Non-reducing End Groups Research Articles

Autolysis of isolated cell walls of Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg Strain 168. Separation of the products and characterization of the mucopeptide fragments.

1. Cell walls were isolated from Bacillus licheniformis N.C.T.C. 6346 and Bacillus subtilis Marburg strain 168 trp grown on casein hydrolysate into exponential phase. Autolysis was carried out and the soluble products, separated by chromatography on DEAE-cellulose, from the two wall preparations are broadly similar in composition and are in agreement with autolysis proceeding with hydrolysis of amide bonds between l-alanine and N-acetylmuramic acid residues in the mucopeptide components. 2. Peptides originating from the mucopeptide components were isolated and shown to be a monomer peptide, l-alanyl-d-glutamyl-meso-diaminopimelic acid and a dimer peptide containing two monomer peptides linked through a residue of d-alanine. Approximately one amide group is present for each equivalent tripeptide unit and is probably substituted on diaminopimelic acid residues. 3. Oligosaccharides originating from the mucopeptide components were isolated and after hydrolysis contained almost equimolar amounts of glucosamine and muramic acid and only very small amounts of amino acids. The number-average chain length, estimated by the release of non-reducing end groups of N-acetylglucosamine with exo-beta-N-acetylglucosaminidase, is approximately ten hexosamine residues for oligosaccharides isolated from either organism. The oligosaccharides are polydisperse. 4. N-Acetylglucosamine residues are the only reducing terminals detectable in the oligosaccharides isolated from B. subtilis or B. licheniformis cell-wall autolysates. The number-average chain lengths of the oligosaccharides were determined by estimation of the content of these residues and are higher than those found by enzymic assay. Possible reasons for the discrepancy are discussed.

Structural features of a lipopolysaccharide isolated from Escherichiacoli 086:Kneg.

Lipopolysaccharide (LPS) prepared from Escherichiacoli 086:Kneg., in 5.5% yield contained D-galactose, D-glucose, L-fucose, L-glycero-D-manno-heptose, D-glucosamine, D-galactosamine, 2-keto-3-deoxy-octulosonic acid (KDO) and lipid A. The molecule appeared to be homogeneous as tested by free boundary electrophoresis, ultracentrifugation, and immunodiffusion against ′O′ specific E. coli antiserum. Methylation studies of the LPS and also of the degraded polysaccharides obtained by partial acid hydrolysis showed that the molecule was highly branched. Sixty percent of the D-galactose units were non-reducing terminal groups, the remainder were linked (1 → 3) and (1 → 2) and 3-O-β-D-galactopyranosyl-D-galactose was identified as a product of mild acid hydrolysis of the parent LPS. Fucose occurred in the polysaccharide as (1 → 4) linked units. Methylation results showed that the D-glucose units were linked (1 → 3) and (1 → 4). Partial acid hydrolysis yielded cellobiose, cellotriose, and laminaribiose, showing that the glucose units formed a glucan chain within the polysaccharide and that the glucosidic linkages were in the β-D-configuration. Approximately one half of the L-glycero-D-manno-heptose units occurred as non-reducing end groups, the other half were linked at C-3 and either one of C-6 or C-7. One half of the D-galactosamine units was linked (1 → 3) with the remainder occurring as double branch points. D-Glucosamine residues occurred exclusively in the lipid A moiety in a (1 → 4) linked core structure.

Immunochemical studies on a laminaribiosyl-azoprotein conjugate

Laminaribiose ( 3-o-β- d-glucopyranosyl- d-glucose ) was coupled to bovine serum albumin (BSA) by an azophenyl linkage to provide the synthetic antigen BSA-p-phenylazo-β-laminaribioside. The behavior of antisera prepared in rabbits immunized with the β-laminaribiosyl conjugate was examined by immunodiffusion, quantitative precipitation and hapten inhibition. Anticonjugate absorbed with carrier protein showed the greatest reactivity with the homologous β-laminaribiosyl-BSA antigen, but also showed some cross precipitation with β-cellobiosyl, β-sophorosyl and β-gentiobiosyl-BSA conjugates. Glucobioses linked through the β-(1 → 2), β-(1 → 3), β-(1 → 4) and β-(1 → 6) positions, as well as laminaridextrins and tri and tetrasaccharides of β-linked glucose possessing a laminaribiose moiety either at a nonreducing end location or at a subterminal location, were assayed for their ability to inhibit antilaminaribioside precipitation. Hapten inhibition data showed anticonjugate a possess a high degree of specificity directed against the terminal nonreducing β-laminaribiosyl end group.

