Bacitracin Inhibits the Synthesis of Lipid-linked Saccharides and Glycoproteins in Plants

The particulate enzyme fraction from mung bean (Phaseolus aureus) seedlings catalyzes the incorporation of mannose from GDP-[¹⁴C]mannose into mannosyl-phosphoryl-dolichol and of N-acetylglucosamine from UDP-[³H]N-acetylglucosamine into N-acetylglucosamine-pyrophosphoryl-polyisoprenol. Bacitracin inhibits the transfer of both of these sugars into the lipid-linked saccharides with 50% inhibition being observed at 5 mm bacitracin. This antibiotic did not inhibit the transfer of glucose from UDP-[¹⁴C]glucose into steryl glucosides or the incorporation of glucose into a cell wall glucan. Bacitracin also inhibited the in vivo incorporation of [¹⁴C]mannose into mannosyl-phosphoryl-dolichol and into glycoprotein by carrot (Daucus carota) slices. While bacitracin also inhibited the incorporation of lysine into proteins by these slices, protein synthesis was less sensitive than glycosylation. Thus at 2 mm bacitracin glycosylation was inhibited 92%, while protein synthesis was inhibited only 50%.     

Structural Analysis of N-Glycans of Storage Glycoproteins in Soybean (Glycine max. L) Seed

The structures of N-linked sugar chains (N-glycans) of storage glycoproteins in soybean seeds have been identified. Eight pyridylaminated (PA-) N-linked sugar chains were derived and purified from hydrazinolysates of the storage glycoproteins by reverse-phase HPLC and size-fractionation HPLC. The structures of the PA-sugar chains purified were first identified by two-dimensional PA-sugar chain mapping and ion-spray mass analysis, considering the results of sugar composition analysis or sequential exoglycosidase digestion. The deduced structures were further analyzed by ion-spray tandem mass spectrometry and 500 MHz ¹H-NMR spectrometry. The eight structures fell into two categories; the major class (96.6%) was a typical high mannose-type, the minor class was a xylose containing-type (Man₃Xyl₁GlcNAc₂, Man₃Fuc₁Xyl₁GlcNAc₂; 3.4%).     

Glycoproteins of Sindbis Virus: Preliminary Characterization of the Oligosaccharides

The carbohydrate content of Sindbis virus was determined by gas chromatographic analysis. The two viral glycoproteins were found to be approximately 8% carbohydrate by weight. Mannose is the sugar present in the largest amount. Smaller amounts of glucosamine, galactose, sialic acid, and fucose were also detected. Each of the two viral glycoproteins appears to contain two structurally unrelated oligosaccharides. Two of the three Sindbis-specific glycoproteins found in infected chick cells were shown to contain short, unfinished oligosaccharides.     

Poly(N-Acetyllactosaminyl) Oligosaccharides of Chromaffin Granule Membrane Glycoproteins

Poly(N-acetyllactosaminyl) oligosaccharides have been identified, on the basis of their susceptibility to endo-beta-galactosidase, in a large-molecular-size glycopeptide fraction derived from chromaffin granule membrane glycoproteins. The glycoproteins containing poly(N-acetyl-lactosaminyl) oligosaccharides were selectively labeled by treatment of chromaffin granule membranes with endo-beta-galactosidase to expose N-acetylglucosamine residues, followed by incubation with galactosyltransferase and UDP-[14C]galactose. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and fluorography demonstrated specific labeling in the 41-47 kilodalton (kD) region and in a distinct band at 90 kDa. Two-dimensional SDS-PAGE revealed that the poly(N-acetyllactosaminyl) oligosaccharides are predominantly present in glycoprotein IV, together with lesser labeling of glycoproteins II and III, whereas they are absent from dopamine beta-hydroxylase and carboxypeptidase H, which are the major glycoproteins of chromaffin granule membranes.     

Glycoprotein Biosynthesis in Cotyledons of Pisum sativum L: Involvement of Lipid-linked Intermediates

Particulate preparations from developing cotyledons of Pisum sativum L. cv. Burpeeana catalyze glycosyl transfer from UDP-[¹⁴C]N-acetylglucosamine and GDP-[¹⁴C]mannose. Radioactivity is transferred to lipid components soluble in chloroform-methanol (2:1) and chloroform-methanol-water (1:1:0.3) and into a water-insoluble and lipid-free residue.     

