This is the first in a series of papers dealing with the structure of cell walls isolated from suspension-cultured sycamore cells (Acer pseudoplatanus) . These studies have been made possible by the availability of purified hydrolytic enzymes and by recent improvements in the techniques of methylation analysis. These techniques have permitted us to identify and quantitate the macromolecular components of sycamore cell walls. These walls are composed of 10% arabinan, 2% 3,6-linked arabinogalactan, 23% cellulose, 9 % oligo-arabinosides (attached to hydroxyproline), 8% 4-linked galactan, 10% hydroxyproline-rich protein, 16 % rhamnogalacturonan, and 21% xyloglucan.The structures of the pectic polymers (the neutral arabinan, the neutral galactan, and the acidic rhamnogalacturonan) were obtained, in part, by methylation analysis of fragments of these polymers which were released from the sycamore walls by the action of a highly purified endopolygalacturonase. The data suggest a branched arabinan and a linear 4-linked galactan occurring as side chains on the rhamnogalacturonan. Small amounts or pieces of a xyloglucan, the wall hemicellulose, appear to be covalently linked to some of the galactan chains. Thus, the galactan appears to serve as a bridge between the xyloglucan and rhamnogalacturonan components of the wall.The rhamnogalacturonan consists of an a-(1 -; 4)-linked galacturonan chain which is interspersed with 2-linked rham. nosyl residues. The rhamnosyl residues are not randomly distributed in the chain but probably occur in units of rhamnosyl-(1 -4 4)-galacturonosyl-(1 -e 2)-rhamnosyl. This sequence appears to alternate with a homogalacturonan sequence containing approximately 8 residues of 4-linked galacturonic acid. About half of the rhamnosyl residues are branched, having a substituent attached to carbon 4. This is likely to be the site of attachment of the 4-linked galactan.The hydroxyprolyl oligo-arabinosides of the hydroxyproline. rich glycoprotein contain 3-linked, 2-linked, and terminal arabinosyl residues. The structure of the hydroxyprolyl oligoarabinosides deduced from our methylation studies agrees with the structure reported for similar oligosaccharides. Cellulose, hemicellulose, pectic polysaccharide, structural protein, and lignin have been identified as the major components of the plant cell wall. These components have been discussed in several recent reviews (4,9,26,35,47,52,63,64,68). Lignin is a characteristic component of secondary walls and is therefore not further discussed.Crystalline cellulose fibers make up an important part of the framework of the cell walls of all higher plants. Electron microscopy and x-ray diffraction have led to a rather detailed description of the structure of this wall component (35,57,69
Degradative enzymes have been used to obtain defined fragments of the isolated cell walls of suspension-cultured sycamore cells. These fragments have been purified and structurally characterized. Fragments released from endopolygalacturonasepretreated cell walls by a purified endoglucanase and the fragments extracted from these walls by urea and alkali provide evidence for a covalent connection between the xyloglucan and pectic polysaccharides. Fragments released by a protease from endopolygalacturonase-endoglucanase-pretreated cell walls provide evidence for a covalent connection between the pectic polysaccharides and the structural protein of the cell wall.Based on these interconnections and the strong binding which occurs between the xyloglucan and cellulose, a tentative structure of the cell wall is proposed.The polymer composition and partial structures of the pectic and hemicellulosic components of isolated sycamore cell walls are described in the preceding papers (6, 26). There remains the problem of how these components are connected to make a functional wall matrix. It has been suggested that the wall is held together by noncovalent interactions between the various macromolecular components (20). However, the evidence presented in this and in the preceding papers suggests that, with the exception of cellulose, the macromolecules of the wall are covalently cross-linked, and that even the linkage between cellulose and the other cell wall polymers has the strength of a covalent bond.
Proteomic technologies are being used to discover and identify disease-associated biomarkers. The application of these technologies in the search for potential diagnostic/prognostic biomarkers in the serum of patients has been limited by the presence of highly abundant albumin and immunoglobulins that constitute approximately 60-97% of the total serum proteins. The purpose of the study was to evaluate whether treatment of human serum with Affi-Gel Blue alone or in combination with Protein A (Aurum serum protein mini kit, Bio-Rad) before two-dimensional gel electrophoresis (2-DE) analysis removed high abundance proteins to allow the visualization of low abundant proteins. Serum samples were treated with either Affi-Gel Blue or Aurum kit and then subjected to 2-DE using 11 cm, pH 4-7 isoelectric focussing strips for the first dimension and 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis for second dimension. Protein spots were visualized using a fluorescent protein dye (SYPRO Ruby, Bio-Rad). Comparison between treatment methods showed significant removal of albumin by both Affi-Gel Blue and Aurum kit and considerable differences in the protein profile of the gels after each treatment. Direct comparison between treatments revealed twenty-eight protein spots unique to Affi-Gel Blue while only two spots were unique after Aurum kit treatment. Unique spots in Affi-Gel Blue and Aurum kit treated serum were not visualized in untreated serum. Sixteen hours of Affi-Gel Blue treatment resulted in enhanced visualization of fifty-three protein spots by two-fold, thirty-one by five-fold, twelve by ten-fold and six by twenty-fold. In parallel after Aurum kit treatment two-, five-, ten- and twenty-fold enhancements of thirty, thirteen, eight and five protein spots, respectively, were observed. The pattern of increased visualization of protein spots with both treatment methods was similar. In conclusion, treatment of serum samples with Affi-Gel Blue or Aurum kit before 2-DE analysis can be used to remove high abundance proteins in order to increase the detection sensitivity of proteins present in low abundance.
The molecular structure, chemical properties, and biological function of the xyloglucan polysaccharide isolated from cell walls of suspension-cultured sycamore (Acer pseudoplatanus) cells are described. The sycamore wall xyloglucan is compared to the extracellular xyloglucan secreted by suspension-cultured sycamore cells into their culture medium and is also compared to the seed "amyloid" xyloglucans.Xyloglucan-or fragments of xyloglucan-and acidic fragments of the pectic polysaccharides are released from endopolygalacturonase-pretreated sycamore walls by treatment of these walls with 8 M urea, endoglucanase, or 0.5 N NaOH. Some of the xyloglucan thus released is found to cochromatograph with the acidic pectic fragments on diethylaminoethyl Sephadex. The chemical or enzymic treatments required for the release of xyloglucan from the walls and the cochromatography of xyloglucan with the acidic pectic fragments indicate that xyloglucan is covalently linked to the pectic polysaccharides and is noncovalently bound to the cellulose fibrils of the svcamore cell wall.The molecular structure of sycamore xyloglucan was characterized by methylation analysis of the oligosaccharides obtained by endoglucanase treatment of the polymer. The struc. ture of the polymer is based on a repeating heptasaccharide unit which consists of 4 residues of 8-1-4-linked glucose and 3 residues of terminal xylose. A single xylose residue is glycosidically linked to carbon 6 of 3 of the glucosyl residues.In the first paper of this series (24), recently developed techniques for the methylation analysis of polysaccharides were used in order to study the structure of the primary cell wall of suspension-cultured sycamore cells and in order to characterize structurally the defined fragments released from these walls by a highly purified endopolygalacturonase. This study indicated
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