Phosphorylation of chondroitin sulfate in proteoglycans from the swarm rat chondrosarcoma

Proteoglycans isolated from the Swarm rat chondrosarcoma were shown to contain 35 mol of phosphate/mol of proteoglycan. While 20% of this phosphate was released by digestion with dilute alkali in the presence of sodium borohydride and is presumably of the phosphoserine/phosphothreonine type, 78% of the phosphate copurified with the peptide-free chondroitin sulfate chains. When chondroitin sulfate chains purified by ethanol precipitation or Sephacryl S200 column chromatography were digested with chondroitinase AC and the digests chromatographed on Bio-Gel P-4, the phosphate co-migrated with a carbohydrate fragment that contained 2 glucuronic acid (one as delta 4,5-unsaturated sugar), 1-galactosamine, 2-galactose, and 1-phosphate residue/xylitol. A second fragment of similar composition but lacking phosphate was also recovered in a ratio of about 3 to 1 relative to the phosphorylated fragment. The phosphate in the chondroitin sulfate linkage region fragment had the alkaline phosphatase sensitivity as well as 31P NMR spectra of a monophosphate esterified to a secondary sugar alcohol. The phosphate was localized on the C-2 of the chain initiating xylose since these residues as xylitol showed a delayed release during acid hydrolysis and the xylitol was recovered intact after periodate oxidation. In the chondrosarcoma, 2-phosphoxylose appears to be a normal synthetic product since [32P]phosphate was readily incorporated into the proteoglycan and the incorporated isotope had similar biochemical properties as the unlabeled phosphate.     

Effect of periodate oxidation on the structure and properties of glucose oxidase.

In order to elucidate the molecular structure of glucose oxidase (beta-D-glucose: oxygen 1-oxidoreductase, EC 1.1.3.4) and the roles of its carbohydrate moiety, chemical, physiochemical and immunological experiments were performed with enzyme samples before and after periodate oxidation. Hydrodynamic parameters indicated that the native enzyme was a globular protein with values of 1.21 for the frictional ratio and 43 A for the Stokes radius. The enzyme contained about 12% carbohydrate by weight, of which the main component was mannose. The periodate treatment decreased the carbohydrate content to about 40% of its original value. Slight modifications were detected in the absorbance spectrum and the content of arginyl residue. However, no significant alteration was brought about by this treatment in the catalytic parameters, immunological reactivities of the gross structure, not in the secondary and quaternary structures of the protein moity. Thermal denaturation temperature (about 72.5 degrees C) and the enthalpy of denaturation (about 450 kcal/mol) were common to the native and the periodate-oxodozed enzymes. The native was found to be quite resistant to sodium dodecyl sulfate and fairly stable to urea and heating. The periodate-oxidized enzyme was also stable to heat treatment, but it showed a diminished stability when denaturing agents were present. Kinetic analyses of the thermal inactivation processes showed that the entropy of activation was greatly decreased by the denaturing agents, especially in the case of the periodate-oxidized enzyme. It is concluded that the carbohydrate moiety of the enzyme plays a role in increasing the stability of the protein moiety, but does not directly participate in the catalytic activity, the immunological reactivity, or in maintaining the conformation of the enzyme protein.     

On the oligosaccharide moiety of angiotensin-converting enzyme.

Two glycopeptide fractions in a pronase digest of rabbit pulmonary angiotensin-converting enzyme were resolved by gel filtration. GP-I, the minor component (～1 mole/mol enzyme) contained mannose, galactose, glucose $\text{N}$-acetylglucosamine, $\text{N}$-acetylgalactosamine and sialic acid in an approximate molar ratio of 1:5:3:4:1:2 and molar equivalents of aspartic acid, threonine and serine. GP-II, the major oligosaccharide unit (～ 12 moles/mol enzyme, ～ 90% of total carbohydrate), contained fucose, mannose, galactose, $\text{N}$-acetylglucosamine, sialic acid and aspartic acid in a molar ratio of 1:4:4:4:1:1. Although accounting for about one-quarter of the weight of the enzyme, GP-II did not compete with the intact glycoprotein for binding to goat antienzyme antibodies. Some structural features of GP-II were deduced by periodate oxidation and digestion with various glycosidases.     