Involvement of the Golgi Apparatus in the Synthesis and Secretion of Hydroxyproline-rich Cell Wall Glycoproteins

Pulse labeling of carrot root phloem parenchyma (Daucus carota L. cv. Nantes) tissue with ¹⁴C-proline followed by fractionation of the cytoplasmic organelles on sucrose gradients was used to determine the identity of the membranous organelles involved in the secretion of the hydroxyproline-rich glycoproteins of the cell wall. Identification of the organelles was done through electron-microscopical observations and through the localization of marker enzymes on the sucrose gradients. Enrichment of the organelles involved in secretion was determined by measuring the percentage of the incorporated radioactivity present as ¹⁴C-hydroxyproline. The Golgi apparatus (dictyosome) was found to be a major site of glycoprotein transport. This identification was based on the observed enrichment of dictyosomes paralleling the purification of newly synthesized cell-wall glycoproteins. A marker enzyme for the Golgi apparatus, inosinediphosphatase, banded with the newly synthesized cell wall glycoproteins on sequential isopycnic and rate zonal sucrose gradients. Marker enzymes for the endoplasmic reticulum and the plasma membrane were clearly separated from the dictyosome-rich fraction. UDP-arabinose arabinosyl transferase, an enzyme involved in the glycosylation of the peptide moiety of this glycoprotein, also banded with the dictyosomes on both kinds of gradients. The results suggest an important role of the Golgi apparatus in the biosynthesis and the secretion of the cell wall glycoproteins of higher plants.     

Polysaccharides of Soybean Seeds

A polysaccharide fraction extracted from soybean seeds with boiling water was examined by several fractionation methods on ultracentrifugal criteria. Four components were found by a column chromatography using TEAE-cellulose or by a paper electrophoresis. Acetone-precipitation, fractionation by conversion into acetyl derivative, and copper-complex-precipitation were unsatisfactory to fractionate into the components. The major component (70%) isolated was an arabinogalactan containing residues of arabinose and galactose in the approximate proportion of 1 : 2 and having molecular weight of 3.3×10⁵.     

种子中的棉子糖半乳糖苷系列寡糖研究进展

棉子糖半乳糖苷系列寡糖广泛分布在许多种植物种子中,并存在于干燥后仍能保持活力的组织内,如禾谷类种子的胚及糊粉层,豆类及其他双子叶植物的子叶和胚轴组织等。棉子糖半乳糖苷系列寡糖在禾谷类种子的非自溶性中央胚乳中不合成,但存在于蓖麻种子的自溶性胚乳细胞中。棉子糖半乳革系列寡糖在种子发育后期累积,并持续到种子大量成熟直到脱水阶段。棉子糖半乳糖苷系列寡糖主要包括棉子糖、水苏糖和毛蕊花糖,是种子中最广泛的低分子量α－半乳糖苷。许多植物正常性种子的发育伴随着棉子糖半乳糖苷系列寡糖的累积,这些糖的累积已被认为在种子脱水耐性获得、种子活力、糖的运输及植物的抗冷驯化等过程中起重要作用。本文从种子的脱水耐性获得、植物的冷驯化、细胞内定位及生物合成等方面综述了棉子糖半乳糖苷系列寡糖的研究进展。     

Polysaccharides of Soybean Seeds

Methylation of hot-water-extract fraction of soybean seed polysaccharides by the Haworth’s method and then by the Purdie’s method followed by fractional extraction afforded a fully methylated arabinogalactan which gave on complete hydrolysis 2,3,5-tri-, and 2,3-di-O-methyl l-arabinose and 2,3,6-tri-, and 2,6-di-O-methyl d-galactose in the approximate proportion of 1 : 1 : 3 ~ 4: 1, and on controlled acid methanolysis 2,3,5-tri-, and 2,3-di-O-methyl l-arabinose in about equal proportion. Residual undegraded polysaccharide gave mainly 2,3,6-tri-O-methyl d-galactose on remethylation and hydrolysis.     

