Cell wall glycoproteins and polysaccharides of mature runner beans

A novel use of chlorite-HOAc treatment (delignification procedure) for the isolation of hydroxyproline (HP) rich “glycoproteins” from the depectinated cell wall material of mature runner beans is described. This procedure can be used for the isolation of wall proteins even from heavily lignified tissues. Its main disadvantage is that some of the constituent amino acids are either destroyed or modified; the nature of these changes was studied using gelatine, lysozyme and “cytoplasmic proteins” of mature beans. The main amino acids to be affected were tyrosine, cystine, methionine and lysine. The chlorite-HOAc solubilized proteins were separated by PhOH-H₂O fractionation into two distinct “glycoprotein fractions”. The major fraction (isolated from the aqueous layer) contained most of the HP of the solubilized proteins. The sugars obtained on hydrolysis of both “glycoproteins” were galactose, arabinose, glucose, xylose, rhamnose and uronic acid. Most of the proteins remaining in the holocellulose could readily be extracted with cold alkali and were relatively poor in HP.     

Solubilization of the Cytoplasmic Membrane of Escherichia coli by Triton X-100

Treatment of a partially purified preparation of cell walls of Escherichia coli with Triton X-100 at 23 C resulted in a solubilization of 15 to 25% of the protein. Examination of the Triton-insoluble material by electron microscopy indicated that the characteristic morphology of the cell wall was not affected by the Triton extraction. Contaminating fragments of the cytoplasmic membrane were removed by Triton X-100, including the fragments of the cytoplasmic membrane which were normally observed attached to the cell wall. Treatment of a partially purified cytoplasmic membrane fraction with Triton X-100 resulted in the solubilization of 60 to 80% of the protein of this fraction. Comparison of the Triton-soluble and Triton-insoluble proteins from the cell wall and cytoplasmic membrane fractions by polyacrylamide gel electrophoresis after removal of the Triton by gel filtration in acidified dimethyl formamide indicated that the detergent specifically solubilized proteins of the cytoplasmic membrane. The proteins solubilized from the cell wall fraction were qualitatively identical to those solubilized from the cytoplasmic membrane fraction, but were present in different proportions, suggesting that the fragments of cytoplasmic membrane which are attached to the cell wall are different in composition from the remainder of the cytoplasmic membrane of the cell. Treatment of unfractionated envelope preparations with Triton X-100 resulted in the solubilization of 40% of the protein, and only proteins of the cytoplasmic membrane were solubilized. Extraction with Triton thus provides a rapid and specific means of separating the proteins of the cell wall and cytoplasmic membrane of E. coli.     

Cell wall glycoproteins and polysaccharides of parenchyma of Phaseolus coccineus

Fractionation of the cell wall material of parenchyma of mature runner beans with and without chlorite-HOAc treatment, clearly showed that at least two main types of wall proteins were present. One relatively rich in hydroxyproline (HP) associated with α′-cellulose, from which most (90%) of it could be readily liberated by chlorite-HOAc treatment and the other relatively poor in HP associated with hemicellulose A. The chlorite HOAC solubilized “glycoprotein” contained a high proportion of arabinose and galactose. It was purified by PhOH-H₂O fractionation and the molar ratios of HP, arabinose, galactose, xylose, rhamnose, glucose and uronic acid in the purified glycoprotein (“glycoprotein X”) were 1:2·6:2·4:0·2:0·2:0·1:0·3. The principal amino acids of glycoprotein X were HP (43·5 mol%), serine and proline which together comprised 66 mol% of the total. These results suggest that the HP-rich wall glycoprotein is associated with cellulose microfibrils and approximates in conformation to polyhydroxyproline carrying arabinose and galactose oligosaccharide side chains.     

