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Acidic capsular polysaccharide of Vibrio parahaemolyticus A55 (05:K15) was esterified with ethylene oxide and then reduced with sodium borohydride. Hydrolysis of the reduced material gave two hexosamines identical with mannosamine (2-amino-2-deoxy-mannose) and gulosamine (2-amino.2-deoxy-gulose) on paper and ion-exchange chromatograms. This fact suggested that the K15 antigen contained mannosaminuronic acid (2-amino-2-deoxy-mannuronic acid) and gulosaminuronic acid (2-amino-2-deoxy-guluronie acid). Direct acid hydrolysis of the antigen gave two 2-amino-2-deoxy-hexuronic acids. These two were identified as mannosaminuronic and gulosaminouronic acids by paper chromatography and paper electrophoresis, being compared with synthesized standard samples. Titration of acid form of the polysaccharide indicated that about 91.5% of the polysaccharide was composed of the N-acetyl-hexosaminuronic acids. [ABSTRACT FROM AUTHOR]

From the lipopolysaccharides of Vibrio cholerae strains Inaba and Ogawa, an unusual amino sugar was isolated. By sequential oxidation with sodium metaperiodate and hypoiodide and by oxidation with periodate followed by detection of the acetaldehyde formed, the amino sugar was characterized as a 2-amino-dideoxyaldose. The hydroehloride of the amino sugar was compared with those of 2-amino-2,6-dideoxy-D-glucose, 2-amino-2,6-dideoxy-L-galactose, 2-amino-2,6-dideoxy-D-mannose, 2-amino-2,6.di-deoxy-D-gulose, and 2-amino-2,6-dideoxy-D-allose in an amino acid analyzer. The amino sugar alcohols were studied by paper electrophoresis in molybdats buffer. From the results obtained the amino sugar was identified as 2.amino-2,6-dideoxyglucose (quinovosamine). Its optical rotation indicated it to be of the D-configuration. [ABSTRACT FROM AUTHOR]

Mild acid degradation of lipopolysaccharides from Pseudomonas aeruginosa O10a and O10a,b (Lányi classification) resulted in O-specific polysaccharides built up of trisaccharide repeating units containing 2-acetamido-2,6-dideoxy-D-glucose (N-acetylquinovosamine, DQuiNAc), 2-acetamido-2,6-dideoxy-D-galactose (N-acetylfucosamine, DFucNAc), and 5-acetamido-3,5,7,9-tetradeoxy-7-[(R)-3-hydroxybutyramido]-L-glycero-L-mannononulosonic acid. The latter is a di-N-acyl derivative of a new sialic-acid-like sugar which was called by us pseudaminic acid (PseN2). A 3-hydroxybutyric acid residue was also found in natural carbohydrates for the first time. In the O10a,b polysaccharide pseudaminic acid carried an O-acetyl group at position 4. For selective cleavage of the O10a polysaccharide, solvolysis with hydrogen fluoride was employed which, owing to the relatively high stability of the glycosidic linkage of pseudaminic acid, led to the disaccharide with this sugar on the non-reducing terminus. Performing the solvolysis in methanol afforded the methyl glycoside of this disaccharide which proved to be more advantageous for further analysis. Carboxyl-reduction made the glycosidic linkage of pseudaminic acid extremely labile, and mild acid hydrolysis of the carboxyl-reduced O10a polysaccharide afforded the trisaccharide with a ketose derivative on the reducing terminus. Establishing the structure of the oligosaccharide fragments obtained and interpreting the 13C nuclear resonance spectra of the polysaccharides allowed to determine the following structure for their repeating units: → 4)DFucNAc(α1 → 3)DQuiNAc(β1 → OCHCH2CO → 7)PseN2Ac(β2 I 41 (R) CH3 OR serotype O10a R = H serotype O10a,b R = Ac In the polysaccharides the N-acetylquinovosamine residue is attached not to pseudaminic acid itself, but to its N-acyl substituent, 3-hydroxybutyryl group, and thus the monomers are linked via both glycosidic and amidic linkages. [ABSTRACT FROM AUTHOR]

