Determination of Anomeric Configuration of Furanoside Derivatives by Carbon-13 NMR

A 24 kDa protein was isolated from tartary buckwheat seeds by using chromatography of Superdex 75 gel filtration and Resource Q ion-exchange column. SDS-PAGE and Sephacryl S-200 gel filtration were used to provide information about the molecular mass of the protein purified from tartary buckwheat. The protein was composed of 215 amino acid residues and showed strong IgE binding activity in an ELISA test to the sera colleted from two patients allergic to buckwheat. These results suggested that the purified 24 kDa protein from tartary buckwheat seeds was an important functional protein and was relatively specific for buckwheat-allergic patients. It should be a very useful tool in the diagnosis of buckwheat allergy in the future.     

一种苦荞麦种子蛋白酶抑制剂的纯化、特性及其抗虫活性

蛋白酶抑制剂广泛存在于生物体内, 是自然界含量最为丰富且具有一定防御作用的蛋白种类之一. 本文采用离子交换层析和凝胶层析等方法,从苦荞麦种子中分离出一种胰蛋白酶抑制剂（TBTI-Ⅱ）. SDS-PAGE分析表明,TBTI Ⅱ的分子量约9.0 kD,由80个氨基酸残基组成,分子中含有较多的 Glu, Asp 和Arg. TBTI-Ⅱ具有较高热稳定性.当在100℃加热处理10 min后,仍保留有67.6%的抑制剂活性. 动力学测定显示,来自苦荞麦中的TBTI-Ⅱ对胰蛋白酶的抑制作用常数(Ki)为1.01×10－4 mol/L. 另外,将含有不同活力单位的苦荞麦蛋白酶抑制剂掺入到棉铃虫的饲料中进行饲养试验显示,TBTI-Ⅱ具有明显的抑制棉铃虫生长的作用. 这些结果表明,来自苦荞麦种子中的小分子蛋白酶抑制剂可能是一种潜在的抗虫因子.     

TBa多克隆抗体的制备及其重组蛋白免疫活性的鉴定

目的:TBa(tartary buckwheat allergen)是苦荞麦中的主要过敏原。为了研究TBa的结构与功能关系并鉴定其重组蛋白的特异性免疫活性。方法:采用之前从种子中分离纯化的天然TBa作为抗原免疫小鼠,制备抗血清,建立了类似竞争酶联免疫吸附检测TBa的分析方法。结果:对重组TBa的免疫活性鉴定表明,采用该方法制备的多克隆抗体能满足重组TBa的免疫活性鉴定,这为下一步研究其免疫学功能奠定了基础。     

The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein.All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds.     

苦荞和甜荞查尔酮合成酶基因的克隆及序列比较

以苦荞品种‘西农9920’和甜荞品种‘西农9976’为材料,根据其它植物查尔酮合成酶(chalcone synthase,CHS)基因DNA序列的保守区域设计的一对简并引物,进行PCR扩增,从2种荞麦基因组中克隆出了长度均为860 bp的CHS基因片段,对其进行回收、克隆,挑选阳性克隆测序;序列分析表明这2个片段含有CHS基因的N端和C端的结构域,分别为苦荞和甜荞的CHS基因片段,命名为FtCHS和FeCHS。对获得的2种荞麦CHS基因的DNA序列进行比较分析,发现两者间存在多达43处单碱基多态性,这些单碱基多态性可能是苦荞和甜荞种子中类黄酮含量差异的重要原因之一。苦荞和甜荞CHS与其它植物CHS的氨基酸序列的进化分析表明,其与同为蓼科的掌叶大黄和石竹科的满天星的同源性较近。     