Isolation and characterization of cell walls from the mesocarp of mature grape berries (Vitis vinifera)

Cell walls have been isolated from the mesocarp of mature grape (Vitis vinifera L.) berries. Tissue homogenates were suspended in 80% (v/v) ethanol to minimise the loss of water-soluble wall components and wet-sieved on nylon mesh to remove cytoplasmic material. The cell wall fragments retained on the sieve were subsequently treated with buffered phenol at pH 7.0, to inactivate any wall-bound enzymes and to dislodge small amounts of cytoplasmic proteins that adhered to the walls. Finally, the wall preparation was washed with chloroform/methanol (1:1, v/v) to remove lipids and dried by solvent exchange. Scanning electron microscopy showed that the wall preparation was essentially free of vascular tissue and adventitious protein of cytoplasmic origin. Compositional analysis showed that the walls consisted of approximately 90% by weight of polysaccharide and less than 10% protein. The protein component of the walls was shown to be rich in arginine and hydroxyproline residues. Cellulose and polygalacturonans were the major constituents, and each accounted for 30–40% by weight of the polysaccharide component of the walls. Substantial varietal differences were observed in the relative abundance of these two polysaccharides. Xyloglucans constituted approximately 10% of the polysaccharide fraction and the remainder was made up of smaller amounts of mannans, heteroxylans, arabinans and galactans. Received: 26 November 1996 / Accepted: 30 January 1997     

Characterization of the cell wall of the unicellular cyanobacterium Synechocystis PCC 6714

Cell walls free of cytoplasmic- and thylakoid membranes were isolated from Synechocystis PCC 6714 by sucrose density gradient centrifugation and extraction with Triton X-100. The Triton-insoluble cell wall fraction retained the multilayered fine structure. Peptidoglycan, proteins, polysaccharides, lipopolysaccharides, lipids and carotenoids were found as constituents of the cell wall. Polypeptide and lipid patterns of cell walls were completely different from that of the cytoplasmic/thylakoid membrane fraction. The purified cell walls contained about twelve outer membrane proteins. The two major polypeptides (M_r 67,000 and 61,000) were found to be associated with the peptidoglycan by ionic interactions.Myxoxanthophyll (major carotenoid), related carotenoid-glycosides and zeaxanthin were the predominating carotenoids of the cell wall of Synechocystis PCC 6714 over echinenone and -carotene. A polar unknown carotenoid was observed, the absorption spectrum of which resembled that of myxoxanthophyll. It was exclusively found in cell walls, but not in the cytoplasmic/thylakoid membrane fraction.Abbreviations Hep heptose - DGDG digalactosyldiglyceride - MGDG monogalactosyldiglyceride - SL sulfolipid - PC phosphatidylcholin - PG phosphatidylglyceride Dedicated to Prof. Dr. G. Drews on the occasion of his 60th birthday     

MECHANICAL BEHAVIOR OF PLANT TISSUES AS INFERRED FROM THE THEORY OF PRESSURIZED CELLULAR SOLIDS

The mechanical behavior of plant tissues and its dependency on tissue geometry and turgor pressure are analytically dealt with in terms of the theory of cellular solids. A cellular solid is any material whose matter is distributed in the form of beamlike struts or complete “cell” walls. Therefore, its relative density is less than one and typically less than 0.3. Relative density is the ratio of the density of the cellular solid to the density of its constitutive (“cell wall”) material. Relative density depends upon cell shape and the density of cell wall material. It largely influences the mechanical behavior of cellular solids. Additional important parameters to mechanical behavior are the elastic modulus of “cell walls” and the magnitude of internal “cell” pressure. Analyses indicate that two “stiffening” agents operate in natural cellular solids (plant tissues): 1) cell wall infrastructure and 2) the hydrostatic influence of the protoplasm within each cellular compartment. The elastic modulus measured from a living tissue sample is the consequence of both agents. Therefore, the mechanical properties of living tissues are dependent upon the magnitude of turgor pressure. High turgor pressure places cell walls into axial tension, reduces the magnitude of cell wall deformations under an applied stress, and hence increases the apparent elastic modulus of the tissue. In the absence of turgid protoplasts or in the case of dead tissues, the cell wall infrastructure will respond as a linear elastic, nonlinear elastic, or “densifying” material (under compression) dependent upon the magnitude of externally applied stress. Accordingly, it is proposed that no single tangent (elastic) modulus from a stress-strain curve of a plant tissue is sufficient to characterize the material properties of a sample. It is also suggested that when a modulus is calculated that it be referred to as the tissue composite modulus to distinguish it from the elastic modulus of a noncellular solid material.     