Polysaccharides of Soybean Seeds

Partial acid hydrolysate of the “hot-water-extract” fraction of soybean seed polysaccharides contained a homologous series of galacto-oligasaccharides as a major component group. Two of them were isolated by column chromatography. They gave, on methylation followed by acid hydrolysis, 2,3,4,6-tetra-, and 2,3,6-tri-O-methyl D-galactose, and were, therefore, 1,4-linked galacto-di- and trisaccharides, respectively. They were hydrolyzed with human saliva to liberate D-galactose but not with brewer’s yeast. The alditols derived from these oligosaccharides showed infrared absorptions at 885 and 895 cm^?1, respectively. These two results were strong evidences for the presence of β-linkages in the molecules of the oligossacharides. The optical rotation and the melting point of the disaccharide agreed with those of the β-1, 4-linked galactodisaccharide hitherto reported. Thus d-galacto-pyranosyl residues in the arabinogalactan are probably connected mainly by β-1,4-linkage.     

beta-Conglycinins in Developing Soybean Seeds

The temporal sequence of development of the major proteins of seeds of soybean (Merr.) has been studied during development of cotyledons from flowering to maturity. A well-defined difference occurred in the times of appearance and the periods of maximum accumulation of α, α′-, and β-subunits of betaconglycinin. Whereas α- and α′-subunits appeared 15 to 17 days after flowering, accumulation of β-subunit did not commence until 22 days after flowering. Such alterations in subunit composition infer that changes also occurred in the amino acid composition of betaconglycinin during maturation, particularly in the content of methionine which is low in the β-subunit.     

种子中的棉子糖半乳糖苷系列寡糖研究进展

棉子糖半乳糖苷系列寡糖广泛分布在许多种植物种子中,并存在于干燥后仍能保持活力的组织内,如禾谷类种子的胚及糊粉层,豆类及其他双子叶植物的子叶和胚轴组织等。棉子糖半乳糖苷系列寡糖在禾谷类种子的非自溶性中央胚乳中不合成,但存在于蓖麻种子的自溶性胚乳细胞中。棉子糖半乳糖苷系列寡糖在种子发育后期累积,并持续到种子大量成熟直到脱水阶段。棉子糖半乳糖苷系列寡糖主要包括棉子糖、水苏糖和毛蕊花糖,是种子中最广泛的低分子量α_半乳糖苷。许多植物正常性种子的发育伴随着棉子糖半乳糖苷系列寡糖的累积,这些糖的累积已被认为在种子脱水耐性获得、种子活力、糖的运输及植物的抗冷驯化等过程 中起重要作用。本文从种子的脱水耐性获得、植物的冷驯化、细胞内定位及生物合成等方面综述了棉子糖半乳糖苷系列寡糖的研究进展。     

In Vitro Synthesis of the alpha and alpha' Subunits of the 7S Storage Proteins (Conglycinin) of Soybean Seeds

Messenger RNAs (mRNAs), isolated from immature soybean (Glycine max L., Merr.) seeds, that bound to oligo(dT)-cellulose were fractionated by centrifugation in sucrose density gradients containing dimethyl sulfoxide. mRNAs with sedimentation values between 21S and 25S coded for the in vitro translation of polypeptides with electrophoretic mobilities similar to those of the α′ and α subunits of the 7S seed storage protein. High pressure liquid chromatographic analyses of the trypsin-induced fragments (“column fingerprinting”) verified that the polypeptides produced in vitro were closely related to authentic α′ and α subunits.     

