Heterogeneity of Arabinogalactan-Proteins on the Plasma Membrane of Rose Cells

Arabinogalactan-proteins (AGPs) have been purified from the plasma membrane of suspension-cultured Paul's Scarlet rose (Rosa sp.) cells. The two most abundant and homogeneous plasma membrane AGP fractions were named plasma membrane AGP1 (PM-AGP1) and plasma membrane AGP2 (PM-AGP2) and had apparent molecular masses of 140 and 217 kD, respectively. Both PM-AGP1 and PM-AGP2 had [beta]-(1-3)-, [beta]-(1,6)-, and [beta]-(1,3,6)-galactopyranosyl residues, predominantly terminal [alpha]-arabinofuranosyl residues, and (1,4)- and terminal glucuronopyranosyl residues. The protein moieties of PM-AGP1 and PM-AGP2 were both rich in hydroxyproline, alanine, and serine, but differed in the abundance of hydroxyproline, which was 1.6 times higher in PM-AGP2 than in PM-AGP1. Another difference was the overall protein content, which was 3.7% (w/w) in PM-AGP1 and 15% in PM-AGP2. As judged by their behavior on reverse-phase chromatography, PM-AGP1 and PM-AGP2 were not more hydrophobic than AGPs from the cell wall or culture medium. In contrast, a minor plasma membrane AGP fraction eluted later on reverse-phase chromatography and was more negatively charged at pH 5 than either PM-AGP1 or PM-AGP2. The more negatively charged fraction contained molecules with a glycosyl composition characteristic of AGPs and included at least two different macromolecules. The results of this investigation indicate that Rosa plasma membrane contains at least four distinct AGPs or AGP-like molecules. These molecules differed from each other in size, charge, hydrophobicity, amino-acyl composition, and/or protein content.     

一种类SOD物质的基本性质与结构研究

玫瑰果（Ｒｏｓａｒｕｇｏｓａｖａｒ．ｃｈａｍｉｓｏｎｉａｎａ）果实中有一种具有高ＳＯＤ活性的成分,系统研究表明,这是一个小肽,定名为类ＳＯＤ物质。用有机溶剂分级、聚几内酰胺、ＤＥＡＥ１１、Ｚｅｒｏｌｉｔ２２５柱分离纯化,提纯物经紫外光谱、圆二色谱分析、氨基酸分析、邻菲咯啉定铁、ＤＮＳＣｌ法检测末端氨基酸等实验,证实类ＳＯＤ物质的等电点为５．２,每分子肽含一个Ｆｅ原子,有７０个氨基酸左右,分子量约为７０００,可能是一种结合多肽,末端氨基酸被封闭,富含ｃｙｓ,呈无规卷曲状态,这与其很高的温度稳定性及ｐＨ稳定性是一致的。     

Immunochemical Characterization of Cell Wall Protein Antigen Purified from the Cell Wall Autolysate of Clostridium botulinum Type A

The cell wall protein antigen was solubilized from the isolated cell walls of Clostridium botulinum type A by autolysis and purified by diethylaminoethyl-cellulose column chromatography followed by gel filtration on Sephadex G-150. The two fractions showed a high degree of the serological activity and produced a main fused precipitin line in immunodiffusion tests against the homologous antiserum. The fact that antigenic fractions contained various kinds of amino acids but no detectable amounts of amino sugars or carbohydrates suggests that the antigens were principally composed of proteins. The protein antigen possessed multiple antigenic components on immunoelectrophoresis. As serological activity, the antigen was heat-stable and resistant to tryptic digestion but sensitive to the actions of pronase, nagarse or pepsin. The protein antigen appeared to be responsible for the common antigenicity among the proteolytic strains of C. botulinum.     

Structure of Plant Cell Walls : XIX. Isolation and Characterization of Wall Polysaccharides from Suspension-Cultured Douglas Fir Cells