Cell wall hydroxyproline-polysaccharide associations in Lupinus hypocotyls

Extraction of lupin hypocotyl cell walls with guanidine thiocynate, both before and after dilute acid treatment does not dissolve the hydroxyproline indicating that compounds containing this amino acid are probably covalently linked to insoluble wall constituents other than through acid labile arabinofuranose-hydroxyproline links. Dilute alkali does extract all of the wall hydroxyproline largely as non-dialysable material. Sequential extraction of cell walls with alkali at two temperatures (2° and 22–25°) removes most of the hemicellulose at the lower temperature but only dissolves the hydroxyproline at the higher temperature. Other studies show that the hydroxyproline containing polymer is co-precipitated with hemicellulose-B arabino-xylan. When cell walls from elongating and non-elongating hypocotyl sections are compared using this sequential extraction, the hemicellulose-B arabino-xylan containing hydroxyproline from the non-elongating wall has a much higher proportion of arabinoseand galactose than the same polymer from the elongating wall. Much more of the hydroxyproline from the elongating wall is dialysable. These results indicate more bonding of the hydroxyproline-containing glycoprotein within the wall of non-elongating tissue consistent with its suggested role in stopping cell elongation. It is suggested that the glycoprotein is linked to insoluble wall constituents such as cellulose through galactose or by direct protein to cellulose links.     

Molecular composition and origin of substrate-attached material from normal and virus-transformed cells

The proteins and polysaccharides which are left adherent to the tissue culture substrate after EGTA-mediated removal of normal, virus-transformed, and revertant mouse cells (so-called SAM, or substrate-attached material), and which have been implicated in the cell-substrate adhesion process, have been characterized by SDS-PAGE and other types of analyses under various conditions of cell growth and attachment. The following components have been identified in SAM: 3 size classes of hyaluronate proteoglycans; glycoprotein C_o (the LETS glycoprotein); protein C_a (a myosin-like protein); protein C_b (MW 85,000); protein C₁ (MW 56,000, which is apparently not tubulin); protein C₂ (actin); proteins C₃–C₅ (histones) which are artifactually bound to the substrate as a result of EGTA-mediated leaching from the cell; and proteins C_c, C_d, C_e, and C_f. The LETS glycoprotein (C_o) and C_d appear in newly-synthesized SAM (which is probably enriched in “footpad” material – “footpads” being focal areas of subsurface membranous contact with the substrate) in greater relative quantities than in the SAM accumulated over a long period of time (which is probably enriched in “footprint” material – remnants of footpads left behind as cells move across the substrate). C_o and C_d turn over very rapidly following short radiolabeling periods during chase analysis. The SAM's deposited during a wide variety of cellular attachment and growth conditions contained the same components in similar relative proportions. This may indicate well-controlled and coordinate deposition of a cell “surface” complex involving the hyaluronate proteoglycans, the LETS glycoprotein, actin-containing microfilaments with associated proteins, and a limited number of additional proteins in the substrate adhesion site. Evidence indicates that SAM is the remnant of “footpad” vesicles by which the cell adheres to the substrate and that EGTA treatment weakens the subsurface cytoskeleton, allowing these footpad vesicles to be pinched off from the rest of the cell. Three different models of cell-substrate adhesion are presented and discussed.     