Characterization of Fucosyl Oligosaccharides Associated with Synaptic Membrane and Synaptic Junctional Glycoproteins

Rats were administered [3H]fucose by intracranial injection and synaptic membranes (SMs) isolated 18 h later. Oligosaccharides associated with SM glycoproteins were prepared by hydrazinolysis and analyzed by a combination of affinity chromatography on concanavalin A (Con A)-agarose, ion-exchange chromatography on DEAE-cellulose, and gel permeation chromatography. Most (94%) of the [3H]fucose-labelled oligosaccharides were present in the fraction that did not bind to Con A. Of these 41% did not bind to DEAE-cellulose, indicating the absence of negatively charged groups and the remainder were resolved into four fractions of increasing acidity. Gel permeation chromatography of the fractions from the DEAE-cellulose column suggested that the major oligosaccharides corresponded to fucosylated triantennary structures containing varying amounts of sialic acid although more highly branched structures containing peripheral branches lacking one or more sugars may also have been present. Comparison of fucosyl oligosaccharides associated with SMs prepared from 10- and 28-day-old animals indicated that although the general oligosaccharide content was similar at both ages, membranes from younger animals were characterized by an increase in the proportion of highly acidic structures. Fucosylated glycans derived from synaptic junctional (SJ) glycoproteins were also characterized by a greater percentage of highly acidic structures than SMs. The results indicate that SMs and SJs are characterized by specific complements of fucosylated glycoprotein oligosaccharides.     

DNA Synthesis in Storage Organs of Germinating Seeds: An Autoradiographic Analysis

Storage organs of different germinating seeds were tested forDNA synthesis, which, apart from the normal replication precedingcell division, may also be involved in some regulatory processas suggested by a previous work. Both persistent cotyledons,programmed to grow into green, leaf-like organs (cotyledonsof lettuce, radish and Haplopappus gracilis), and storage organswhich senesce and die after depletion of reserves and wheregrowth and greening are only slight or non-existent (cotyledonsof garden pea, cowpea, soybean, aleurone layers of barley andendosperm of castor bean), were tested. The results showed that widely variable patterns of behaviourcan occur, making it difficult to establish a possible and generallyvalid explanation of nuclear DNA synthesis in storage organsduring germination. Possible interpretations are discussed. Key words: DNA synthesis, Storage organs, Seed germination     

大豆低聚糖对小鼠体液免疫功能的影响试验研究

通过利用大豆低聚糖对小鼠灌服后溶血素生成量的测定,阐明大豆低聚糖对小鼠体液免疫功能的影响。     

大豆种子储藏性磷对幼苗适应低磷胁迫的作用

选用种子大小不同、磷效率不同的两个大豆品种‘巴西10号’(B10)和‘本地2号’(L2),在不同供磷条件下进行营养液浇灌沙培,从大豆萌发至2片三出复叶完全展开期测定植株主要器官总磷、可溶性磷浓度、子叶可溶性蛋白、酸性磷酸酶比活性、植酸酶比活性的变化动态,探讨储藏性磷在大豆幼苗期适应磷胁迫中的作用。结果发现:(1)磷效率不同的两个大豆品种的种子中磷含量差异显著。(2)大豆萌发和幼苗生长过程中子叶的磷逐渐转入根、茎、叶中,并以转入叶中的磷最多,其中磷高效品种B10在发芽过程中子叶磷向各个器官转移的总磷量要高于磷低效品种L2,且持续时间长。(3)大豆萌发和幼苗生长过程中外源供磷水平显著影响子叶磷的转移,在外源供磷充足条件下各器官中总磷均高于低供磷条件,子叶中磷和外源磷存在补偿关系。(4)磷高效品种B10子叶中酸性磷酸酶活性在低磷条件下显著高于高磷条件,但磷低效品种L2在高、低磷间无显著差异。研究表明,大豆种子储藏性磷在幼苗期耐低磷能力建立方面具有重要作用。     

酶法合成功能性低聚糖

功能性低聚糖具有无毒、无残留、稳定性强等特点,作为新型绿色添加剂被广泛应用在食品、饲料、医药行业。国际市场上10余种低聚糖产品中除大豆低聚糖、棉籽糖外,主要采用酶法制备。用于合成功能性低聚糖的酶包括糖苷酶、糖基转移酶和磷酸化酶。本文综述了功能性低聚糖种类、性质和制备方法,分析了酶法合成低聚糖的优缺点,阐述了磷酸化酶种类、催化特性和低聚糖产物。多酶法合成策略和目标酶的分子改造将是酶法合成功能性低聚糖的发展方向。