Immunochemical Relationships of Certain Capsular Polysaccharides of Klebsiella, Pneumococci and Rhizobia

Abstract Pyruvic acid is an immunologic determinant in the capsular polysaccharides of Klebsiella K32, pneumococcal types IV and XXVII, and eight strains of Rhizobia. All of these react heavily in anti-Pn XXVII. A cross-reaction between Pn IV and Pn XXVII is described and shown not to be due to pyruvic acid. Klebsiella K9 is characterized by non-reducing end-groups of D-glucuronic acid (or of 4-O-methyl-d-glucuronic acid) but differs in other respects from K2. Cross-reactions of K47 show it to be the only one of those tested with non-reducing end-groups of l-rhamnose. K52 reacts heavily in anti-Pn XXVIII. When depyruvylated, the rhizobial polysaccharides show much greater reactivity, and, occasionally, different reactivities in anti-Pn sera other than XXVII, in which depyruvylation virtually abolishes precipitation. The rhizobial polysaccharides may contain their pyruvic acid linked as a ketal to d-galactose; the linkage in S IV is more labile.

Immunochemistry of the capsular polysaccharide of an acinetobacter.

The capsular polysaccharide of a strain, BD4, of Acinetobacter calco-aceticus (A.c-a), composed of L-rhamnose and D-glucose, 4:1, precipitates all streptococcal group B antisera tested, as well as streptococcal group G antisera and antipneumococcal (Pn) type XXIII serum. Nonreducing end groups of L-rhamnose, known to be major determinants of the antigens giving rise to these antisera, are thus identified in A.c-a, which also precipitates anti-Pn VI sera. Both the rhamnose and glucose in the capsular polysaccharide (S) of Pn VI are linked 1,3-, as is the rhamnose in Pn S II, which also precipitates anti-Pn VI because of this linkage. A.c-a and S II precipitate the same fraction of anti-Pn VI, identifying a second portion of the rhamnose of A.c-a as 1,3-linked. This is confirmed by the stability of a portion of the rhamnose on oxidation of A.c-a with periodate, and 1,3-linked glucose or a periodate-stable branch point is also indicated in the same way. Two features of the fine structure of A.c-a have thus been established and a third indicated.

Immunochemical Relationships of Certain Streptococcal Group and Type Polysaccharides to Pneumococcal Capsular Antigens

Summary The quantitative data on cross-reactions of streptococcal group-like and group F polysaccharides permit these conclusions: z1 apparently contains non-reducing end-groups of L-rhamnose; z2 precipitates antipneumococcal type VII serum relatively independently of the type II substance occurring in the same cell but has groupings in common with TIV; z3 appears to contain 1,3-linked L-rhamnose; z4 overlaps markedly with TIV and z6 in anti-PnVII and reacts as if it contained end-groups of D-galactose. Type I polysaccharide overlaps somewhat with z1 in anti-PnVII and with TIII in anti-PnX; galactose is probably involved. Precipitation of anti-PnVII with TII removes some of the antibody reactive with z2; after both are removed there is no further precipitation with z1. Possibly PnSVII and z1, z2 and TII all contain 1,2-linked L-rhamnose or a few non-reducing end-groups of this sugar. The reactions of TII in anti-PnVIII and -XIX show for the first time that the long-known cross-reaction between PnVIII and XIX is due to multiple residues of 1,4-linked D-glucose. Like TI, TIII cross-reacts in anti-PnX and also precipitates anti-PnXIII, XIV and XX, the last not being due to glycogen-like linkages of D-glucose. The strong reactions of TIV with anti-PnVII and XVI are accounted for by multiples of non-reducing end-groups of β-D-galactose, as is perhaps also the heavy precipitation in anti-PnXVII. TIV also precipitates anti-PnVIII and -XIX, at least partly for the same reason as TII. z6 shows almost complete overlapping with z1 in anti-PnVII and -XXIII.