Protein profiling in potato (Solanum tuberosum L.) leaf tissues by differential centrifugation

Foliar diseases, such as late blight, result in serious threats to potato production. As such, potato leaf tissue becomes an important substrate to study biological processes, such as plant defense responses to infection. Nonetheless, the potato leaf proteome remains poorly characterized. Here, we report protein profiling of potato leaf tissues using a modified differential centrifugation approach to separate the leaf tissues into cell wall and cytoplasmic fractions. This method helps to increase the number of identified proteins, including targeted putative cell wall proteins. The method allowed for the identification of 1484 nonredundant potato leaf proteins, of which 364 and 447 were reproducibly identified proteins in the cell wall and cytoplasmic fractions, respectively. Reproducibly identified proteins corresponded to over 70% of proteins identified in each replicate. A diverse range of proteins was identified based on their theoretical pI values, molecular masses, functional classification, and biological processes. Such a protein extraction method is effective for the establishment of a highly qualified proteome profile.     

Effect of Ethylenediaminetetraacetic Acid, Triton X-100, and Lysozyme on the Morphology and Chemical Composition of Isolated Cell Walls of Escherichia coli

Extraction of a partially purified preparation of cell walls from Escherichia coli with the nonionic detergent Triton X-100 removed all cytoplasmic membrane contamination but did not affect the normal morphology of the cell wall. This Triton-treated preparation, termed the “Triton-insoluble cell wall,” contained all of the protein of the cell wall but only about half of the lipopolysaccharide and one-third of the phospholipid of the cell wall. This Triton-insoluble cell wall preparation was used as a starting material in an investigation of several further treatments. Reextraction of the Triton-insoluble cell wall with either Triton X-100 or ethylenediaminetetraacetic acid (EDTA) caused no further solubilization of protein. However, when the Triton-insoluble cell wall was extracted with a combination of Triton X-100 and EDTA, about half of the protein and all of the remaining lipopolysaccharide and phospholipid were solubilized. The material which remained insoluble after this combined Triton and EDTA extraction still retained some of the morphological features of the intact cell wall. Treatment of the Triton-insoluble cell wall with lysozyme resulted in a destruction of the peptidoglycan layer as seen in the electron microscope and in a release of diaminopimelic acid from the cell wall but did not solubilize any cell wall protein. Extraction of this lysozyme-treated preparation with a combination of Triton X-100 and EDTA again solubilized about half of the cell wall protein but resulted in a drastic change in the morphology of the Triton-EDTA-insoluble material. After this treatment, the insoluble material formed lamellar structures. These results are interpreted in terms of the types of noncovalent bonds involved in maintaining the organized structure of the cell wall and suggest that the main forces involved are hydrophobic protein-protein interactions between the cell wall proteins and to a lesser degree a stabilization of protein-protein and protein-lipopolysaccharide interactions by divalent cations. A model for the structure of the E. coli cell wall is presented.     

The relationship between xyloglucan endotransglycosylase and in-vitro cell wall extension in cucumber hypocotyls

It has been proposed that cell wall loosening during plant cell growth may be mediated by the endotransglycosylation of load-bearing polymers, specifically of xyloglucans, within the cell wall. A xyloglucan endotransglycosylase (XET) with such activity has recently been identified in several plant species. Two cell wall proteins capable of inducing the extension of plant cell walls have also recently been identified in cucumber hypocotyls. In this report we examine three questions: (1) Does XET induce the extension of isolated cell walls? (2) Do the extension-inducing proteins possess XET activity? (3) Is the activity of the extension-inducing proteins modulated by a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂)? We found that the soluble proteins from growing cucumber (cucumis sativum L.) hypocotyls contained high XET activity but did not induce wall extension. Highly purified wall-protein fractions from the same tissue had high extension-inducing activity but little or no XET activity. The XET activity was higher at pH 5.5 than at pH 4.5, while extension activity showed the opposite sensitivity to pH. Reconstituted wall extension was unaffected by the presence of a xyloglucan nonasaccharide (Glc₄-Xyl₃-Gal₂), an oligosaccharide previously shown to accelerate growth in pea stems and hypothesized to facilitate growth through an effect on XET-induced cell wall loosening. We conclude that XET activity alone is neither sufficient nor necessary for extension of isolated walls from cucumber hypocotyls.     