自交可育甜荞、金苦荞、米苦荞不同品系总黄酮、粗蛋白及其蛋白组分含量研究北大核心CSCD

为探究荞麦新品种的营养保健价值,该文对自交可育甜荞、金苦荞、米苦荞共56个不同品系荞麦种子的粗蛋白、总黄酮、蛋白组分含量及其果实性状的变异进行了研究。结果表明:(1)甜荞、金苦荞、米苦荞种子中粗蛋白含量平均值分别为13.19%、15.44%、11.75%,总黄酮含量平均值分别为0.14%、2.50%、2.09%,清蛋白含量的平均值分别为5.22%、6.13%、4.56%,球蛋白含量的平均值分别为1.29%、1.15%、0.91%,醇溶蛋白含量的平均值分别为0.42%、0.58%、0.55%,谷蛋白含量的平均值分别为2.66%、3.36%、2.80%,三种荞麦的蛋白组分均符合清蛋白>谷蛋白>球蛋白>醇溶蛋白。(2)果实性状中,甜荞果实千粒重、果实面积、果实直径的变异系数最大,米苦荞果实周长、果实长宽比、果实长、果实宽和50 mL容重的变异系数最大。(3)相关分析表明甜荞种子粗蛋白含量与果实长宽比、果实长,金苦荞种子粗蛋白含量与果实周长、果实长,米苦荞种子粗蛋白含量与果实宽、总黄酮含量与果实面积、果实宽、果实直径、50 mL容重的相关性均达到了显著或极显著水平。(4)该研究筛选出甜荞(1808-166贵甜2号优系)、金苦荞(多苦74、多苦78)、米苦荞(1906-136黑米荞麦、43-2)等高蛋白含量或高黄酮含量的荞麦品系。该研究结果对荞麦优良品种的选育和荞麦新产品的开发具有一定指导意义。     

Purification and differential expression of enolase from maize

Enolase was purified from maze ( Zea mays L. inbred B73)seeds to a 55 and 56 kDa protein doublet based upon sodium dodecyl sulfate – polyacrylamide gel electrophoresis. Purification included ammonium sulfate-precipitation, gel filtration, Mono Q, and Phenyl Superose chromatography. Two-dimensional gels further resolved the 56 kDa protein into three isoselectric forms. Polyclonal antibodies raised against the purified proteins, were found to bind specifically to both the 55 and 56 kDa proteins during purification. Theses antibodies did not recognized a 56 kDa protein when the strain was complemented with maize enolase (pZM245). Maize enolase antibodies recognized a extracts indicated that the 55 kDa form of enolase was more abundant in roots. Enolase protein levels remained unchanged in maize roots after 24 h of anaerobiosis, even though the specific activity of enolase increased to twice its initial levels. A plastid form of enolase in maize could not be found as either enolase activity or protein (with immunoblots).     

苦荞全基因组11S种子储藏蛋白基因的鉴定及表达分析

以苦荞（Fagopyrum tataricum（L.）Gaertn）全基因组数据为平台,采用生物信息学方法,挖掘出9个11S种子储藏蛋白基因,并对其定位、蛋白结构、系统发育及表达模式进行了分析。结果表明,苦荞9个11S种子储藏蛋白基因编码的蛋白长度为189~914 aa,等电点位于5.18~9.82之间,分子量为21.27~103.33 kD;定位分析结果显示,这些成员位于苦荞基因组的6条连锁群上（Megascaffold2/5以及scaffold77/344/395/861）;序列比对分析发现,除了1个11S种子储藏蛋白sample1_00009513-RA具有1个cupin保守结构域外,其余8个都含有2个cupin结构域,并且在cupin保守结构域中,苦荞和拟南芥（Arabidopsis thaliana（L.）Heynh）共有14个保守的氨基酸残基;蛋白结构预测表明,苦荞11S种子储藏蛋白的结构具有2种类型;苦荞与其它6个物种[拟南芥、花生（Arachis hypogaea Linn.）、大豆（Glycine max（Linn.）Merr.）、杏仁（Armeniaca vulgaris Lam.）、胡桃（Juglans regia L.）和芝麻（Sesamum indicum Linn.）]11S种子储藏蛋白以及苦荞过敏蛋白（TBb和TBt）系统发育分析结果表明,这些蛋白可以分为3类,共具有4对旁系同源蛋白和3对直系同源蛋白;与已报道的苦荞过敏性储藏蛋白以及其它5个物种（花生、大豆、杏仁、胡桃和芝麻）的11S过敏蛋白比较发现,5个11S种子储藏蛋白（sample1_00013128-RA、sample1_00013130-RA、sample1_00021677-RA、sample1_00021668-RA和sample1_00021674-RA）与苦荞2个过敏蛋白的同源性较高,同时它们与胡桃11S过敏蛋白的同源性最高,但尚需进一步实验来确定这5个成员是否为食物过敏原;RNA-Seq转录组数据显示,4个基因（sample1_00018411-RA、sample1_00026786-RA、sample1_00021674-RA、sample1_00022718-RA）在2种荞麦属植物的灌浆期种子中表达水平较高,且在‘大苦1号’中的表达水平要高于‘大甜1号’。     

Purification of CMP-N-acetylneuraminic acid synthetase from bovine anterior pituitary glands