Some structural features of the mucilage from the bark of Ulmus fulva (slippery elm mucilage)

Slippery elm mucilage contains residues of l-rhamnose, d-galactose, 3- O-methyl- d-galactose, and d-galacturonic acid. The methylated polysaccharide yields 3- O- and 4- O-methyl- l-rhamnose, 2,3,4,6-tetra- and 2,3,6-tri- O-methyl- d-galactose, and 2,3,4-tri- and 2,3-di- O-methyl- d-galacturonic acid, in addition to trace amounts of 2,3,4-tri and 3,4-di- O-methyl- l-rhamnose and 2,4,6-tri- O-methyl- d-galactose. Borohydride reduction of the periodate-oxidised polysaccharide yields a polyalcohol, which, on partial hydrolysis with acid, affords O-(3- O-methyl- d-galactopyranosyl)-(1 → 4)- O-(3- O-methyl- d-galactopyranosyl)-(1 → 4)- O-(3- O-methyl- d-galactopyranosyl)-(1 → 4)- l-rhamnose. Mild, acid hydrolysis yields a Smith-degraded polysaccharide. Methylation analyses are reported for the polyalcohol and for the Smith-degraded polysaccharide. It is concluded that the polysaccharide contains chains of 3- O-methyl- d-galactose residues attached to the C-4 positions of certain l-rhamnose residues, and that 3- O-methyl- d-galactose residues occur in some cases as non-reducing end-groups. d-Galactose is attached as single residues or as 4- O-substituted residues to the C-3 positions of some l-rhamnose residues. This evidence indicates that the polysaccharide is more highly branched than was at one time supposed.

Cross-Reactions of the Group-Specific Polysaccharides of Streptococcal Groups B and G in Anti-Pneumococcal Sera with Especial Reference to Type XXIII and its Determinants

Summary Quantitative data are given on the extensive cross-reactivity of the group polysaccharides of streptococcal groups B and G in antipneumococcal type XXIII horse serum and on inhibition-reactions in this antiserum. L-Rhamnose, known to be a principal determinant of the two streptococcal substances, is even more easily split off from the specific polysaccharide of pneumococcal type XXIII. This, and the ready inhibition of both the S XXIII anti-XXIII and group B anti-XXIII reactions by L-rhamnose, show that this sugar, probably in the form of non-reducing end-groups, is also a major determinant of S XXIII. D-Galactose is also involved in the specificity of S XXIII and may reinforce the cross-reaction.

Acid‐catalyzed methanolysis of tri‐O‐methylamylose and tri‐O‐methylcellulose

AbstractPast experimental evidence has indicated that the acid‐catalyzed hydrolysis of polysaccharides does not proceed randomly, and it has been suggested that hydrolysis is more rapid for the glycosidic bonds by which the nonreducing endgroups are attached. To test this hypothesis, amylose and cellulose were permethylated and subjected to methanolysis. It was found that in both the methanolysis of tri‐O‐methylamylose and tri‐O‐methylcellulose the production of methyl 2,3,4,6‐tetra‐O‐methyl‐α,β‐D‐glucopyranoside was complete before the production of methyl 2,3,6‐tri‐O‐methyl‐α,β‐D‐glucoside was finished. Since the former compound could only come from the original nonreducing end units and the latter from all other units, these results were interpreted as giving support to the idea of a preferential scission of the bonds at the nonreducing ends, even though the release of original end units was not complete until 70–85% of the glycosidic bonds had been cleaved. It was concluded that methanolysis proceeds by a modification of the hydrolysis mechanism and that methanolysis is therefore a poor model for hydrolysis.

