decrease was greater than during the full-sunlighttreatment. The ratio of Chl a/b for Chl a and b bound to LHCIIwas about 1.2, irrespective of leaf age or irradiance treatment.When the amounts of Chl bound to LHCII were calculated fromthe total leaf content of Chl and the ratio of Chl a/b, assuminga ratio of Chl a/b bound to LHCII of 1.2, they were well correlatedwith the amounts of LHCII protein. Changes in the amounts of LHCII synthesized during the two irradiancetreatments were examined using an ¹⁵ tracer. Incorporation of¹⁵N into LHCII declined dramatically during both treatmentsfrom full expansion through senescence, suggesting that therewas little synthesis of LHCII protein during that time. In addition,the amount of LHCII synthesized during senescence was lowerduring the shade treatment than during the 100% sunlight treatment.These results indicate that the absence of an apparent changein levels of LHCII with shade treatment during senescence wascaused by the very low rate of turnover of LHCII protein. (Received June 17, 1992; Accepted September 28, 1992)     

大量分离叶绿素a和b的方法

在原有分离叶绿素a和b的DEAE SepharoseCL 6B和SepharoseCL 6B柱层析法的基础上 ,用较大的层析柱 ,在保持分离效果的前提下 ,提高流速 ,并对层析柱进行原位再生处理 ,实现循环分离 ,可节省装柱和平衡时间 ,快速大量分离纯化叶绿素并可循环再生。 10 0 g菠菜叶可分离得到叶绿素a约 5 0mg ,叶绿素b约 15mg。     

Isolation and Characterization of a Light-Harvesting Chlorophyll a/b Protein Complex Associated with Photosystem I

A chlorophyll a/b protein complex has been isolated from a resolved native photosystem I complex by mildly dissociating sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The chlorophyll a/b protein contains a single polypeptide of molecular weight 20 kilodaltons, and has a chlorophyll a/b ratio of 3.5 to 4.0. The visible absorbance spectrum of the chlorophyll a/b protein complex showed a maximum at 667 nanometers in the red region and a 77 K fluorescence emission maximum at 681 nanometers. Alternatively, by treatment of the native photosystem I complex with lithium dodecyl sulfate and Triton, the chlorophyll a/b protein complex could be isolated by chromatography on Sephadex G-75. Immunological assays using antibodies to the P₇₀₀-chlorophyll a-protein and the photosystem II light-harvesting chlorophyll a/b protein show no cross-reaction between the photosystem I chlorophyll a/b protein and the other two chlorophyll-containing protein complexes.     

Synthesis and Breakdown of the Apoproteins of Light-Harvesting Chlorophyll a/b Proteins in Chlorophyll b-deficient Mutants of Rice

Turnover, in the light, of apoproteins of light-harvesting chlorophylla/6-proteins for Photo-system I and II (LHC-I and LHC-II, respectively)was studied with the wild-type and three chlorophyll 6-deficientmutants of rice. (1) Synthesis of the 24 and 25 kDa apoproteinsof LHC-II and the 20 and 21 kDa apoproteins of LHC-I was examinedby incubating leaf segments with [³⁵S]-methionine. The threerice mutants, chlorina 2, which totally lacks chlorophyll b,and chlorina 11 and 14, which are partially deficient in chlorophyllb, synthesized the apoproteins as rapidly as did the wild typerice. (2) Pulse-chase experiments showed that breakdown of theapoproteins proceeded slowly, such that only a small proportionof the newly synthesized apoproteins was lost during the 48h of the chase in normal rice leaves. By contrast, large fractionsof the labelled apoproteins were rapidly degraded within thefirst several hours of the chase period in the chlorina mutants.The greater the deficiency in chlorophyll b of the mutant, thelarger were the rate and extent of the protein breakdown. Thisresult indicates that chlorophyll b is needed to stabilize theapoproteins of LHC-II and LHC-I. (3) However, even in chlorina2, there were small fractions of the apoproteins with lifetimesas long as those of apoproteins in the wild-type rice, suggestingthat the newly synthesized apoproteins are partially protectedby a factor(s) other than chlorophyll b. (4) The rate of turnoverof the apoproteins was significantly reduced in the dark andstrongly inhibited by prior treatment of leaf segments withchloramphenicol. (Received November 24, 1988; Accepted March 17, 1989)     

中药巴戟天中水溶性多糖的分离及结构鉴定(英文)