Chemical composition of the cell walls of Lolium multiflorum endosperm

Cell walls isolated from Lolium multiflorum endosperm grown in liquid suspension culture contain 90% carbohydrate (as anhydro-glucose), 0·3 nitrogen, 1·9% lipid and 4·3% ash. The relative proportions of neutral sugars present in hydrolysates of the wall polysaccharides are glucose, 50%; arabinose, 19%; xylose, 26% and galactose, 5%. Extraction of the wall with 7 M urea solubilizes a polysaccharide representing 19% of the wall and composed of glucose and minor amounts of pentoses. This fraction has been examined by acid and enzymic hydrolysis and by periodate oxidation, and was shown to be a β-1,3; 1,4-glucan with approx. 79% 1,4-linkages. A specific β-glucan hydrolase has been used to determine the content of this mixed-linked glucan in isolated endosperm cell walls.     

The effect of condition of oxidation of the carbohydrate component of peroxidase on the composition and properties of insulin-peroxidase conjugate

The influence of sodium metaperiodate concentration on kinetics and conversion degree of peroxidase carbohydrate moiety as well as the effect of the oxidation degree of the carbohydrate moiety on the composition, structure and properties of insulin-peroxidase conjugates were studied. The initial rate of peroxidase's oxidation is directly proportional to the periodate concentration; the oxidation rate constant of peroxidase carbohydrate moiety is 1.23 x 10(-3) M-1 min-1. At the molar ratio of metaperiodate to peroxidase 150:1 or higher, the maximal quantity of aldehyde groups (62 +/- 2) in the peroxidase molecule is formed and the oxidation of each carbohydrate chain leads to the formation of eight aldehyde groups. The molecular mass composition of the insulin-peroxidase conjugates was studied by HPLC. The conjugates proved to be multicomponent mixtures of oligomers (53, 83, 128, 174, 268, 440 kD and higher). The insulin-peroxidase molar ratio in the fractions of the conjugates with molecular masses higher than 83 kD is 8:1. It was shown that the affinity of insulin-peroxidase conjugates to antibodies depends on the oxidation degree of peroxidase used for production of conjugates.     

Chemical Composition of the Cell Walls of Clostridium botulinum Type A

Cell walls were isolated by sonic disruption of log-phase cells of Clostridium botulinum type A strain 190L and purified by treatment with sodium dodecyl sulfate (SDS) followed by digestion with proteases. Electron microscopy revealed that the cell walls thus obtained were free of both cytoplasmic membrane and cytoplasmic fragments. The purified cell wall contained 8.7% total nitrogen, 15.0% total hexosamines, 22.4% reducing groups, 8.3% carbohydrate, and 3.1% glucose. The content of total phosphorus was very low (0.02%), and therefore it was expected that teichoic acid might be absent in the cell wall. The wall peptidoglycan contained glutamic acid, alanine, diaminopimelic acid, glucosamine and muramic acid in the molar ratios of 1.00:1.85:0:85:1.06:0.67. A low amount of galactosamine was also present, but no other amino acids were found in significant quantities. The SDS-treated cell walls were not attacked by lysozyme, but after extraction with hot formamide they were completely dissolved by the enzyme and released reducing groups. The lysozyme digest was separated into two constituents, the saccharide moiety and the peptide moiety on Sephadex G-50.     

Cell wall components of gramicidin S resistant Staphylococcus aureus]

Comparative study of two staphylococcus aureus 209P strains--resistant and susceptible to gramicidin S demonstrated that peptidoglycanes of two strains differ by ratio glycine/serine at peptide bridges. Besides peptidoglycanes significantly differ by amidation of alfa-carboxyles of glutamic acid in muropeptide. This peptidoglycane modification of resistant cells along with enhanced content of etherized D-alanine in teichoic acid provides lower negative charge of cell wall components. It may influence the cell wall ability to react with positively charged gramicidin molecules. It was shown that isolated cell walls and peptidoglycane of resistant cells binds significantly less gramicidin than cell walls and peptodoglyce of susceptable cells. Simultaneous determination of gramicidin binding by intact S. aureus cells and their killing revealed that lower ability of resistant cells to bind gramicidin is significant but not critical factor of gramicidin resistance.     