Two lipopolysaccharide preparations were obtained from Escherichia coli 058 by extraction with 45% aqueous phenol and fractional precipitation with cetyltrimethyl ammonium bromide (Cetavlon). Chemical analysis and polyacrylamide gel electrophoresis in the presence of sodium dodecylsulfate showed that the two preparations differed only in the extent of the O-specific polysaccharide moiety. The O-specific polysaccharide was characterized with proton magnetic resonance and infrared spectroscopy, optical rotation and paper electrophoresis. Using gas-liquid chromatography and ion-exchange chromatography, it was shown to contain D-mannose, 2-acetamido-2-deoxy-D-glucose, 3-O-(R-1'-carboxyethyl)-L-rhamnose (rhamnolactylic acid), and O-acetyl groups in the molar ratios of 2:1:1:1. The polysaccharide and oligosaccharides obtained from it were subjected to methylation and chromic acid oxidation. The results obtained indicated that the polysaccharide consists of tetrasaccharide repeating units in which the trisaccharide β-GlcNAc1-4αMan-1-4(2/3-O-Ac)-Man is substituted at C-3 of the non-acetylated mannose with rhamnolactylic acid. The repeating units are joined through α-mannosyl-1-3-glucosamine bonds. This structure is identical with that of the cell wall polysaccharide of Shigella dysenteriae type 5. [ABSTRACT FROM AUTHOR]

A linear lattice of equivalent binding sites with nearest neighbor cooperativity is used as a basic model for a linear biopolymer displaying cooperative binding of small ligands. Complicating effects due to the dimerization of free ligands are taken into account. The theory implies two types of intrinsic binding processes: (1) the binding of an isolated ligand (nucleation), and (2) the binding of a ligand in the immediate neighborhood of an already bound one (aggregation). The ratio of the respective association constants determines the degree of cooperativity. It is generally shown how the equilibrium properties of the system can be derived by means of the matrix method. Sonic properties of special practical significance, such as the equilibrium concentrations of free and bound monomer ligands, are discussed in more detail. On the basis of a pertinent kinetic model chemical relaxation of strong cooperative bindinng is also examined. The paper gives particular emphasis to the formulation of quantitative relations which are most useful for an experimental application of the the oretical results. Appropriate procedures to plot measurable data and to evaluate basic parameters are proposed. They are applied to specific experimental systems in a subsequent set of papers. [ABSTRACT FROM AUTHOR]

Proton-nuclear-magnetic-resonance spectroscopy is a powerful tool for investigating the solution structure of biopolymers provided that a substantial number of proton resonances are assigned in the spectrum. For large proteins the assignments have usually been made by the comparative one-dimensional NMR investigations of the parent and derivative proteins in different physicochemical conditions. In this paper, we show that the more powerful two-dimensional methods could be successfully applide to proteins of the size of human adult hemoglobin (Mr = 64 500). J-Correlated and NOE-correlated spectroscopy, together with topological relationships in the known crystalline structure, enabled us to assign a large number of resonances. The majority of the assigned resonances correspond to the heine substituents and to amino acids in the heme pockets of the two subunits. These results thus provide an extensive set of intrinsic probes for mapping the conformation of the ligand-binding site and its functional changes. Comparison of the observed ring-current shifts of the assigned resonances with those calculated from the known crystallographic coordinates suggests a close similarity between the heme-pocket tertiary conformation in solution and in the crystalline state. A significant difference was noted for Leu141 in β subunits which, in solution, appears to have stronger contacts with the heine groups than in the crystalline form. The present results also demonstrate that two-dimensional-NMR methods could be successfully applied to the investigation of the structure of large biomolecules in solution (Mr ≤ 65 000). [ABSTRACT FROM AUTHOR]

The primary structure of Klebsiella serotype K10 capsular polysaccharide has been investigated using mainly the techniques of methylation, partial hydrolysis, and 1H and 13C NMR spectroscopy. The polysaccharide was found to consist of hexasaccharide repeating units having the following structure: [This symbol cannot be presented in ASCII format] [ABSTRACT FROM AUTHOR]

Westphal's procedure was found the most suitable for the isolation of lipopolysaccharide fractions from Brucella melitensis cells; only the fraction obtained in the aqueous layer (LPS) is studied in this paper. Beside LPS, an extracellular polysaccharide fraction (PS) was extracted from the culture medium of the bacteria, and the chemical composition of both fractions, LPS and PS, has been compared. In both cases, nucleic acid contaminants were eliminated either by alcohol precipitation or by use of cetavlon. These treatments also removed an acidic polysaccharide containing mainly galactosaminuronic acid, which might be Vi antigen. The main difference between LPS and PS is the presence of a small amount of lipid A in LPS (ca. 1%). As in the case of lipid A isolated from LPS of Enterobacteria, Brucella lipid A contains glucosamine and fatty acids, but β-hydroxymyristic acid could not be detected. The behaviour of LPS and PS fractions on DEAE-cellulose columns is very similar, and the small differences that could be observed may be explained by solubility changes due to the lipid moiety present in LPS. Both fractions contain 2-keto-3-hydroxy-octulosonic acid, a mixture of D- and L-glyceroD-mannoheptoses, a high amount of glucose, galactose, a 3,6-dideoxyhexose (colitose or abequose), and a methylated sugar behaving as a methylated derivative of dideoxyhexose. Small amounts of rhamnose in PS and of arabinose in LPS seem to be due to contaminants. [ABSTRACT FROM AUTHOR]