CMP-beta-N-acetylneuraminic acid (CMP-neuNAc) is the substrate for the sialylation of glycoconjugates by sialyltransferases in microbes and higher eukaryotes. CMP-neuNAc synthetase catalyzes the formation of this substrate, CMP-neuNAc, from CTP and neuNAc. In this report we describe the purification of CMP-neuNAc synthetase from bovine anterior pituitary glands. The enzyme was purified by ion exchange, gel filtration, and affinity chromatography. The protein was homogeneous on SDS-PAGE with a molecular weight of 52 kDa, a subunit size similar to that of the E.coli K1 (48.6 kDa). The identity of the 52 kDa protein band was confirmed by native gel electrophoresis in that the position of the enzyme activity in gel slices coincided with the position of major bands in the stained gel. Photoaffinity labeling with 125I-ASA-CDP ethanolamine resulted in the modification of a 52 kDa polypeptide that was partially protected against modification by the substrate CTP. Enzyme activity in crude fractions could be adsorbed onto an immunoadsorbent prepared from antibody against the purified 52 kDa protein. Taken together these data suggest that the 52 kDa polypeptide purified by this procedure described in this report is indeed CMP-neuNAc synthetase. The active enzyme chromatographed on a gel filtration column at 158 kDa suggesting it exists in its native form as an oligomer.     

Purification and subunit composition of a GTP-binding protein from maize root plasma membranes

When frozen plasma membranes isolated from maize seedling roots are thawed, a significant portion of GTP-binding activity goes into solution. The GTP-binding protein was purified by ion exchange chromatography on Mono-Q and gel filtration on Superose 6. Its molecular weight was estimated at 61 kDa by gel filtration. The same molecular weight was obtained upon solubilization of the GTP-binding protein with cholic acid followed by gel filtration in the presence of this detergent. SDS-PAGE demonstrated that the isolated GTP-binding protein consists of two types of subunit of molecular weights 27 kDa and 34 kDa.     

Purification and preliminary characterisation of a thiamine-binding protein from spruce seeds

A thiamine-binding protein was isolated from spruce seeds (Picea abies L.), Karst.) in a nearly homogeneous form using a combination of ammonium sulphate fractionation, DEAE-cellulose ion-exchange chromatography, high performance gel filtration on TSK G3000SW column and fast protein ion-exchange chromatography with MonoQ column. SDS-polyacrylamide gel electrophoresis revealed the basic subunit of 23 kDa. However, the native protein was an oligomer with a molecular mass of about 130 kDa as estimated by gel filtration on analytical Superdex-200 column. The estimated isoelectric point was about 5.1. Thiamine was bound with a capacity of 8.5 nmol per mg protein, suggesting a simple 1:1 molar stoichiometry of thiamine-protein interaction. The dissociation constant of the complex was 8 μM in 0.05 M phosphate buffer at optimal pH 8.6. Several thiamine analogues were also bound to this protein but always with a lower affinity than thiamine. From this chemical probing, the binding site on the spruce protein seems to fit the model, previously deduced for the purified buckwheat-seed protein and generalised in a study of seed extracts of species sampled from major classes of Spermatophyta.     

苦荞TBW16重组表达及靶向结合蛋白的初步分析

苦荞16kD过敏蛋白(Tartary buckwheat 16kD allergen,TBW16)是定位于种子胚中的过敏蛋白,其生物学功能未知。该研究以苦荞种子灌浆期cDNA文库中获得苦荞过敏原TBW16基因的序列为基础,构建TBW16成熟蛋白的原核表达载体pET47b-TBW16,实现了其在大肠杆菌BL21Star(DE3)中的高效表达。结果表明:该过敏原以包涵体的形式表达,经包涵体复性及金属离子螯合层析纯化了目标蛋白;以1,4丁二醇二缩水甘油醚环氧基介导的蛋白偶联技术固定化TBW16于Sepharose CL 6B上,采用亲和层析分离与TBW16靶向结合的蛋白,MALDI-TOF质谱鉴定显示,苦荞TBW16过敏原靶向结合蛋白与细菌膜孔蛋白高度同源,该研究结果为分析苦荞TBW16生物学功能奠定了基础。     

Purification of a lectin from the marine red alga Gracilaria ornata and its effect on the development of the cowpea weevil Callosobruchus maculatus (Coleoptera: Bruchidae)