Structural studies on the capsular polysaccharide of pneumococcus Type V

Further details of the structure of the capsular polysaccharide (SV) of Pneumococcus Type V have been elucidated by methylation studies and by oxidation with periodate. It is proposed that the polysaccharide contains multiple residues of 2-acetamido-2,6-dideoxy-3- O-( Dglucopyranosyluronic acid)- L-galactose, to which are attached residues of D-glucose and 2-acetamido-2,6-dideoxy- L-talose N-acetyl- L- pneumosamine). Residues of 2-acetamido-2,6-dideoxy- L-galactose N-acetyl- L-fucosamine) constitute branch points, and pneumosamine residues are present mainly as non-reducing end-groups. The majority of the D-glucose and D-glucuronic acid residues are 1,4- and 1,2-linked, respectively. The immunochemistry of SV is reviewed in the light of these findings.

N-Acetylmannosamine digestion by human oral bacteria.

LEACH1 showed that N-acetylneuraminic acid was rapidly metabolized by human saliva and ascribed this to the presence of an inducible aldolase produced by contaminating micro-organisms since such destruction was prevented by the addition of ‘Ledermycin’ or by heating the saliva in boiling water. This appears to be the second step in a process whereby salivary mucoproteins having N-acetylneuraminic acid residues as their non-reducing end groups act as inducer molecules to oral bacteria, producing first a neuraminidase and later an aldolase which cleaves the N-acetylneuraminic acid liberated by the neuraminidase into N-acetylmannosamine and pyruvic acid. Such a sequence has been found by us2 to occur when (α1-glycoprotein, which is present in sputum, is used as an inducer molecule in cultures of Klebsiella aerogenes. We wish to report on the third step whereby N-acetylmannosamine itself is digested.

Cellulose Studies

In an attempt to establish unambiguously the presence of the nonreducing end group in cellulose when treated under nondegrading conditions, different celluloses were sub jected to exhaustive methylation, using sodium hydroxide and dimethyl sulfate in toluene. Other methods of methylation were also investigated. Samples with 43.8% methoxyl content were obtained which were found suitable for this investigation. Identification and quantitative estimation of tetramethyl glucose, which originates from the nonre ducing end of the cellulose chain, involved hydrolysis, extraction of the tetramethyl glucose from the main component, trimethyl glucose, and chromatographic analysis. Paper, thin-layer, and gas chromatography were used in the analysis and special techniques were developed for each method. Identification and quantitative estimation by gas chromatography were accomplished for the methyl glucosides and the trimethyl silyl derivatives of the methylated glucoses. Isolation of crystalline tetramethyl glucose and the preparation of its anilide derivative provided unambiguous proof of the presence of nonreducing end groups in cellulose when treated under nondegrading conditions. It was observed that methyl tetramethyl glucoside is volatile at room temperature and atmospheric pressure. This is advanced as a probable reason for the previously reported absence of nonreducing end groups in cellulose. The quantitative method of tetramethyl glucose estimation, in relation to the tri methyl glucose content, was used for the determination of the number average degree of polymerization of cellulose. For methyl celluloses of different origins, these were found to be: 908% ± 18% for cotton cellulose, 465% ± 21% for a commercial methyl cellulose, and 197% ± 20% for viscose rayon cellulose.

ISOLATION AND SOME STRUCTURAL FEATURES OF A POLYSACCHARIDE FROM BIRCH SAP

A polysaccharide, precipitated by cetyltrimethylammonium hydroxide from the non-dialyzable portion of birch xylem sap, contained D-galactose, D-mannose, and D-glucose in a molar ratio of 2:1.4:1 and small amounts of D-glucuronic acid. Periodate oxidation studies indicated that the polysaccharide was branched and contained a high proportion of periodate-resistant monosaccharides. This was confirmed by methylation and hydrolysis which yielded 2,3,4,6-tetra-O-methyl-D-glucose (2.0 moles); 2,4,6-tri-O-methyl-D-mannose (0.9 mole); 2,4,6-tri-O-methyl-D-galactose (3.8 moles); unidentified tri-O-methyl hexose (1.2 moles); and 3,5-di-O-methyl-D-mannose (1.8 moles). The latter, a new di-O-methyl-D-mannose, snowed the presence of D-mannofuranose units in the polysaccharide. This, together with the 1 → 3 linked α-D-mannopyranose units, makes this polysaccharide unique among plant products. Immunochemical tests with antipneumococcal and antiparatyphoid B horse sera confirmed the presence of D-glucuronic acid and D-glucopyranose non-reducing end groups and supported the other structural conclusions by showing that there were few, if any, 1 → 4 and 1 → 6 linked D-mannopyranose units or D-galactopyranose non-reducing end groups.