The body wall of the sea cucumber contains high amounts of sulfated glycans, which differ in structure from glycosaminoglycans of animal tissues and also from the fucose-rich sulfated polysaccharides isolated from marine algae and from the jelly coat of sea urchin eggs. In Ludwigothurea grisea, glycans can be separated into three fractions which differ in molecular mass and chemical composition. The fraction containing a high-molecularmass component has a high proportion of fucose and small amounts of amino sugars, whereas another fraction contains primarily a sulfated fucan. The third fraction, which represents the major portion of the sea cucumber polysaccharides, contains besides fucose, approximately equimolar proportions of glucuronic acid and amino sugars, and has a sulfate content higher than that in the other two fractions. Both D and L-isomers of fucose are found in these polysaccharides, and the sulfate is linked to the 0-3 position of the fucose residues. The attachment position of the sulfate groups to the glucuronic acid units and amino sugars is still undetermined. It is possible that these compounds are involved in maintaining the integrity of the sea cucumber's body wall, in analogy with the role of other macromolecules in the vertebrate connective tissue. [ABSTRACT FROM AUTHOR]

The sulfated polysaccharides in the tunic of Styela plicata occur as three fractions that differ markedly in molecular mass and chemical composition. The high-molecular-mass fraction has a high galactose content and a strong negative optical rotation while the low-molecular-mass fractions have a higher proportion of amino sugars and glucose. The galactose occurs in these polysaccharides entirely in the L-enantiomeric form. Although L-galactose is a constituent of several polysaccharides, this is the first report of sulfated polysaccharides that contain high amounts of L-galactose, and that lack the o enantiomorph of this sugar. Furthermore, the structure of the high-molecular-mass fraction, which is composed mainly ora core of α-L-galactopyranose residues, sulfated at position 3, linked glycosidically though position 1→4, and with non-sulfated L-galactopyranose non-reducing end-units, is unique among other previously described sulfated glycans. These data arc of considerable interest as they show an unusual example of possible variants of polyanionie glycans with structure function in living tissues. [ABSTRACT FROM AUTHOR]

A polymeric fraction containing the putative O-antigen has been isolated from the lipopolysaccharide of the reference strain (CDC 4534-60) for serogroup O9 of Serratia marcescens. The major component of the fraction was a polymer with a disaccharide repeating-unit of L-rhamnose (Rha) and 2-acetamido-2-deoxy-D-galactose (GalNAc) with the following structure: →3)D-GalpNAc(β1→3)L-Rhap(α1→. Evidence for the presence in the fraction of a similar, minor polymer containing 4-substituted rhamnose residues was provided by the NMR spectra, methylation analysis, and Smith degradation. [ABSTRACT FROM AUTHOR]

Lipopolysaccharides were isolated from the phenol layer on aqueous phenol extraction of cells of Pseudomonas aeruginosa O11 (Lányi classification), strains 170021 and 170040. On mild acid degradation of the lipopolysaccharides, with the subsequent gel-filtration on Sephadex G-50, neutral O-specific polysaccharides made up of 6-deoxysugars alone were obtained. Two 2-acetamido-2,6-dideoxy-L-galactose (LFucNAc), 2-acetamido-2,6dideoxy-D-glucose (DQuiNAc) and L-rhamnose (LRha) residues were found to be the components of the strain 170 021 polysaccharide repeating units; those of strain 170040 contained the same monosaccharides, but, instead of 2-acetamido-2,6-dideoxy-D-glucose residue, that of 2-acetamido-2,6-dideoxy-D-galactose (DFucNAc) was present. On the basis of the 13C nuclear magnetic resonance data, methylation analysis and three successive Smith degradations the following structures were determined for the polysaccharide repeating units: strain 170021. → 2)LRha(α1 → 3)LFucNAc(α → 3)LFucNAc(α1 → 3)DQuiNAc(β1 →; strain 170040, → 2)LRha(α1 → 3)LFucNAc(α1 → 3)LFucNAc(α1 → 3)DFucNAc(β1 → differing from one another by configuration of C-4 of 2-acetamido-2,6-dideoxy-D-hexopyranose only. [ABSTRACT FROM AUTHOR]