Structural studies on periodate-oxidized chicken ovomucoid. Spectral behaviour of the tyrosine residues.

About 50% of the carbohydrate moiety of ovomucoid was destroyed by periodate oxidation. The oxidation was carried out for 6 h or 24 h. The data obtained showed that in the carbohydrate chain 2-5 glucosamines and 1-2 neutral sugar residues were decomposed with the consumption of 16 mol and 29 mol of periodate respectively. Periodic oxidation slightly changed the inhibitory activity of the ovomucoid, but altered its spectral properties. An increase of the absorption maximum at 278 nm was noted, as well as a tendency for normalization of phenolic ionization and an increase of the relative fluorescence. The reactivity of tyrosine residues towards tetranitromethane is also changed. It was suggested that even in native ovomucoid the tyrosines could be regarded as 'dissolved' in the 'carbohydrate solvent'. This contact could be achieved by the hydrogen bonds in the formation of which the NHCOCH3 groups of the glucosamine residues play an essential role. Peroxidate oxidation seems to lead to an alteration of the nature of the 'sugar solvent' and disturbs the conformation of the sugar chain.     

Immunochemistry of the Group-Specific Polysaccharide of Nocardia brasiliensis.

Estrada-Parra, Sergio (Escuela Nacional de Ciencias Biológicas, México, D.F., México), Abel Zamora, and L. F. Bojalil. Immunochemistry of the group-specific polysaccharide of Nocardia brasiliensis. J. Bacteriol. 90:571-574. 1965.-The group-specific polysaccharide of Nocardia brasiliensis was further purified, yielding an amorphous white material with the following characteristics: [alpha](D) (20) = + 48; nitrogen, 0.5%; phosphorus, 0.1%; and ash as sodium, 0.8%. The polymer is made of d-arabinose and d-galactose in a molar ratio of 3:1, and no other sugars were detected. Mild hydrolysis liberates mainly arabinose. The polysaccharide consumes 3.46 mumoles of periodate per mg of polymer in 15 days at 4 C (this value remains constant after 4 more days). Oxidation results in destruction of two of the arabinose, with the formation of two glycerols after borohydride reduction and hydrolysis. The polysaccharide oxidized by periodate and reduced under mild acid hydrolysis at 20 C yields glycerol and a polymer formed by galactose and arabinose (in a ratio of 1:1) which is resistant to a second oxidation. Therefore, the polysaccharide is probably formed by a main chain of glactose linked 1,3 and arabinose linked 1,2 or 1,3 or both, and nonreducing side chains of arabofuranose residues. The intact polysaccharide cross-reacts with sera from patients with active tuberculosis, and this, as well as the homologous reaction, is abolished by oxidation with periodate.     

Isolation and structural elucidation of a water-soluble polysaccharide (PS-I) of a wild edible mushroom, Termitomyces striatus

A heteropolysaccharide (PS-I), isolated from the hot aqueous extract of an edible mushroom, Termitomyces striatus, is composed of d-glucose, d-galactose, d-mannose and l-fucose in a molar ratio 2:1:1:1. Structural investigation of the native as well as the Smith-degraded polysaccharide was carried out using methylation analysis, periodate oxidation studies and 1D and 2D NMR spectroscopy, and the repeating unit of the polysaccharide is established as follows: [carbohydrate structure: see text]     