Studies on inherited variants of blood proteins: III. Sequential action of neuraminidase and galactose oxidase on transferrin B 1–2B 2

Transferrin B 1–2B 2 was isolated by sequential chromatography of human serum on diethylaminoethylcellulose and hydroxylapatite. The protein was homogeneous as indicated by starch-gel electrophoresis, immunoelectrophoresis, and iron-binding studies. After incubation with neuraminidase, the transferrin served as substrate for galactose oxidase, which can react with bound galactose when it occurs as a nonreducing end group. This is evidence that neuraminic acid is bound terminally to galactose in the carbohydrate moiety of transferrin B 1–2B 2.

THE STRUCTURE OF LINSEED MUCILAGE: PART II

Linseed mucilage has been separated into an acidic and a neutral fraction. The acidic fraction was further separated, by the use of cupric acetate solution, into two fractions, CuI and CuII. Fraction CuI contained L-rhamnose, L-galactose, and D-galacturonic acid. The methylated reduced polysaccharide gave on hydrolysis 2,3,4-tri-O-methyl-L-rhamnose, 3,4-di-O-methyl-L-rhamnose, 4-O-methyl-L-rhamnose, 2,3,4,6-tetra-O-methyl-D-galactose, 2,3,6-tri-O-methyl-D-galactose, and 2,3-di-O-methyl-D-galactose (?); L-galactose was lost during the methylation process. Periodate oxidation studies on the material indicated that the polymer was composed of a main chain of L-rhamnose units with most of the L-galactose units attached as non-reducing end groups. Fraction CuII contained L-rhamnose, L-fucose, L-galactose, and D-galacturonic acid. The methylated reduced polysaccharide gave on hydrolysis 2,3,4-tri-O-methyl-L-fucose, 2,3,4,6-tetra-O-methyl-L-galactose, 2,3,6-tri-O-methyl-D-galactose, 4-O-methyl-L-rhamnose, L-rhamnose, and possibly 2,3-di-O-methyl-D-galactose and 3-O-methyl-D-galactose. Periodate oxidation studies and a degradation by the Smith procedure indicated the presence of a L-rhamnose backbone with L-fucose and L-galactose units attached as non-reducing end groups.The neutral fraction yielded a periodate-oxidizable material after one Smith-type degradation. Periodate oxidation studies indicated that the degraded material was branched. Methylation of the degraded polysaccharide followed by hydrolysis yielded 2,3,4-tri-O-methyl-D-xylose, 2,3-di-O-methyl-D-xylose, 2,4-di-O-methyl-D-xylose, 4-O-methyl-D-xylose, D-xylose, and traces of 2,3,4-trt-O-methyl- or 2,5-di-O-methyl-L-arabinose, 2,4-di-O-methyl-D-xylose, and 3-O-methyl-D-xylose. The main backbone of the degraded polysaccharide appeared to consist of (1 → 4)-linked D-xylose units. Linkages of the (1 → 3) type were also present. The smaller fragments from the Smith-type degradation, L-arabinose, 2-O-α-L-arabinosyl glycerol, and glycerol were characterized. A partial acid hydrolysis of the neutral fraction yielded a number of oligosaccharides.

Monosaccharide sequence in Pneumococcus type XIV polysaccharide.

PREVIOUS work1 has established that in Pneumococcus type XIV polysaccharide there are two non-reducing end groups of D-galactopyranose, one D-galactopyranose residue linked 1 : 3, one D-glucose residue linked 1 : 4 and two 1 : 4 : 6-linked 2-acetamido 2-deoxy D-glucose residues for every six monosaccharide units. Although the majority of these linkages are (β-, some α-D-galactose units may be present. The two major disaccharides left after treatment of the type XIV polysaccharide with N hydrochloric acid at 100° for 2½ hr. were 4-O-β-D-glucosyl 2-acetamido 2-deoxy D-glucose and 3-O-β-(2-acetamido 2-deoxy)-D-glucosyl D-galactose. Less drastic hydrolysis has enabled other disaccharides to be isolated and the structure of these is the subject of the present communication.