Partially purified enzymatic fractions from extracts of Escherichia coli B/r catalyse transfer of the isotope label from [adenine-2,8-3H]NAD+ to some bacterial proteins, as well as to hen egg-white lysozyme. The radio-active group in the modified lysozyme was identified as mono(ADP-ribose). Several bacterial proteins were labelled in vivo with 32P; the presence of the label in the form of an ADP-ribosyl group was shown in one of them. [ABSTRACT FROM AUTHOR]

A polysaccharide containing D-galactose (Gal), 2-acetamido-2-deoxy-D-galactose (GalNAc), 2-acet-amido-2-deoxy-D-glucose (GlcNAc), and 3-deoxy-3-(D-3-hydroxybutyramido)-D-quinovose (Qui3NR) was isolated from lipopolysaccharide (LPS) obtained from cells walls of the reference strain for Acinetobacter baumannii O23. By means of NMR studies, methylation analysis, and chemical degradations, the repeating unit of the polymer was identified as a branched pentasaccharide with the structure 1. The same polymer was apparently also present in LPS of the reference strain for serogroup O12, together with a second polymer based on a branched tetrasaccharide with the structure 2. This second polymer has previously been isolated as the O16 antigen of A. baumannii [Haseley, S. R., Diggle, H. J. & Wilkinson, S. G. (1996) Carbohydr. Res. 293, 259–265] and is probably present as a minor component of the LPS of A. baumannii O11 [Haseley, S. R. & Wilkinson, S. G. (1996) Eur. J. Biochem. 237, 266–271]. [ABSTRACT FROM AUTHOR]

A major polysaccharide containing D-galactose, D-glucose and 2-acetamido-2-deoxy-D-galactose was obtained after mild acid hydrolysis of the water-soluble material released by treatment of cell walls from Acinetobacter baumannii strain O11 with hot, aqueous phenol. By means of NMR studies, Smith degradation and N-deacetylation/deamination, the repeating unit of the polymer was identified as a branched pentasaccharide of the structure shown. Also present was a minor polymer containing glucose, 2-acetamido-2-deoxyglucose and 2-acetamido-2-deoxygalactose, the structure of which was not elucidated. On serological testing, the polymeric material was shown to correspond to the O-antigenic moiety of the parent extract (assumed to be lipopolysaccharide) and circumstantial evidence indicated that O11 specificity was conferred by the major polymer. [This Equation can not be represented as ASCII.txt] [ABSTRACT FROM AUTHOR]

A polysaccharide containing 2-acetamido-2-deoxy-D-glucose (GlcNAc), 2-acetamido-2-deoxy-L-fucose (FucNAc), and 2-acetamido-2-deoxy-D-galacturonic acid (GalNAcA) was isolated from an aqueous phenol extract of lipid-free, isolated cell walls of the reference strain for Acinetobacter baumannii sero-group O5, by mild acid hydrolysis of the extract and chromatography of the water-soluble products on Sephadex G-50. By means of NMR studies, methylation analysis, carboxyl reduction and chemical degradations, the repeating unit of the polymer was identified as a branched tetrasaccharide of the structure shown. The serologically active polymer is believed to correspond to the side chain of the O5 lipopolysaccharide: α-L-FucpNAc 1 ↓ 4 → 3)-β-D-DlcpNAc-(1 → 3)-α-D-GalpNAc-(1→3)-α-L-FucpNAc-(1→ [ABSTRACT FROM AUTHOR]