Contrast in Enzyme activities Between the Two Kinds of Epidermal Cells in Garlic Protective Sheath During Senescing

During the transformation of the protective sheath of garlic from a fleshy scale into a tough skin, most cells of the outer epidermis undergo wall thickening by secondary deposition, yet among them, a few cell interspersed after the fashion of stematal distribution, remain unthickened at the early stage of development (Plate Ⅰ, Fig. 1). While the unthickened cell maintains its original shape and protoplasmic contents, the nucleus and cytoplasmic constituents in the thickening cell begin to disassemble themselves to some extent and “nuclear extrusion” from one cell into the other (Plate Ⅰ, Fig. 2) can frequently be detected under microscope after the specimen is treated with a nuclear stain (Plate Ⅰ, Fig. 3). During middle stage of senescence cytochemical localization of several enzyme activities in the thickening and the unthickened cells (Plate Ⅰ, Fig. 5–8) have revealed striking contrast between the two kinds of cells: The unthickened cell is rich in ATPase (as well as in cytochrome oxidase and peroxidase) activity; whereas the thickening cell fails to show any. Conversely, the thickening cell is full of APase (and in polyphenol oxidase) activity but the unthickened cell shows none or very weak. Nevertheless, the conspicuous differences of the two kinds of cells do not last long the unthickened cell also transforms itself into the thickening one in succession at the late stage of senescence. Finally, the unthickened cell is composed of secondary deposition of wall materials and a narrow empty cell cavity (Plate Ⅰ, Fig. 4). Prominent differences in cellular structure and in enzymic activity at middle stage and their final trans- formation into the same rind structure toward the end of senescence seem to indicate that the unthickened cell is reserved to collect a large part of the evacuating cellular contents from adjoining thickening cells and to transpose them to the tissue underneath epidermis, hence the name “post cell” is given.     

A simple, rapid and non-destructive procedure to extract cell wall-associated proteins from Frankia

A simple cell fractionation procedure was developed to extract cell wall-associated proteins from the nitrogen-fixing actinomycete Frankia. The method was based on washing Frankia mycelia in 62.5 mM Tris-HCl (pH 6.8) buffer supplemented with 0.1% Triton X-100 as solubilizing agent. Cell wall-associated proteins were efficiently extracted in less than 10 min, recovering approximately 94.5+/-7.44 microg protein per extraction procedure from exponentially growing cells corresponding to 50 ml of culture. The amount of cell lysis occurring during the cell wall extraction was estimated to be 1.50+/-0.51%. Three peptidoglycan hydrolases with apparent molecular masses of 4.7, 12.1, and 17.8 kDa were detected by zymography in the cell wall-associated protein fraction. On the contrary, no cell wall lytic enzyme was detected in the cytoplasmic protein fraction. These results indicate that the present method enables a specific extraction of cell wall-associated proteins. Moreover, fluorescein isothiocyanate (FITC) labelling of the cell surface proteins showed an efficient removal of cell wall-associated proteins. Growth of the treated Frankia cells (i.e. cells from which the cell wall-associated proteins were removed) in semi-solid media suggested that these cells were still viable. This technique is of importance for functionality studies of cell wall-associated proteins, particularly for bacteria where traditional cell fractionation methods are difficult to be applied.     