A lectin from the marine red alga Gracilaria ornata (Gracilariaceae, Rodophyta) was purified and characterized. The purification procedure consisted of extracting soluble proteins in 0.025 M Tris-HCl buffer, pH 7.5, followed by ammonium sulfate precipitation (70% saturation), ion exchange chromatography on DEAE-cellulose and affinity chromatography on mucin-Sepharose 4B. The purified G. ornata lectin (GOL) showed a single protein band with an apparent molecular mass of 17 kDa when submitted to SDS-polyacrylamide gel electrophoresis under reducing conditions. The native molecular mass of GOL determined by gel filtration on a Sephadex G-100 column was 17.4 kDa and its carbohydrate content was estimated to be 2.9%. Therefore, GOL is a monomeric glycoprotein. The purified lectin agglutinated trypsin-treated erythrocytes from rabbit and chicken but not from human. Its activity was not inhibited by any of the mono- and disaccharides tested but by the complex glycoproteins porcine stomach mucin, lactotransferrin, asialofetuin and bovine and porcine thyroglobulins. Isoelectric focusing showed that GOL is an acidic protein with a pI of 5.4 with analysis of its amino acid composition revealing high contents of Asx, Glx, Ser, Glu, Ala and Cys. When incorporated in artificial seeds, GOL significantly affected the development of Callosobruchus maculatus larvae, indicating the possibility of using this lectin in a biotechnological strategy for insect management of stored cowpea seeds.     

染料结合法测定荞麦种子蛋白质含量的研究

以考马斯亮蓝G250为染料,结合经典的凯氏定氮法测定结果,对影响染料结合法测定蛋白质含量的振荡时间、温度、考马斯亮蓝溶液浓度等因素进行了研究,并分析了由考马斯亮蓝染料和蛋白质结合后染料结合量(OD值差)与凯氏法测得的蛋白质百分含量之间的相关性。结果表明:测定的适宜条件是:温度15℃,处理时间50min,考马斯亮蓝溶液的浓度是0.06mg/mL。此条件形成的络合物较稳定,重复性较好,并且所测的染料结合量与凯氏定氮法测得的蛋白质含量间呈极显著的一元线性回归和相关关系。栽培甜荞和栽培苦荞的回归方程分别为:y=15.364x+3.865和y=10.769x+6.287,这两个回归方程差异显著,不能合并,分别适合于快速估计甜荞和苦荞种子的蛋白质含量。     

Isolation of a novel legumin-like lectin with potent hemagglutinating activity from seeds of the Chinese chestnut Castanea mollisima

A novel mannose- and glucose-specific lectin with high hemagglutinating activity was isolated from seeds of the Chinese chestnut Castanea mollisima. The lectin possessed a molecular mass of 140 kDa and was made up of two subunits, one with a molecular mass of 31 kDa and another with a molecular mass of 32 kDa. They exhibited substantial homology in N-terminal sequence to the storage protein legumin. The lectin was unstable in the presence of acid and alkali and at temperatures above 50 degrees C, but it was unaffected by various salts. The lectin was purified with a procedure involving ion exchange chromatography on CM-Sepharose, Q-Sepharose and Resource Q and gel filtration on Superose 12.     

Purification of an anionic isoperoxidase from peach seeds and its immunological comparison with other anionic isoperoxidases

A soluble anionic isoperoxidase (EC 1,11,1,7) was purified from peach ( Prunus persica L. Batsch cv. Merry) seeds. Purification was achieved by DEAE-Sephacel, Sephacryl S-300 and CM-cellulose chromatography. The purified isoperoxidase de-carboxylated indole-3-acetic acid (S_0.5 0.13 m M , Hill coefficient 1.7). Molecular mass, determined by gel filtration and sodium dodecyl sulfate polyacrylamide gel electrophoresis, was ca 60 kDa. Polyclonal antibodies were raised in rabbit against this isoperoxidase. Using immunoprecipitation this isoenzyme was found to be immunologically different from other soluble anionic isoperoxidases isolated from peach seeds.     

Isolation of a low molecular mass vasoactive intestinal peptide binding protein.

A vasoactive intestinal peptide (VIP) binding protein was purified in active form by detergent solubilization of lung membranes, gel filtration, VIP-Sepharose affinity chromatography, reverse phase high performance liquid chromatography, and anion exchange chromatography. The mass of this protein was estimated at 18 kDa by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 17 kDa by gel filtration. The binding of VIP by this protein was inhibited by Mg2+, covalent cross-linking of [Tyr10-125I]VIP to the protein produced two radioactive bands at 22 and 26 kDa identified by electrophoresis, and the purified protein exhibited saturable and high affinity binding of VIP and the related neuropeptide, rat growth hormone releasing factor.