The partial purification and characterization of cell wall polysaccharides isolated from suspension-cultured Douglas fir (Pseudotsuga menziesii) cells are described. Extraction of isolated cell walls with 1.0 m LiCl solubilized pectic polysaccharides with glycosyl-linkage compositions similar to those of rhamnogalacturonans I and II, pectic polysaccharides isolated from walls of suspension-cultured sycamore cells. Treatment of LiCl-extracted Douglas fir walls with an endo-α-1,4-polygalacturonase released only small, additional amounts of pectic polysaccharide, which had a glycosyl-linkage composition similar to that of rhamnogalacturonan I. Xyloglucan oligosaccharides were released from the endo-α-1,4-polygalacturonase-treated walls by treatment with an endo-β-1,4-glucanase. These oligosaccharides included hepta- and nonasaccharides similar or identical to those released from sycamore cell walls by the same enzyme, and structurally related octa- and decasaccharides similar to those isolated from various angiosperms. Finally, additional xyloglucan and small amounts of xylan were extracted from the endo-β-1,4-glucanase-treated walls by 0.5 n NaOH. The xylan resembled that extracted by NaOH from dicot cell walls in that it contained 2,4- but not 3,4-linked xylosyl residues. In this study, a total of 15% of the cell wall was isolated as pectic material, 10% as xyloglucan, and less than 1% as xylan. The noncellulosic polysaccharides accounted for 26% of the cell walls, cellulose for 23%, protein for 34%, and ash for 5%, for a total of 88% of the cell wall. The cell walls of Douglas fir were more similar to dicot (sycamore) cell walls than to those of graminaceous monocots, because they had a predominance of xyloglucan over xylan as the principle hemicellulose and because they possessed relatively large amounts of rhamnogalacturonan-like pectic polysaccharides.     

Preparation and Characterization of Intact Globular Cell Wall Peptidoglycan from Micrococcus lysodeikticus

The intact globular cell wall peptidoglycan was prepared from Micrococcus lysodeikticus without any mechanical disruption. The purification procedure consists mainly of proteolytic digestion and extractions with hot 5% TCA and with 0.01 N NaOH. The purified preparation showed an amphoteric, heteroporous three-dimensional network structure of the peptidoglycan, which amounted to 77% of the dry weight of the preparation, retaining the chemical and morphological integrity as judged by an electron microscopic observation and chemical analyses.     

Purification, Characterization, and Fractionation of the delta-Aminolevulinic Acid Synthesizing Enzymes from Light-Grown Chlamydomonas reinhardtii Cells

The synthesis of δ-aminolevulinate from glutamate by Chlamydomonas reinhardtii membrane-free cell homogenates requires Mg²⁺, ATP, and NADPH as cofactors. The pH optimum is about 8.3. When analyzed by a Fractogel TSK gel filtration column the δ-aminolevulinate synthesizing enzymes, including glutamate-1-semialdehyde aminotransferase, elute with an apparent molecular weight of about 45,000. The enzymes obtained from the gel filtration column were separated into three fractions by affinity column chromatography. One fraction binds to heme-Sepharose, one to Blue Sepharose, while the enzyme converting the putative glutamate-1-semialdehyde to δ-aminolevulinic acid is retained by neither column. All three fractions are necessary for the conversion of glutamate to δ-aminolevulinate. The δ-aminolevulinate synthesizing enzymes from Chlamydomonas are sensitive to inhibition by heme but not sensitive to inhibition by protoporphyrin.     

Synthesis of Arabinose-Containing Cell Wall Precursors in Suspension-Cultured Tobacco Cells II. Partial Purification and Some Physical Characterization of an Intracellular Precursor of Cell Wall Glycoproteins

Arabinose-containing macromolecules accumulate in the Golgiapparatus prior to their secretion into the cell wall. Theseintracellular macromolecules of suspension-cultured tobaccocells were labelled with ¹⁴C-arabinose, extracted and partiallycharacterized. The major component is a glycoprotein, in whicharabinose is linked to hydroxyproline residues as oligosaccharides,among them mainly as disaccharides. A pulse-chase experimentshowed the major glycoprotein to be a precursor of cell-wallmaterials. The glycoprotein has a density of 1.65 g/cm³, indicatinga high content of carbohydrate (90%), of which arabinose, galactoseand uronates are the major components. The glycoprotein is highlyacidic (pI1.3), probably due to the presence of uronate. Thelarge stokes' radius, equivalent to a 5 ? 10⁵-dalton protein,and a small S value (6.5 S) indicate a swollen structure forthe molecule. These data indicate a close similarity of theglycoprotein to an extracellular arabinogalactan protein secretedinto the culture medium. Present address: Institute for Plant Virus Research, TsukubaScience City, Yatabe, Ibaraki 305, Japan. (Received April 12, 1982; Accepted October 20, 1982)     

Characterization of Solubilized Products from the Cell Wall of Streptococcus pneumoniae during Autoplast Formation

The structure of the cell wall of Streptococcus pneumoniae R36a was investigated by means of chemical analysis of the solubilized products formed during autoplast formation. By autoplast formation, almost all of the cell wall components were solubilized as the end products within several hours. Analysis of the solubilized products revealed that the pneumococcal cell wall consists of three macromolecular components, teichoic acid-glycopeptide I (TA-GP I), teichoic acid-glycopeptide II (TA-GP II), and glycopeptide (GP III). The molecular size of TA-GP I was larger than that of TA-GP II. TA-GP I and TA-GP II were constituted of similar components, galactosamine, 2-acetamido-4-amino-2, 4, 6-trideoxyhexose, glucose, ribitol, choline, phosphate, and peptidoglycan components, but the ratio of teichoic acid to glycopeptide in TA-GP II was higher than that in TA-GP I. TA-GP II was solubilized more slowly than TA-GP I and GP III during autoplast formation. The assembly of the cell wall by TA-GP I and II, and GP III is discussed in connection with the action of autolysin.     