Purification of a plant cell wall fibronectin-like adhesion protein involved in plant response to salt stress

The structural role of extracellular-matrix (ECM) has been recognized in both plants and animals as a support and anchorage-inducing cell behavior. Unlike the animal ECM proteins, the proteins that have been identified in plant ECM have not yet been purified from whole plants and cell wall. As several immunological data indicate the presence of animal ECM-like proteins in plants cell wall, especially under salt stress or water deficit, we propose a protocol to purify a fibronectin-like protein from the cell wall of epicotyls of young germinating peas. The process consists of a combination of gelatin and heparin affinity chromatography, close to the classical one used for human blood plasma fibronectin purification. Proteins with affinity for gelatin and heparin, immunologically related to human fibronectin, are found in the cell wall of epicotyls grown under salt stress or not. Total amount of purified proteins is 3-4 times more enriched in salt stressed epicotyls. SDS-PAGE and Western blot with antibodies directed against human blood plasma fibronectin give evidence that the cell wall proteins purified by gelatin/heparin affinity chromatography are closely related to human fibronectin. The present protocol leads us to purify 17 (control) or 65 (salt stress) micrograms of protein per g of fresh starting material. Our results suggest that plant cell wall proteins can provide better anchorage of the cell to its cell-wall during salt stress or water deficit and could be considered not only as cell adhesion but also as signaling molecules.     

Generation of high‐quality protein extracts from formalin‐fixed,paraffin‐embedded tissues

A wealth of information on proteins involved in many aspects of disease is encased within formalin‐fixed paraffin‐embedded (FFPE) tissue repositories stored in hospitals worldwide. Recently, access to this “hidden treasure” is being actively pursued by the application of two main extraction strategies: digestion of the entangled protein matrix with generation of tryptic peptides, or decrosslinking and extraction of full‐length proteins. Here, we describe an optimised method for extraction of full‐length proteins from FFPE tissues. This method builds on the classical “antigen retrieval” technique used for immunohistochemistry, and allows generation of protein extracts with elevated and reproducible yields. In model animal tissues, average yields of 16.3 μg and 86.8 μg of proteins were obtained per 80 mm² tissue slice of formalin‐fixed paraffin‐embedded skeletal muscle and liver, respectively. Protein extracts generated with this method can be used for the reproducible investigation of the proteome with a wide array of techniques. The results obtained by SDS‐PAGE, western immunoblotting, protein arrays, ELISA, and, most importantly, nanoHPLC‐nanoESI‐Q‐TOF MS of FFPE proteins resolved by SDS‐PAGE, are presented and discussed. An evaluation of the extent of modifications introduced on proteins by formalin fixation and crosslink reversal, and their impact on quality of MS results, is also reported.     

Cell wall metabolism in ripening fruit. VIII. Cell wall composition and synthetic capacity of two regions of the outer pericarp of mature green and red ripe cv. Jackpot tomatoes

Pericarp discs were excised from mature green and red ripe tomato (Lycopersicon esculentum Mill. cv. Jackpot) fruit and kept in sterile tissue culture plates for 4 days, including 2 days of incubation with D-[U-¹³C]-glucose. Cell walls were prepared and differentially extracted with dimethylsulfoxide (DMSO), trans-1,2-diaminocyclohexane-N,N,N′,N′-tetraacetic acid (CDTA). Na₂CO₃, 4 M KOH and 8 M KOH. Cell wall noncellulosic neutral sugar (NS) composition and cell wall synthetic capacity (i.e. incorporation of density label into cell wall sugars) were determined by using a gas chromatograph coupled to a flame ionization detector and a mass spectrometer, respectively. In the crude cell wall, there was significantly less galactose (Gal) and glucose (Glc) in the “outer”2-mm pericarp region, including the cuticle, compared to the “inner”2-mm region immediately below it (closer to the locules). In the CDTA-soluble pectin, rhamnose (Rha), arabinose (Ara) and Gal accounted for approximately 90% of the total NS. The ratios of these sugars were very similar in the total (¹²C plus ¹³C) sugars, and also in the newly synthesized ([¹³C]-labeled) sugars, suggesting that newly synthesized NS associated with the chelator-extractable pectic fraction has a composition very similar to that of preexisting NS. In the 4 M KOH-soluble material, xylose (Xyl) and Glc accounted for approximately 70% of the total NS. The ratio of these sugars was very similar in the total sugars, but much lower in the newly synthesized portion. This suggests that the hemicellulosic polymers synthesized during the ripening process are different in type and/or proportion from those present in the developing fruit. Because the outer pericarp of tomatoes contains at least two distinct tissue types and these have a distinct cell wall composition, analysis of tomato cell wall polysaccharide composition by homogenization of the entire outer pericarp will obscure subtle changes associated with ripening/softening within specific tissue types.     