Composition of the cell wall of Chlorella fusca

Isolated cell walls of mature Chlorella fusca consisted of about 80% carbohydrate, 7% protein, and 13% unidentified material. Mannose and glucose were present in a ratio of about 2.7:1 and accounted for most of the carbohydrate. Minor components were glucuronic acid, rhamnose, and traces of other sugars; galactose was absent. After treatment with 2 M trifluoroacetic acid or with 80% acetic acid/HNO₃ (10/1, v/v), a residue with a mannose/glucose ratio of 0.3:1 was obtained, probably representing a structural polysaccharide. An X-ray diffraction diagram of the walls showed one diffuse reflection at 0.44 nm and no reflections characteristic of cellulose. Walls from young cells contained about 51% carbohydrate, 12% protein, and 37% unidentified material. Mannose and glucose were also the main sugars; their absolute amounts per wall increased 6–7 fold during cell growth. Walls isolated with omission of a dodecylsulphate/mercaptoethanol/urea extraction step had a higher protein content and, with young walls, a significantly higher glucose and fucose content. These data and other published cell wall analyses show a wide variability in cell wall composition of the members of the genus Chlorella.Abbreviations GLC gas liquid chromatography - TFA trifluoroacetic acid     

Isolation and partial characterization of the major sialoglycoprotein of human T-lymphoblastoid cells of a MOLT-4B cell line.

A sialoglycoprotein with an approx. mol.wt. of 95000 was isolated from human lymphoblastoid cells of a MOLT-4B cell line, which was of human T-lymphocyte origin, by ion-exchange chromatography, affinity chromatography on a column of wheat-germ agglutinin-Sepharose and preparative slab-gel electrophoresis. The localization of this glycoprotein on the cell surface was indicated by surface labelling by the periodate/NaB3H4 and lactoperoxidase-catalysed iodination methods. Carbohydrate analyses of this glycoprotein revealed that its total carbohydrate content is 28% (w/w), and it contains fucose, galactose, mannose, N-acetylglucosamine, N-acetylgalactosamine and sialic acid in molar proportions 1.0:4.0:3.7:3.5:1.2:2.5, suggesting that it has two types of sugar chain, i.e. sugar chains like those of serum glycoproteins and sugar chains of the type found in mucins. Actually, alkaline borohydride treatment of this glycoprotein yielded tri- and tetra-saccharide, the latter containing 1 molecule of fucose in addition to each molecule of galactose, N-acetylgalactosamine and sialic acid. This glycoprotein bound to Ricinus communis agglutinin and concanavalin A as well as to wheat-germ agglutinin.     

A water-insoluble (1-->3)-beta-D-glucan from the alkaline extract of an edible mushroom Termitomyces eurhizus

A water-insoluble glucan, TEINS has been isolated from the hot alkaline extract of an edible mushroom Termitomyces eurhizus. The total carbohydrate content of the polysaccharide fraction was found to be 98.4%, and it was found to contain only glucose as the monosaccharide constituent. On the basis of total acid hydrolysis, a methylation experiment, periodate oxidation and (13)C NMR experiment, the repeating unit of the polysaccharide was established as: -->3)-beta-D-Glcp-(1-->.     

Cell wall components in Staphylococcus aureus with double resistance to gramicidin S and actinomycin D]

Cell walls of Staphylococcus aureus R9/80 resistant to gramicidin S and actinomycin D were investigated. The strain was isolated after passages of a previously isolated strain of S. aureus with resistance to gramicidin and definite changes in the cell walls, a medium with increasing concentrations of actinomycin being used for the passages. The data on the study of the cell walls of the strain with the double resistance were compared with the results of the investigation of the cell walls of the strain susceptible to gramicidin, the gramicidin resistant strain (initial for strain R9/80) and the actinomycin adapted strain that also showed changes in the cell walls. The cell walls of the resistant strains had no significant changes in the peptidoglycane and glucosamine levels, as well as in the peptidoglycane amino acid composition. Teichoic acids of all the strains had different levels of substitution of ribite by D-alanine (a factor influencing the negative charge of teichoic acids and the wall at large). It was noted that all the strains resistant to the tested antibiotics had lower levels of teichoic acids in the cell walls. The resistant cells showed some increase of the lipid component in the walls: from 1.6% in the susceptible strain to 2.1-2.9% in the resistant cells. The main trend of the changes in the resistance development was revealed to be the thickening of the cell wall and its consolidation. The development of resistance to gramicidin, actinomycin and to both the antibiotics provoked respectively a 2.4-, 4- and 5.4-fold increase of the content of the main cell component. i.e. peptidoglycane in the cell biomass. The barrier role of the cell walls in the resistant strains and their ability to bind the antibiotic is discussed.     