Soluble Signals from Cells Identified at the Cell Wall Establish a Developmental Pathway in Carrot

Cells in a plant differentiate according to their positions and use cell-cell communication to assess these positions. Similarly, single cells in suspension cultures can develop into somatic embryos, and cell-cell communication is thought to control this process. The monoclonal antibody JIM8 labels an epitope on cells in specific positions in plants. JIM8 also labels certain cells in carrot embryogenic suspension cultures. We have used JIM8 and secondary antibodies coupled to paramagnetic beads to label and immunomagnetically sort single cells in a carrot embryogenic suspension culture into pure populations. Cells in the JIM8(+) population develop into somatic embryos, whereas cells in the JIM8(-) population do not form somatic embryos. However, certain cells in JIM8(+) cultures (state B cells) undergo asymmetric divisions, resulting in daughter cells (state C cells) that do not label with JIM8 and that sort to JIM8(-) cultures. State C cells are competent to form somatic embryos, and we show here that a conditioned growth medium from a culture of JIM8(+) cells allows state C cells in a JIM8(-) culture to go on and develop into somatic embryos. JIM8 labels cells in suspension cultures at the cell wall. Therefore, a cell with a role in cell-cell communication and early cell fate selection can be identified by an epitope in its cell wall.     

Synthesis of Cell Wall Galactans from Flax (Linum usitatissimum L.) Suspension-Cultured Cells

Cell walls of flax suspension-cultured cells contain both ß1     

Cell Fractionation of Anterior Pituitary Glands from Beef and Pig

Fresh anterior pituitary glands from beef and pig were separated by differential centrifugation into subcellular fractions. Nuclei and debris were obtained at 700 g for 15 minutes, secretory granules at 7000 g for 20 minutes, mitochondria at 34,000 g for 15 minutes, and microsomes at 78,000 g for 3 hours. Electron micrographs were taken of the individual fractions. Each fraction was analyzed for nitrogen, pentosenucleic acid (PNA), and phospholipide. Beef and pig anterior lobes were quite similar in their intracellular composition as seen in the subcellular fractions. Succinic dehydrogenase was localized in mitochondria, while alkaline phosphatase was concentrated in the microsomes. A proteinase with pH optimum at 8.2 was exclusively localized. in microsomal and supernatant fractions. Acid phosphatase, acid ribonuclease, and acid proteinase were distributed among the subcellular fractions in another pattern, indicating the presence of a particle type distinct from mitochondria and microsomes. The distribution of cytoplasmic PNA paralleled that of alkaline phosphatase.     

Characterization of the Cell Wall Microdomain Surrounding Plasmodesmata in Apple Fruit

In fleshy fruits ripening is generally associated with a loss in tissue firmness resulting from depolymerization of wall components and separation of adjacent cells. In the regions of the wall that contain plasmodesmata, the usual sequences of ripening events, i.e. depolymerization of the middle lamellae and splitting of the walls, are not observed. In the present study we attempted to characterize in apple (Malus domestica Borkh.) fruit the structural microdomain of the cell wall that surrounds the plasmodesmata by in muro visualization of the cell wall components. Anionic sites of galacturonic acids were labeled with cationic gold. Low-esterified homogalacturonans were labeled with the monoclonal antibody JIM 5. In addition, a polyclonal antibody directed toward [beta](1->3)-glucopyranose was used to target callose in situ. The results indicated that the plasmodesmata-wall complexes were surrounded by a pectic microdomain. This domain was composed of low-esterified homogalacturonans that were not involved in calcium cross-bridging but were probably surrounded by a cationic environment. These structural features may result in the prevention of normal cell wall separation in regions containing plasmodesmata. However, observations by low-temperature scanning electron microscopy suggested that splitting of these walls ruptured the plasmodesmata and ultimately resulted in the spatial separation of adjacent cells.     