The precursors of the legumin-like storage protein from developing white lupin seeds (35 days after flowering) are trimers composed of protomers of Mr 72000 or 67000. Some subunits of these oligomers contain processed precursor polypeptides, namely α polypeptides of either 52000 or 44000 linked through disulphide bonds to a β polypeptide of 21000, typical of the mature legumin. The prolegumin is glycosylated. Legumin oligomers purified from the same seeds are both trimers and hexamers; some of their subunits are still made of precursor polypeptides. The hexamer contains less precursor polypeptide than the trimer. A low level or absence of precursor appears to be a condition of hexamer assembly. The heterogenous prolegumin and legumin oligomers represent intermediates in the processing of the prolegumin to mature legumin. Hydrophobic-interaction chromatography on TSK-phenyl-5PW and titration with the hydrophobic probe 8-anilino-1-naphthalenesulphonate indicate that the legumin is less hydrophobic than the prolegumin. This is attributed to structural rearrangements at processing of the propolypeptide, made evident by the behaviour in CD and by the second-derivative ultraviolet spectra of the two proteins. The total protein extract of developing cotyledons at 40 days after flowering contains endopeptidases, similar to those existing in the resting seeds, which cause a limited cascade degradation of the prolegumin and legumin. [ABSTRACT FROM AUTHOR]

The following structure of the repeating unit of the O-specific polysaccharide of Proteus mirabilis O27, containing amides of D-glucuronic and D-galacturonic adds with L-lysine and L-alanine, respectively, N-acetyl-Dglucosamine and phosphoethanolamine, has been determined: ... The main methods used for structural analysis of the polysaccharide were selective solvolysis with anhydrous hydrogen fluoride and Smith degradation, as well as NMR spectroscopy and mass spectrometry. The immunodominant role of the lateral N-acetyl-D-glucosamine and phosphoethanolamine in manifesting the serological specificity of P. mirabilis O27 has been established. [ABSTRACT FROM AUTHOR]

The polysaccharide chain of Proteus vulgaris Ol 9 lipopolysaccharide contains o-galactose, N-acetyl-D-glucosamine, N-acetyl-D-galactosamine and N-acetyl-L-fucosamine in the ratio 1:1:1:1. The structure of the polysaccharide was established by full acid hydrolysis and methylation analysis, as well as by non-destructive methods, i.e. the computer-assisted evaluation of the 13C-NMR spectrum and computer-assisted evaluation of the specific optical rotation by Klyne's rule. The polysaccharide is regular and built up of tetrasaccharide repeating units of the following structure: →3)-α-L-FucNAcp-(1→3)-β-D-GlcNAcp-(1→3)-α-D-Galp-(1→4)-α-D-GalNAcp-(1→ The O19-antiserum cross-reacts with lipopolysaccharide from P. vulgaris O42, the structure of which is still unknown. No cross-reactions were observed with O-polysaccharides Pseudomonas aeruginosa O7 and Salmonella arizonae O59 in spite of some structural similarities. [ABSTRACT FROM AUTHOR]

Only two S-adenosyl-L-methionine synthetase forms exist in rat liver: high-M, S-adenosyl-L-methionine synthetase and low-Mr S-adenosyl-L-methionine synthetase, which have been purified to apparent homogeneity as judged by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. High-Mr S-adenosyl-L-methionine synthetase had an apparent molecular mass, determined by gel filtration, of 210 kDa and was a tetramer constituted by 48,5-kDa subunits, estimated by sodium dodecyl sulfate/polyacrylamide gel electrophoresis. The apparent molecular mass of low-Mr S-adeaosyl-L-methionine synthetase, as estimated by gel filtration, was 110 kDa and was constituted by two subunits of 47 kDa. An antiserum against low-Mr S-adenosyl-L-methionine synthetase cross-reacted with the two forms. Reverse-phase HPLC runs of tryptic digestions of high-M, and low- Mr & adenosyl-L-methionine synthetase showed that the peptide maps of the two forms were very similar, if not ideatical. High-Mr S-adenosyl-L-methonine synthetase activity was inhibited by S-adenosyl- L-methionine and pyrophosphate. Depending on the dose used, S-adenosyl-L-methionine activated or inhibited low-Mr, S-ad enosyl- L-methionine synthetase and pyrophosphate had no effect on this form. The two synthetases showed a different specific activity at the physiological concentration of methionine. This report shows that even though the two forms are constructed of the same polypeptide chains, they are regulated in a different manner by methionine and by the products of the reaction. [ABSTRACT FROM AUTHOR]