中药巴戟天(Morinda officinalis)的根经过水提、醇沉、脱色和离子交换得到水溶性多糖(MOHP-1),经过FIIR、HPLC、NMR和GC-MS分析,最后确定MOHP-1是由果糖以(2→1)糖苷键连接的菊淀粉性多糖,其结构为α-Glcp-(1→2)-[β-Fruf-(2→1)-β-Frucf]n。     

中国红豆杉枝叶中的紫杉烷二萜

从中国红豆杉(Taxus chinensis)枝叶的乙醇提取物中分离得到8个紫杉烷二萜,通过波谱分析分别确定为：14p羟基巴卡亭Ⅵ(1),巴卡亭Ⅵ(2),巴卡亭Ⅳ(3),1β-去羟基巴卡亭Ⅳ(4),云南红豆杉酯甲(5),2α-去乙酰-2α-苯甲酰基-13α-乙酰基云南紫杉亭(6),5α-羟基-2α,7βp,9α,10β,13α-五乙酰氧基紫杉-4(20),11-二烯(7)和Taxaein(8)。其中化合物1为新化合物,并报道八个化合物在丙酮中测得的核磁共振信号,化合物8的数据属首次报道。     

Isolation of Three Crystalline Proteins from Beef Liver After Partial Purification by Acetone Fractionation

The isolation of three proteins in crystalline form from ground beef liver is described. These proteins are FTBL protein (Arch. Biochem. Biophys. 188, 251–265 (1978), crotonase, and catalase. Crotonase is isolated by crystallization from a 32 acetone extract of the ground liver. FTBL protein and catalase can subsequently be isolated from the same extract. For optimal yield and ease of isolation, FTBL protein is isolated from a 46.5% acetone extract from which catalase can subsequently be crystallized by dialysis.

The isolation of FTBL protein as well as the isolation of catalase involves a preliminary fractional precipitation and solution before crystallization can be achieved. Isopropanol can be substituted for acetone in the isolation of the above three proteins and in the case of catalase, results in an exceptionally high yield.

Methods for the recrystallization of the proteins are presented and the role of organic solvents in recrystallization is discussed.     

Migration of Electronic Energy from Chlorophyll b to Chlorophyll a in Solutions

Absorption, emission, and fluorescence excitation spectra of pure solutions of chlorophyll a (Chl a) and chlorophyll b (Chl b) in diethyl ether and of equimolecular mixed solutions of the two pigments, were determined at room temperature as functions of concentration (in the range from 5 × 10^-6 M to 4 × 10^-3 M) and of wavelength of the exciting light (in the regions 380-465 and 550-650 nm). The efficiency of energy transfer from Chl b to Chl a, derived from these data, was found to depend on the wavelength of exciting light. Furthermore, the transfer efficiency calculated from sensitization of Chl a fluorescence by Chl b was substantially smaller than that calculated from quenching of Chl b fluorescence by Chl a. Both these effects are tentatively explained as evidence of superposition of a “fast” energy transfer (taking place before the Boltzmann distribution of vibrational energy had been reached) upon the “delayed” transfer, which takes place after vibrational equilibration. The first-named mechanism is made possible by overlapping of the absorption bands of the two pigments; the second, by overlapping of the emission band of Chl b and the absorption band of Chl a. The first mechanism can lead to repeated transfer of excitation energy between pigment molecules, the second only to a one-time transfer from the donor to the acceptor. Both mechanisms could be of the same, second-order type, with the transfer rate proportional to r^-6. An alternative is for the fast mechanism to be of the first order, with the transfer rate proportional to r^-3, but spectroscopic evidence seems to make this alternative less probable.     

Development and Distribution of a Lectin from the Stems and Leaves of Dolichos biflorus

The stems and leaves of the Dolichos biflorus plant contain a lectin that cross-reacts with antiserum against the seed lectin. This cross-reactive material (CRM) was followed during early seedling growth, stem elongation, and seed development using a specific radioimmunoassay.

No CRM was detected in developing seeds, but very low levels were found in dormant and imbibed seeds. As germination proceeds, the CRM accumulates at the apex of both etiolated and green seedlings in the epicotyl and leaves. Lower amounts of CRM are found in the cotyledons and hypocotyl, but no CRM was detected in the roots.