Behavior of aldehyde moieties involved in the activation of suppressor cells by sodium periodate

The treatment of mouse spleen cells with periodate at the optimal mitogenic concentration (1 mM) induces the activation of suppressor cells of the in vitro antibody response and leads to the formation of aldehydes on the carbohydrate termini of the surface sialoglycoconjugates. These aldehyde moieties are found on the C8 (N-AN 8) and the C7 (N-AN 7) derivatives of sialic acid. Immediate borohydride reduction prevents the activation of the suppressor cells. Data from this work show that borohydride reduction must be performed within the first 6 hr to prevent the generation of suppressor cells; 18 hr after the initial periodate oxidation, borohydride treatment did not reverse the in vitro suppressive activity of periodate-treated cells. The kinetics of the disappearance of aldehydes from the cell surface were studied by using [3H]borohydride labeling and chromatographic analysis of sialic acid derivatives. About 70 to 80% of the aldehyde moieties were found to be present 6 hr after periodate oxidation. After 18 hr, 50 to 70% of the aldehyde had disappeared from the lymphocyte membrane. Oxidized sialyl residues disappear completely after 60 hr of culture. This period corresponds to the de novo synthesis of sialic acid residues on the surface of periodate-activated cells. The two classes of oxidized sialyl-glycoconjugates were found to behave in different ways. In effect, our data showed that the aldehydes remaining at 18 hr are mainly located on the gangliosides, whereas the aldehyde moieties located on high m.w. glycoproteins disappear from the cell surface between 9 and 18 hr. This would suggest that the remaining aldehydes located on gangliosides are not directly involved in the expression of suppressive activity.     

Complementary immunolocalization patterns of cell wall hydroxyproline-rich glycoproteins studied with the use of antibodies directed against different carbohydrate epitopes.

Antisera raised against the major hydroxyproline-rich glycoprotein (HRGP) in carrot (Daucus carota L.) taproot, extensin-1, and a minor HRGP, extensin-2, were characterized by western blot analysis, enzyme-linked immunosorbent assay, and periodate oxidation and found to be directed against carbohydrate epitopes shared by both glycoproteins. The anti-extensin-1 antibodies (gE1) target periodate-sensitive epitopes and may recognize the terminal alpha-1,3-arabinoside of extensin-1. The anti-extensin-2 antibodies (gE2) recognize periodate-insensitive epitopes, possibly binding the reducing, internal beta-1,2-arabinosides on the carbohydrate side chains. Despite the cross-reactivity of these antibodies, immunolocalization studies of carrot taproot and green bean (Phaseolus vulgaris L.) leaf tissues reveal a spatial segregation of gE1- and gE2-labeling patterns. The gE1 antibodies bind only to the cellulose-rich region of the cell wall (J.P. Staehelin and L.A. Stafstrom [1988] Planta 174: 321-332), whereas gE2 labeling is restricted to the expanded middle lamella at three cell junctions. Periodate oxidation of nonosmicated, thin-sectioned tissue abolishes gE1 labeling but leads to labeling of the entire cell wall by gE2, presumably as a result of unmasking cryptic epitopes on extensin-1 in the cellulose layer. Purified extensin-2 protein is more efficient than extensin-1 protein at agglutinating avirulent Pseudomonas strains lacking extracellular polysaccharide. Our results indicate that extensin-2 does not form a heterologous HRGP network with extensin-1 and that, in contrast to extensin-1, which appears to serve a structural role, extensin-2 could participate in passive defense responses against phytopathogenic bacteria.     

Structure of the glycan chain from the surface layer glycoprotein of Clostridium thermohydrosulfuricum L77-66.