The polymorphism of lipid A, the endotoxic principle of the lipopolysaccharides of gram-negative bacteria, has been investigated in the fully hydrated state at temperatures between 5° and 58°C via Fourier-transform infrared spectroscopy. These measurements were supplemented by X-ray diffraction, fluorescence intensity techniques and differential thermal analysis. Up to three distinct phase transitions could be detected, with the main transition temperatures lying at approximately 41°, 46°, 44° and 47°C for Escherichia coli lipid A, Salmonella minnesota lipid A, and the synthetic lipid A compounds 506 and 516, respectively. 4′-Monophosphoryl-lipid A samples exhibited their main transition temperatures at considerably higher temperatures (about 52°C for E. coil lipid A). The analysis of >CH2 stretching absorption bands as well as the wide-angle scattering behaviour of the lipid A samples showed that the main transition apparently involved the completion of hydrocarbon chain melting of lipid A, as typically observed for phospholipids. However, the phase transition behaviour was found to be much more complex than that usually observed for model phospholipid systems. Even below the main transition temperature, considerable amounts of the methylene segments of the acyl chains of lipid A were found to assume gauche conformations. These conformational changes might be related to the occurrence of up to two further transitions located at about 22°, 30°, 27° and 25.5°C (first transition) and at about 34°, 42°, 38.5° and 40.5°C (second transition) for E. coli lipid A, S. minnesota lipid A and the synthetic lipid A compounds 506 and 516, respectively. Furthermore, by the analysis of some characteristic infrared absorption bands related to the hydrophilic backbone, it could be demonstrated that the temperature-induced conformational changes occurring within the hydrocarbon chains were constantly and simultaneously accompanied by detectable rearrangements within the interfacial region and the polar head group of lipid A. The following conclusions were drawn: 1. Up to about 30°C the lipid A assemblies were supposed to adopt virtually bilayered, true lamellar arrangements, as revealed by the analysis of >CH2 scissoring vibrations and X-ray diffraction pattern. However, as indicated by fluorometric techniques, no stable closed vesicles seemed to be formed even under these conditions. 2. At elevated temperatures no straightforward interpretation of the data was possible: while the X-ray data did not indicate any drastic changes in the bilayer arrangement at the molecular level even after completion of the acyl chain melting process, the fluorometric and calorimetric data suggested the existence of more complex supermolecular arrangements at temperatures above the main transition. [ABSTRACT FROM AUTHOR]

Coccoliths of the unicellular marine alga Emiliania huxleyi are formed intracellularly in a specialized vesicle. Closely associated with the CaCO3 crystals of the coccoliths is an acid Ca2+-binding polysaccharide. The latter is considered to fulfil a regulatory function in CaCO3 crystallization. In this study it is demonstrated that the coccolith polysaccharide is able to inhibit CaCO3 precipitation in vitro. The degree of inhibition is dependent on the nature of the cations bound to the acid groups of the polysaccharide. After substitution of Na+ by Ca2+ ions the polysaccharide is far less effective in inhibiting CaCO3 precipitation. The coccolith polysaccharide contains two types of acid groups: uronic acids and sulphate esters. Only the uronic acids are responsible for the inhibition of CaCO3 precipitation. Desulphated polysaccharide inhibits precipitation to the same extent as the native molecule whereas the carboxyl-reduced polysaccharide is unable to block CaCO3 crystallization. Inhibition of CaCO3 precipitation can be suppressed by the presence of ethanol. Presumably the conformation the molecule is altered under these conditions. Alginic acid [poly(mannuronic-iduronic acid)] and poly(galacturonic acid) also inhibit CaCO3 precipitation. Inhibition of precipitation is effected by the coccolith polysaccharide and alginic acid at comparable concentrations. The concentration of poly(galacturonic acid) needed to obtain the same result is about 20-times higher. Contrary to the coccolith polysaccharide, both poly(uronic acid)s inhibit CaCO3 precipitation in the absence as well as in the presence of ethanol. These results suggest that conformational changes of the coccolith polysaccharide may play a role in CaCO3 crystallization in vivo. [ABSTRACT FROM AUTHOR]

A procedure is described in detail by which the algal polysaccharide, t-carrageenan, can be modified and fragmented to products of more regular structure and shorter chain length. Since most chains of this product can form uninterrupted helices they undergo the disorder-order transition without the gelation that is a feature of the behaviour of the natural polymer. The disorder-order transition becomes much sharper and occurs at higher temperatures with increasing chain length. The sign and magnitude of the accompanying shift in optical rotation can be estimated satisfactorily from the solid-state geometry of the helix using our semi-empirical method for correlating optical rotation with the conformations at glycosidic linkages. In solutions of the helical form, but not the coil form, cationic dyes such as methylene blue or acridine orange show induced optical activity and a changed visible spectrum which we attribute to binding and stacking on the helix. This also causes a shift in the temperature of the helix-coil transition. We discuss the possibility that the tendency of dyes to stack could drive polysaccharides into conformations that they would not otherwise adopt, with implications for their use as ‘conformational probes’. [ABSTRACT FROM AUTHOR]

