Cell walls preparation

SEKINE K, OHTA J, ONIS M, TATSUKI T, SHIMOKAWA Y, TOIDA T, KAWASHIM T, HASHIMOTO Y (1995) Analysis of antitumor properties of effecter cells stimulated with a cell wall preparation (WPG) of Bifidobacterium infantis. Biol Pharm Bull. 18 148-53. 

Watermelon cell walls, prepared as follows, were kindly provided by Dr. Niels O. Maness of the Department of Horticulture and Landscape Architecture of Oklahoma State University. Ripe watermelon mesocarp tissues were placed on ice, diced into small pieces, and then homogenized on ice in Tris-saturated phenol to give enzymically inactive watermelon cell walls [6]. The solids were collected on two layers of mira cloth and washed with water until the smell of phenol was gone. The crude cell walls were further washed with chloroform methanol (1 1, W/V) and acetone until a fluffy consistency was obtained. The acetone-washed cell wall residue was dried in an oven at 60 °C and stored in a brown bottle. 

Xylose-rich pectic polysaccharide was extracted from defatted and protein-free cell wall preparation (5) using HCl solution (pH 1.6) at 85° C for 4 h. The extract was adjusted to pH 5.0 with ammonia, concentrated on a rotary evaporator under reduced pressure at 40°C, and precipitated with 5 volumes of 96% ethanol. After washing twice with 80% ethanol and drying in an air circulated oven at 40°C for 2 h, the pellet was ledissolved with distilled water and then precipitated with 4 vols 96% ethanol. Before the pellet was gently ground, the precipitated pellet was washed twice with 70% ethanol and dried at 40 ° in an air circulated oven for 16 h. The resultant white powder was labelled "xylose-rich pectic polysaccharide" and stored in a refrigerator. 

Suberin, being an adcrustation on the cell wall, cannot be separated from cell walls. Instead, suberin-enriched wall preparations can be obtained by digesting away as much carbohydrate polymers as possible using pectinases and cellu-lases [3,7]. Depending on the source of the suberized cell wall preparation, the polyester part may constitute a few percent to 30% of the total mass. 

Removal of the aliphatic materials by hydrogenolysis leaves a residue that contains low amounts of polymethylenic components, suggesting that the suberized material contains some aliphatic components not susceptible to cleavage by such methods [3]. On the other hand, removal of suberin from cork cell wall preparations was examined by CPMAS and the results showed that the aliphatic components were nearly completely removed from this suberin preparation as the spectra showed that the residual material was virtually devoid of methyl... 

Comparison of the Overall Composition of Cell Wall Preparations. As can be seen from Table I, cell wall preparations showed a significant difference in their polymer composition. The endosperm cell walls resembled primary walls, since they were virtually free of lignin but rich in pectic substances. On the other hand, the cell wall preparations obtained from the caryopsis coat and the aleuron tissue were highly lignified, and their pectic content was very low. The germ cell wall showed a somewhat intermediate composition between these two types, probably reflecting the fact that it consists of several different tissues. 

The monosaccharide composition of the hemicelluloses, which were a major fraction of all of these cell wall preparations, also showed significant differences, especially in the amounts of glucose and galactose (Table II). 

Figure 1. Scanning electron micrograph of the cell wall preparations obtained from the different parts of rice grain (7). Caryopsis coat (upper left), aleuron layer (upper right), germ (lower left) and starchy endosperm (lower right). Bars in the picture indicate 5 /zm.

Table I. Composition of the Cell Wall Preparations Obtained from Different Histological Fractions...

Table II. Monosaccharide Composition of Pectic Polysaccharides and Hemi-cellulose Obtained from Different Cell Wall Preparations...

The sugar composition of the pectic polysaccharides obtained from these cell wall preparations (Table II) also suggested differences in the structural features of the different cell wall preparations. Detailed structural information is only available for the pectic polysaccharides obtained from the endosperm cell wall The main fraction of endosperm pectic polysaccharide was separated into two fractions, a neutral sugar-rich fraction and a fraction with a very high content of D-galacturonic acid (8). Structural... 