Chemotactic attraction of Crassostrea virginica hemolymph cells to Staphylococcus lactus.

The nuclear polyhedrosis virus (NPV) of Porthetria dispar was isolated and purified through a two-step zonal centrifugation procedure. The LD₅₀ of the purified NPV was determined by a dose-response assay. Quantitative analyses were made of whole polyhedra and of separated fractions of polyhedral protein, virus rods, and denatured material, i.e., the pellet obtained from low speed centrifugation of dissolved polyhedra, to determine the protein, DNA, and “RNA” (orcinol-positive material) present in this NPV. Approximately one-half the “RNA” was present in the denatured material. Trace elements were also determined, and four, Fe, Mg, Cu, and Zn, of the ten assayed were present in the polyhedral protein fraction, while only Mg and Zn were in the virus rod fraction.     

Gel and gel-free proteomics to identify Saccharomyces cerevisiae cell surface proteins

The study of Saccharomyces cerevisiae cell surface proteins was performed because of their important role in cell wall biogenesis and in the physiology of the yeast. Two different proteomic approaches were carried out. First, proteins loosely associated or S–S linked to structural wall components were released by treatment of whole intact cells with dithiothreitol, separated by 2D-PAGE and identified by mass spectrometry. Second, cell surface-exposed proteins (surfome) were digested with trypsin and DTT from whole intact cells, and analyzed by LC–MS/MS. Ninety-nine different proteins were identified: 67 with DTT treatment and 52 with DTT and trypsin digestion. These proteins were classified in different cellular processes: control of cell wall organization, cell rescue, defence, and virulence, protein fate, protein synthesis and metabolism. Most of the proteins have already been reported as present on the cell surface showing that the yeast cell surface is composed not only by typical but also by atypical cell wall proteins. “Bona fide” cell wall proteins were identified by both protocols but a higher number with the non-gel strategy. However, only 20% of the proteins identified were common to both protocols, thus, for a complete knowledge of the cell surface proteome, several strategies have to be used.     

Characterization of the cell wall and cell wall proteins of Chromatium vinosum.

Highly purified cell walls of Chromatium vinosum were isolated by differential centrifugation, with or without Triton X-100 extraction. The isolated material had a protein composition similar to that of cell walls obtained by sucrose density gradient centrifugation. Twenty-two proteins were reproducibly detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A 42-kilodalton protein was shown to account for 65% of the total cell wall protein. The majority of cell wall proteins were solubilized in sodium dodecyl sulfate at room temperature; however, they existed as high-molecular-weight complexes unless heated to 45 degrees C or above. The cell wall contained one heat-modifiable protein which migrated with an apparent molecular weight of 37,400 when solubilized at 70 degrees C or below, but which migrated with an apparent molecular weight of 52,500 if solubilized at 100 degrees C. The electrophoretic mobility of three proteins was modified by 2-mercaptoethanol. The majority of C. vinosum cell wall proteins had isoelectric points between pH 4.5 and 5.5, and the 42-kilodalton protein focused at pH 4.9. No proteins were detected which were analogous to the lipoprotein or peptidoglycan-associated proteins of the Enterobacteriaceae. Nearest-neighbor analysis with a reducible, cross-linking reagent indicated that three proteins, including the 42-kilodalton protein, associated with themselves. Most of the cell wall proteins were partially accessible to proteases in both intact cells and isolated cell walls. Protease treatment of the whole cell or isolated cell wall digested approximately an 11,000-molecular-weight portion of the 42-kilodalton protein.