The chemical structure of the O-specific polysaccharide of Citrobacter 036 lipopolysaccharide has been established by methylation analysis using gas-liquid chromatography and mass spectrometry. This polysaccharide is a linear homopolymer composed of (β1 → 2)4-linked 4-deoxy-D-arabinohexopyranosyl residues. [ABSTRACT FROM AUTHOR]

The polygalacturonate lyase and pectinesterase activities of Clostridium multifermentans, both produced extracellularly when the organism grows on pectin or polygalacturonate, have been suggested to be associated in a single complex. Both enzymic sites act on their respective substrates by single-chain action patterns, as shown by equivalent release of terminal tritium label and total product throughout the reaction. From these results, the Km and V of the lyase, and the amount of lyase activity present, we calculate the steady-state concentration of lyase substrate expected during action of the two sites on pectin if the sites are independent. No such steady-state concentration of lyase substrate was observed. Therefore, we conclude that the two types of active site act in a coordinated manner; the polysaccharide chain passes from the esterase site to the lyase site without intermediate dissociation and rebinding. This ‘molecular disassembly line’ constituted by the two sites may represent a system of general significance in synthesis and degradation of biological polymers. [ABSTRACT FROM AUTHOR]

A number of ATP analogs were tested for their ability to depolymerize F-actin and interact with it. It was found that those analogues which are hydrolysable are incorporated into the F-actin polymer whereas the non-hydrolysable ones are displaced from the nucleotide binding site during the polymerization process. [ABSTRACT FROM AUTHOR]

Four of the six chromatin preparation methods examined here are shown to produce chromatins whose template properties differ radically from one another. Comparing the template-activity profiles obtainable with endogenous (i.e. chromatin-bound) and exogenous form B rat liver RNA polymerases across a range of salt concentrations, it appears that only one of these chromatins resembles isolated nuclei at all closely. A study of RNA product size on formamide gradients further reveals that only in this single case is there any sign of product-chains larger than 28-S rRNA. However, all these chromatins contain considerable exonucleotytic RNase activity, more marked at low than at high ionic strengths. In addition, prolonged (30-s) shearing is shown to cause drastic changes in the template properties of chromatin, as well as solubilizing it by reduction of fragment size. [ABSTRACT FROM AUTHOR]

From Escherichia coli O8:K87(B?>):H19 an acidic polysaccharide was extracted with 45% aqueous phenol and purified by ultracentrifugation and fractional precipitation with cetyltrymethylammonium bromide (cetavlon). The acidic polysaccharide was homogenous as judged by ultracentrifugation analysis. It has a molecular weight (sO20,w/D) of 280000, a sedimentation constant (sO20,w) of 3.07 x 10-13 sec and a diffusion constant (D) of 0.85 x 10-7 cm per sec. The acidic polysaccharide consists of equimolar amounts of L-fucosamine, glucosamine, glucose, galactose and glucuronic acid. It contains one O-acetyl group per 5 sugar units. Using periodate oxidation, hydrazinolysis, carboxyl reduction, methylation and partial acid hydrolysis it was found that the polysaccharide is built from about 270-300 of pentasaccharide repeating units which have the following structure: β-D-GlcUa-(1,3)-L-FuncNAc-(1,3)-GlcNAc-(1,6)-Gal ... The linkage between the repeating units is a galactosyl-(1,4)-glucuronic-acid bond. The O-acetyl groups are bound in the 2 position of either glucose or galactose of each repeating unit. With the aid of bacterial agglutination, passive hemagglutination and immune precipitation it was established that the acidic polysaccharide represents the K87 antigen of E. coli 08:K87(B?):H19. As determined by seriological inhibition studies, the non-reducing glucose units and )-acetyl groups (on the 2-position of either glucose) are essential parts of the determinant groups(s). [ABSTRACT FROM AUTHOR]

Water insoluble blue starch polymer particles incorporated into agar medium were shown to be hydrolyzed by α-amylase enzyme. When a drop of alpha;-amylase was placed on such a medium the enzyme diffused at the same time hydrolyzing the water insoluble blue starch polymer to from a clear area which was easily visible and easily measured. The size of the hydrolyzed area was studied with respect to the time of hydrolysis, temperature of hydrolysis, and enzyme concentration. The valance of repeated measurements of the diameter of 80 hydrolyzed areas, as well as one area measured ten times, was determined. The variance in diameter measurements of the hydrolyzed areas on different plates and at different distances from the center of the plate was determined [ABSTRACT FROM AUTHOR]