Sugar Composition of Cell Wall Polysaccharides of Suspension-cultured Tobacco Cells

Neutral sugar composition of cell walls of suspension-cultured tobacco cells was examined with the advance of culture age by an anion-exchange chromatography. Isolated cell walls gave on hydrolysis the following sugars: 2% of l-rhamnose, 6% of d-mannose, 26% of l-arabinose, 13% of d-galactose, 8% of d-xylose and 47% of d-glucose as neutral sugars. Little changes in composition of cell wall polysaccharides were recognized with the advance of culture age. Sugar composition of the extra-cellular polysaccharides was similar to that of hemicellulose fraction from cell walls. Pectinic acid gave on hydrolysis 2-O-(α-d-galactopyranosyluronic acid)-l-rhamnose, d-galacturonic acid and its oligosaccharides.     

Structural Basis of Cell Wall Cleavage by a Staphylococcal Autolysin

The major autolysins (Atl) of Staphylococcus epidermidis and S. aureus play an important role in cell separation, and their mutants are also attenuated in virulence. Therefore, autolysins represent a promising target for the development of new types of antibiotics. Here, we report the high-resolution structure of the catalytically active amidase domain AmiE (amidase S. epidermidis) from the major autolysin of S. epidermidis. This is the first protein structure with an amidase-like fold from a bacterium with a gram-positive cell wall architecture. AmiE adopts a globular fold, with several α-helices surrounding a central β-sheet. Sequence comparison reveals a cluster of conserved amino acids that define a putative binding site with a buried zinc ion. Mutations of key residues in the putative active site result in loss of activity, enabling us to propose a catalytic mechanism. We also identified and synthesized muramyltripeptide, the minimal peptidoglycan fragment that can be used as a substrate by the enzyme. Molecular docking and digestion assays with muramyltripeptide derivatives allow us to identify key determinants of ligand binding. This results in a plausible model of interaction of this ligand not only for AmiE, but also for other PGN-hydrolases that share the same fold. As AmiE active-site mutations also show a severe growth defect, our findings provide an excellent platform for the design of specific inhibitors that target staphylococcal cell separation and can thereby prevent growth of this pathogen.     

A Suberized Layer in the Cell Wall of Mucilage Cells of Cinnamomum

The ultrastructure of developing and mature mucilage idioblastsin the shoot apex and leaves of Cinnamomum burmanni and Cinnamomumverum (Lauraceae) is described. In all mucilage cells a suberizedlayer is present in the outer cellulosic cell wall from a veryearly stage in development onwards. This represents the firstultrastructurally documented record of a suberized wall layerin mucilage cells. Oil cells, the other type of secretory idioblastsin Lauraceae, commonly have subenzed wall layers, and the presentresults support suggestions of a possible homology of oil andmucilage cells in the so-called primitive angiosperms Suberized layer, mucilage idioblasts, Cinnamomum burmanni     

Functions of the Cell Wall in the Interactions of Plant Cells: Analysis Using Carrot Cultured Cells

Interactions between cells and between tissues are importantin the development and morphogenesis of higher plants. Attemptsto characterize the role of the cell wall in such interactionshave benefited from the use of carrot (Daucus carota L.) culturedcells in vitro as a model system. The development of carrotcells in culture can be divided into three processes: the acquisitionof embryogenic competence; the development of the embryo; andthe maturation and dormancy of the embryo. Induction of non-embryogeniccallus is accompanied by weakened intercellular attachment,decreased levels of endogenous ABA and a decrease in responsivenessto exogenous ABA. Cell wall polysaccharides are known to beinvolved in various developmental and morphogenetic events.In carrot cultured cells, possible roles in intercellular attachmenthave been proposed for arabinan and xylose in the neutral sugarregions of pectins, and various extracellular proteins havebeen shown to be involved in somatic embryogenesis in vitro.Some of these proteins are also present around and/or in zygoticembryos, possibly being involved in the formation and functionsof zygotic embryos and seeds. A 57-kDa extracellular solubleglycoprotein that binds to insulin-like peptides and an 18-kDaextracellular insoluble cystatin that inhibits the proteinasesof germinating seeds of carrot might be involved in cellularsignal transduction and inter-tissue interaction, respectively,in carrot seeds. ¹ Recipient of the JSPP Young Investigator Award, 1997     

Rapid Method for Characterization of Actinomycetes by Cell Wall Composition

To achieve a rapid identification of the cell wall components of actinomycetes, several modifications of the procedure of Cummins and Harris were developed. Purified cell walls were prepared by extraction of whole cells with 0.1 N NaOH. A 2-hr 2 N HCl hydrolysate was prepared for identification of sugars. A 2-hr 6 N HCl hydrolysate was prepared for amino acid analysis. Two-dimensional paper chromatography was run on 19-cm papers. Total time required for a single analysis was approximately 24 hr. This procedure gave qualitative results which were completely satisfactory for differentiation of certain species in the genus Actinomyces and in related genera.