Cross-reactions of antityphoid and antiparatyphoid B horse sera with various polysaccharides.

A study was made of cross-reactions of synthetic polyglucose and of numerous plant and bacterial gums in an antityphoid and an antiparatyphoid B horse serum. The observed differences permit conclusions to be drawn regarding certain of the linkages likely to be found in the fine structures of each of the corresponding Salmonella polysaccharides:- 1. Cross-reactions of the antityphoid serum with the specific polysaccharide of Type II pneumococcus and with tamarind seed polysaccharide, glycogen and synthetic polyglucose indicate that the acetic acid-degraded O-polysaccharide of S. typhi, strain O 901, may contain part, at least, of its glucose as 1,4,6-branch points or in 1,6-linkage, perhaps adjacent to a terminal, non-reducing, galactopyranose unit. 2. Cross-reactions of both antisera with arabogalactans point to the existence of (probably beta-) 1,3-, 1,6-, and/or 1,3,6-linkages of galactose in both the typhoid and paratyphoid B polysaccharides. 3. The differential reactivities of the galactomannans and yeast mannan suggest that the mannose in the typhoid polysaccharide is linked 1,2- or 1,3- with possible non-reducing mannopyranose end groups attached 1,6-. In the paratyphoid B polysaccharide the linkages are probably galacto-oligomannose 1,4-, or 1,4,6-, or the corresponding linkages of mannose alone.

Oxidation as a source of anomalies in amylose.

PEAT and his collaborators1–3 have found that amylose prepared from potato, sago, maize and tapioca starch cannot be degraded by purified β-amylase or phosphorylase to an extent greater than about 70 per cent. Retrogradation of the amylose, which might have accounted for this, has been ruled out2, leaving the implication that a proportion of the molecules of amylose have either an anomalous non-reducing end-group, or an anomalous group at some point along the chain. Peat, Thomas and Whelan4,5 find that the anomaly can be removed by treating amylose with a β-glucosidase, termed Z-enzyme, and therefore suggest that it may result from the presence of a single glucose residue attached to the main chain by a link of β-configuration.

THE CONSTITUTION OF A POLYURONIDE HEMICELLULOSE FROM WHEAT STRAW

The structure of a polyuronide hemicellulose from wheat straw containing xylose, arabinose, and hexuronic acid units has been investigated. Graded hydrolysis preferentially removed anhydro-L-arabinose units leaving a xylan to which uronic acid units were attached. Methylation and hydrolysis yielded 2,3,4-trimetliyl xylose (2.7%) indicating one nonreducing end group per 37 units; 2,3,5-trimethyl-L-arabinose, 2,3-dimethyl-D-xylose, and 2-methyl-D-xylose were found in a molar ratio 1:5:1. In addition, a methylated uronic acid complex was recovered which on extensive hydrolysis yielded an aldobiuronic acid. The latter, on reduction with sodium borohydride, yielded 2,3,4-trimethyl glucose (1 mole) and 2-methyl xylose (1 mole); the structure 2-methyl 3[2,3,4-trimethyl α-D-glucuronosido] D-xylose was therefore indicated for the methylated aldobiuronic acid.A structure for the hemicellulose is proposed which consists of approximately 32 anhydro-D-xylose units linked β 1,4- in a chain to which five anhydro-L-arabinose and three D-glucuronic acid units are attached as side groups by 1,3-glycosidic bonds. Results of periodate oxidation and estimation of reducing power support the proposed structure.

Decomposition of formic acid by periodate.

SINCE the introduction of periodic acid as a reagent for glycols1, it has been widely employed for elucidating the constitution of many organic compounds2. In recent years valuable information has been obtained in the study of polysaccharides by oxidation with periodic acid3. Under suitable conditions, one molecule of formic acid is liberated from a non-reducing end-group, and two from a reducing end-group4. Determination of the formic acid set free may give an idea of the chain-length of a poly-saccharide, while the consumption of periodate per repeating unit is useful in locating the position of hydroxyl groups therein. It is therefore necessary to know whether or not formic acid itself is attacked by the reagent under experimental conditions.