From E. coli 0100:K?(B):H2 a lipopolysaccharide and an acidic polysaccharide were extracted with saline. The lipopolysaccharide was purified by repeated ultracentrifugation and the acidic polysaccharide by fractional precipitation with cetavlon. Both substances contained rhamnose, galactose, glucosamine, glycerol, and phosphate. The lipopolysaccharide was degraded in 1% acetic acid at 100° and the degraded polysaccharide was fractionated on Sephadex G-50. Thus a fraction was obtained which represents the specific polysaccharide moiety of the lipopolysaccharide. comparison of this fraction with the acidic polysaccharide (chemical analysis, periodate oxidation, alkaline hydrolysis, partial acid hydrolysis) showed that both substances have not only the same composition (rhamnose, galactose, glucosamine, glycerol and phosphate in the molar ratios of 2:1:1:1:1) but also the same structure. Both substances consist of a main chain (composed of rhamnose, galactose and glucosamine) to which glycerol is bound via a phosphodiester bridge from one of its primary hydroxyl groups to one of the rhamnose residues. Some alternatives for the structure are presented. In serological tests (passive haemagglutination, bacterial agglutination, immune precipitation and inhibition of these) it was shown that acidic polysaccharide and lipopolysaccharide have identical reactivities in anti-O and anti-OK antisera. This confirms the structural identity of the polysaccharides. It is concluded that E. coli 0100:K?(B):H2 contains no polysaccharide which represents a K antigen. The O antigen of this strain contains glycerol phosphate as (part of) a determinant group. Precipitating antibodies, directed against this group are present in anti-OK antiserum and not in anti-O antiserum. In this context the phenomenon of O inagglutinability of E. coli atrains, which carry K antigens, is discussed. Possible implications in the definition of K antigens of the B type in human enteropathogenic E. coli strains are presented. [ABSTRACT FROM AUTHOR]

An Escherichia coli ‘permeabilized’ cell system obtained by treatment in high sucrose concentration, because of its depletion from endogenous metabolites, its low nuclease, phosphatase and phosphokinase content, catalyzes a very efficient polymerisation of the common ribonucleoside diphosphates (UDP, CDP, ADP and GDP) presumably through the polynucleotide phosphorylase retained within the cells after osmotic shock. Accordingly incorporation of nucleotides into polymers is insensitive to actinomycin D and does not take place in the presence of Mg2+ ions with E. coli Q13 a strain in which the polynucleotide phosphorylase is a Mn-dependent enzyme. Polymers containing U, C, G, or A as the only constituting nucleosides have been found to accumulate for several hours at a linear rate, in the supernatant phase from osmotically shocked E, coli D10. If, following the arrest of polymer formation the amino acid coded for by the corresponding triplet is added to the incubation medium together with an energy-yielding system, a very efficient incorporation of this amino acid (and of this amino acid only) into an acid-precipitable material takes place. The resulting product (presumably an homopolypeptide) is associated with the cell bodies. Translation of the preformed poly-ribonucleotide is concomitant with its nucleolytic cleavage by a K-dependent nuclease. [ABSTRACT FROM AUTHOR]

Three new double-stranded polydeoxyribonucleotides containing the nucleoside analogs 6-thiodeoxyguanosine and 6-thiodeoxyinosine were synthesized enigmatically. Poly[d(A-C)· d(T-s6G)] and poly[d(A-s6G) · d(T-C)] were prepared by extensive synthesis with DNA polymerase from Micrococcus luteus, while the same enzyme from Escherichia coli failed in extensive polymerization but brought about the synthesis of poly [d(T-s6G)] and poly[d(T-s6)] by the repair mechanism using single-stranded poly [d(A-C)] template and a minute amount of d(T-G)n primer. The analog-containing DNAs were characterized by ultraviolet absorption and circular dichroisrn spectra as well as by their stability against thermal and alkaline denaturation. The substitution by 6-thiodeoxyguanosine or 6-thiodeoxyinosine for deoxyguanosine reduces the stability of the biopolymers considerably. Exchange of the analog-containing strand by single- stranded poly[d(T-G)] takes place rapidly even at ambient temperature. [ABSTRACT FROM AUTHOR]