The amount of CRM in the first and second stem internodes increases during elongation and gradually declines after the completion of elongation. Approximately 80% of the CRM in the stems of 19-day-old Dolichos biflorus plants is associated with the elongating tissues. These results are discussed with respect to the possible roles of lectins in plants.

     

二氧化氯对球形棕囊藻叶绿素a、蛋白质、DNA含量的影响

分析了二氧化氯(1.0、1.5、2.0、2.5mgL-1,作用96h;2.5、6.0、8.0、16.0mgL-1,作用60min)对球形棕囊藻叶绿素a、蛋白质含量、氨基酸组成、核酸含量的影响,用透视电镜对二氧化氯(0.5mgL-1,0.8mgL-1)作用24h后藻细胞形态的变化进行了观察,以探讨二氧化氯去除赤潮藻的机理。结果表明,二氧化氯对球形棕囊藻叶绿素a、蛋白质、DNA的含量均有影响。实验各组(1.0-2.5mgL-1,2.5-16.0mgL-1)球形棕囊藻的叶绿素a、蛋白质、DNA的含量均显著低于对照。二氧化氯浓度超过2mgL-1时,球形棕囊藻的叶绿素a、蛋白质、DNA的含量持续降低,氨基酸相对含量发生明显变化;半胱氨酸、酪氨酸和赖氨酸等的相对含量明显降低,而组氨酸、缬氨酸和苯丙氨酸则明显增加。当二氧化氯(2.5-16.0mgL-1)作用3min后,DNA漏出率在13%-18%之间;电镜观察发现,二氧化氯可使细胞膜破损,引起内容物外泄。这些结果显示,二氧化氯能以单分子形式进入细胞,引起叶绿素、蛋白质结构的变化,并破坏藻细胞膜系统,最终导致藻细胞死亡。     

不同季节灰毡毛忍冬茎叶中绿原酸和木犀草苷含量的分析

目的：比较分析不同季节的灰毡毛忍冬茎叶中绿原酸和木犀草苷的含量,为综合利用茎叶资源提供参考依据.方法：采用蒸制晒干法对灰毡毛忍冬的茎叶进行干燥处理,利用超声波法提取样品中的绿原酸和木犀草苷并通过高效液相色谱法进行含量测定.结果：绿原酸和木犀草苷含量在不同季节的灰毡毛忍冬茎叶中差异明显.绿原酸含量依次为花＞叶＞茎,以秋季（11月份）叶片样品中的绿原酸含量最高（3.031％）,是花蕾中含量的96％;木犀草苷含量总体趋势为叶＞花和茎,以冬季（2月份）叶片样品中的含量最高（0.558％）,是花蕾中含量的4.6倍;二者均高于2010版《中国药典》对山银花和金银花指标物质含量的规定.结论：秋冬季节对灰毡毛忍冬进行休眠期整型修剪,副产物叶片资源丰富,药源成分充足,具有广阔的开发应用前景.     

Conversion of Chlorophyll b to Chlorophyll a and the Assembly of Chlorophyll with Apoproteins by Isolated Chloroplasts

The photosynthetic apparatus is reorganized during acclimation to various light environments. During adaptation of plants grown under a low-light to high-light environment, the light-harvesting chlorophyll a/b-protein complexes decompose concomitantly with an increase in the core complex of photosystem II. To study the mechanisms for reorganization of photosystems, the assembly of chlorophyll with apoproteins was investigated using isolated chloroplasts. When [14C]chlorophyllide b was incubated with chloroplasts in the presence of phytyl pyrophosphate, it was esterified and some of the [14C]chlorophyll b was converted to [14C]chlorophyll a via 7-hydroxymethyl chlorophyll. [14C]Chlorophyll a and b were incorporated into chlorophyll-protein complexes. Light-harvesting chlorophyll a/b-protein complexes of PSII had a lower [14C]chlorophyll a to [14C]chlorophyll b ratio than P700-chlorophyll a-protein complexes, indicating the specific binding of chlorophyll to apoproteins in our systems. 7-Hydroxymethyl chlorophyll, an intermediate molecule from chlorophyll b to chlorophyll a, did not become assembled with any apoproteins. These results indicate that chlorophyll b is released from light-harvesting chlorophyll a/b-protein complexes of photosystem II and converted to chlorophyll a via 7-hydroxymethyl chlorophyll in the lipid bilayer and is then used for the formation of core complexes of photosystems. These mechanisms provide the fast, fine regulation of the photosynthetic apparatus during construction of photosystems.