The thermophilic eubacterium Clostridium thermohydrosulfuricum L77-66 is covered by a crystalline surface layer composed of identical glycoprotein subunits which are arranged in a hexagonal lattice with centre-to-centre spacings of approx. 14.3 nm. Sodium dodecyl sulphate-polyacrylamide gel electrophoresis of cell wall preparations showed the presence of several broadened, carbohydrate-containing bands in a molecular mass range of 90 to 200 kDa. A total carbohydrate content of approx. 14% was determined in the purified surface layer glycoprotein. Chemical deglycosylation of this material by trifluoromethanesulfonic acid resulted in the disappearance of the complex banding pattern. Only a single band with a molecular mass of 82 kDa remained visible upon Coomassie staining. After proteolytic digestion of the surface layer glycoprotein a single glycopeptide fraction with an apparent molecular mass of approx. 25 kDa was obtained by gel filtration. Composition analysis, methylation, periodate oxidation and a combination of homonuclear and 1H-detected heteronuclear shift-correlated nuclear magnetic resonance experiments established the following structure for the glycan chain of the surface layer glycoprotein.     

Investigations on the structure of a hemicellulose fraction isolated from the trunk of a young bael (Aegle marmelos) tree

Purified hemicellulose isolated from a young bael (Aegle marmelos) tree with 2.5m sodium hydroxide contained d-xylose and 4-O-methyl-d-glucoronic acid in the molar ratio of 7.43:1; traces of glucose, galactose, rhamnose, and arabinose were also present. The linkages between the monosaccharide units were determined by methylation analysis of a hemicellulose fraction (II A) and carboxyl-reduced, hemicellulose II A, and the results were corroborated by those from periodate oxidation and Smith degradation. The anomeric configurations of the d-xylopyranosyl residues were determined by chromium(VI) trioxide oxidation of the acetylated, carboxyl-reduced hemicellulose, and the aldobiouronic acid obtained from graded hydrolysis was characterized. These experiments clearly revealed the structure of this hemicellulose.     

Charge asymmetry of the purple membrane measured by uranyl quenching of dansyl fluorescence

Purple membrane was covalently labeled with 5-(dimethylamino) naphthalene-1-sulfonyl hydrazine (dansyl hydrazine) by carbodiimide coupling to the cytoplasmic surface (carboxyl-terminal tail: 0.7 mol/mol bacteriorhodopsin) or by periodate oxidation and dimethylaminoborane reduction at the extracellular surface (glycolipids: 1 mol/mol). In 2 mM acetate buffer, pH 5.6, micromolar concentrations of UO2 +(2) were found to quench the dansyl groups on the cytoplasmic surface (maximum = 26%), while little quenching was observed at the extracellular surface (maximum = 4%). Uranyl ion quenched dansyl hydrazine in free solution at much higher concentrations. Uranyl also bound tightly to unmodified purple membrane, (apparent dissociation constant = 0.8 microM) as measured by a centrifugation assay. The maximum stoichiometry was 10 mol/mol of bacteriorhodopsin, which is close to the amount of phospholipid phosphorus in purple membrane. The results were analyzed on the assumptions that UO2 +(2) binds in a 1:1 complex with phospholipid phosphate and that the dansyl distribution and quenching mechanisms are the same at both surfaces. This indicates a 9:1 ratio of phosphate between the cytoplasmic and extracellular surfaces. Thus, the surface change density of the cytoplasmic side of the membrane is more negative than -0.010 charges/A2.     

Structural features of the sulphated polysaccharide from a green seaweed,Caulerpa taxifolia

A sulphated heteropolysaccharide was isolated from a green seaweed, Caulerpa taxifolia, by extraction with acid and purified via its copper complex. Methylation analysis of both the sulphated and desulphated polysaccharides revealed the presence of 1,4-linked xylose, 1,6-linked galactose, 1,4,6-linked mannose and non-reducing galactose end group units which are all devoid of sulphate groups. In addition 1,4-linked galactose units sulphated at C-3 are also present. Quantitative periodate oxidation showed a consumption of 1.30 and 1.60 moles of oxidant per anhydrosugar unit in the sulphated and desulphated polysaccharides respectively. The oxo-polysaccharides after reduction and hydrolysis revealed the presence of glycerol, erythritol and unoxidized galactose in the mol ratio 11.6:5.1:4.9 and 11.2:5.0:1.0 respectively, besides threitol (3.9 mol) in the desulphated polysaccharide.