Figure 3 shows the structure of the hemicellulosic polysaccharides obtained from the bran cell wall preparations. These structures were deduced from the results of the methylation analysis of purified fractions (13). In contrast to the endosperm cell wall, no / -l,3-,l,4-glucan was obtained from... 

Examination of moist, isolated potato cell walls using atomic force microscopy (AFM) showed the cellulose microfibrils as an interwoven network (Kirby et al., 1996,2006). Although accurate height measurements of cellulose microfibrils have not been obtained using AFM on potato cell walls, they have on similar parenchyma cell-wall preparations from onion (Allium cepa) and Arabidopsis thaliana (Davies and Harris, 2003). These studies showed that the microfibrils were 4-6 nm in diameter, and reduced to 3.2 nm (A. thaliana) when extracted to remove some of the non-cellulosic polysaccharides. 

Small proportions of 4-linked Xylp residues were found in linkage analyses of potato cell-wall preparations (Ring and Selvendran, 1978) and of 1 M and 4 M KOH soluble extracts (Ring and Selvendran, 1981). At least some of these residues may be present in heteroxylans of primary cell walls. Such heteroxylans are known to occur in small proportions in the primary cell walls of other eudicotyledons, but their structure has been characterized in detail only for those in the walls of suspension-cultured sycamore (Acer pseudoplatanus) cells (Darvill et al., 1980). These heteroxylans are glucuronoarabinoxylans with a backbone of p-D-Xyl/ residues linked... 

Significant technical problems face those who wish to study the structure of primary cell-walls. An important consideration is the purity of the cell-wall preparations. Often, cell-wall studies have been carried out on walls obtained from heterogeneous, differentiated tissues containing... 

A further problem is that many of the methods for plant cell-wall preparation, based upon insolubility in aqueous buffers, salt solutions, and organic solvents, undoubtedly remove, or detach, some of the molecules, present in the intact wall, which may have important, structural functions. 

Despite the elaborate washing procedures usually employed, cell-wall preparations may also frequently be contaminated with cytoplasmic constituents that sediment with the walls after tissue homogenization. Starch grains and proteins are particularly difficult to remove in this respect. Both incubation with purified alpha amylase (EC 3.2.1.1)55,101 and extraction with chloral hydrate118 have been utilized for removal of starch from cell-wall preparations. Pronase57,101 has been used to remove proteins. 

Poly(ribitol phosphate) synthetase has been found in particulate fractions from Staphylococcus aureus H, and Lactobacillus plantatrum.lt ll-m The bulk of the activity in Lactobacillus plantarum was in crude, cell-wall preparations, and the enzyme is apparently located in the membrane, although intimate association with the wall itself has been suggested. Unlike the natural teichoic acid, the enzymically synthesized ribitol phosphate polymer was readily extracted with phenol hydrolysis by acid and by alkali gave the expected products, and oxidation with periodate indicated a chain length of 5-9 units, a value which compares well with that of 8 units for the natural polymer in the walls of this organism. 

Harris, P.J., Blakeney, A.B., Henry, R.J., and Stone, B.A. 1988. Gas chromatographic determination of the monosaccharide composition of plant cell wall preparations. J. Assoc. Off. Anal. Chem. 71 272-275. 

Weigh 5 mg (record exact weight) of cell walls or cell-wall fraction (in duplicate) into borosilicate glass tubes (see unit E3.i for cell wall preparation and cell-wall fractionation). 

Cell wall preparations of Colletrichum lindemuthianum Phaseolus vulgaris phenylalanine ammonia lyase cinnamic acid 4-hydrolase chalcone synthase chalcone isomerase (40)... 

Cell wall preparation of yeast Glycine max. phenylalanine ammonia lyase (41)... 

Cell wall preparations of Chaetomium globosum Daucus carota chitinase phenylalanine ammonia lyase (43)... 

Methods of isolating bacterial cell-walls have been used mainly in studies of Gram-positive bacteria, but hydrolysates of similar preparations from Gram-negative cells reveal the sugar components of the somatic antigens. It was from cell-wall preparations of Escherichia coU that l-glycero-T)-manno-heTptose was isolated. ... 

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