The pyridine nucleosidases from Bacillus subtilis and Neurospora crassa. Isolation and structural properties.

The DPNases from Bacillus subtilis and Neurospora crassa as well as the DPNase inhibitor found in B. subtilis were purified to homogeneity using conventional techniques. The three compounds are unusual in that they contain large amounts of carbohydrate, consisting chiefly of galactose, mannose, and aminosugars. The carbohydrate portions in the B. subtilis DPNase. the inhibitor, and the Neurospora enzyme represent 55, 71, and 76%, respectively, of the molecular weights.The B. subtilis DPNase is completely inhibited by a stoichiometric amount of the inhibitor. The enzyme-inhibitor complex is capable of binding DPN⁺, but cannot hydrolyze the dinucleotide.Some preliminary experiments are described using affinity chromatography for the purification of the B. subtilis DPNase.     

Carbohydrate composition of normal and variant human alpha 1-protease inhibitors.

Carbohydrate composition of normal human alpha 1-protease inhibitor (PiM₁) and several variant inhibitors (PiM₂, PiM₃, PiA, PiS, and PiZ) was determined by methanolysis of the samples followed by quantitative analysis of both neutral and amino sugars using gas-liquid chromatography. All normal and variant inhibitors contained nine mannose, seven galactose, ten N-acetylglucosamine, and eight N-acetylneuraminic acid residues per molecule, and no significant difference was found in their carbohydrate compositions. PiA is a variant with the fastest anodal electrophoretic mobility, and PiZ is a variant with the slowest mobility thus far reported. The differences in electrophoretic mobility of these Pi variants are entirely due to their amino acid substitutions determined previously. These amino acid substitutions have no effect on the carbohydrate structure of the protease inhibitor.     

Feeding rats a high fat/carbohydrate ratio diet reduces jejunal S/I activity ratio and unsialylated galactose on glycosylated chain of S–I complex

AimsWe examined whether decreasing jejunal sucrase/isomaltase (S/I) activity ratio by feeding rats a high fat/carbohydrate ratio diet is regulated by changing glycosylated chains on the S–I complex.Main methodsJejunal activities of sucrase, isomaltase and β-1,4-galactosyltransferase were examined in rats fed a high fat/carbohydrate or a low fat/carbohydrate ratio diet. The amount of galactose and mannose in the glycosylated chain on the S–I complex in rats fed both diets was determined using RCA₁₂₀ and Con A lectins, respectively. The effects of reducing unsialylated galactose from the glycosylated chain on the S–I complex were assessed by determining sucrase activity in purified S–I complex treated with β-galactosidase.Key findings and significanceFeeding rats a high fat/carbohydrate ratio diet reduced jejunal S/I activity ratio in mucosal homogenates and purified fractions. The level of unsialylated galactose in glycosylated chains on the S–I complex was reduced by feeding rats a high fat/carbohydrate ratio diet. The form with reduced levels of unsialylated galactose had lower sucrase activity than that with more unsialylated galactose. The reduction of galactose on the S–I complex by β-galactosidase in vitro reduced sucrase activity. Feeding rats a high fat/carbohydrate ratio diet also reduced jejunal β-1,4-galactosyltransferase activity. Taken together, decreasing the S/I activity ratio by feeding rats a high fat/carbohydrate diet is associated with the reduction of unsialylated galactose on the glycosylated chain of the S–I complex.     

Structures of the carbohydrate moiety attached to one site in the first domain of turkey ovomucoid: elucidation by 1H-n.m.r. spectroscopy

1H-N.m.r. spectroscopy was used to elucidate the primary structures of the carbohydrate moiety attached to asparagine at residue 53 in the first domain of turkey ovomucoid, a serine proteinase inhibitor. The carbohydrate moiety is a heterogeneous mixture of three structurally closely related complex-type oligosaccharides. Of the total carbohydrate moiety, 61% is tetra-antennary with terminal galactose and with an intersecting N-acetylglucosamine, and containing an additional N-acetylglucosamine (10') attached to mannose (4'). Another 23% is tri-antennary with terminal galactose and with an intersecting N-acetylglucosamine. The remaining 16% is tri-antennary with terminal galactose (6 and 8 only), with an intersecting N-acetylglucosamine.     

Utilization of Lactose, Glucose, and Galactose by a Mixed Culture of Streptococcus thermophilus and Lactobacillus bulgaricus in Milk Treated with Lactase Enzyme

The mechanism responsible for an increased rate of acid production when yogurt starter cultures are grown in milk treated with lactase enzyme was investigated by studying carbohydrate utilization and acid development by a pure culture of Streptococcus thermophilus and a mixed yogurt starter culture consisting of S. thermophilus and Lactobacillus bulgaricus. In milk containing glucose, galactose, and lactose, glucose and lactose (but not free galactose) were fermented. Fermentation of lactose in control milk was accompanied by the release of free galactose, with the result that carbohydrate utilization was less efficient than in treated milk. This phenomenon also occurred when lactose was fermented by S. thermophilus in broth culture. Carbohydrate utilization by the mixed yogurt culture was more rapid when the lactose in milk was partially prehydrolyzed. Our results suggest that the more rapid acid development that took place when a mixed yogurt starter culture was grown in milk containing prehydrolyzed lactose was the result of a more rapid and efficient utilization of carbohydrate by S. thermophilus when free glucose in addition to lactose was available for fermentation. The evidence presented also suggests that uptake and utilization of glucose and lactose by S. thermophilus are different in broth and milk cultures.     

Carbohydrate compositions of the rabbit plasminogen isozymes.

The carbohydrate compositions of the two affinity-chromatography-resolved isozymes of rabbit plasminogen and plasmin as well as the isoelectric-focusing-resolved subforms of each plasminogen isozyme have been investigated in detail. The first plasminogen isozyme as well as its subforms all possess four to five residues of N-acetylglucosamine, two residues of N-acetylgalactosamine, three residues of mannose and five residues of galactose per molecule of protein. Additionally, we previously reported three residues of sialic acid present on this protein molecule. The corresponding plasmin heavy chain for this isozyme contains essentially all of the carbohydrate, and the plasmin light chain appears devoid of carbohydrate. On the other hand, the second plasminogen isozyme as well as its subforms all possess only trace amounts of N-acetylglucosamine, two residues of N-acetylgalactosamine, less than one residue of mannose and three residues of galactose per molecule of protein. In addition, we have previously reported two residues of sialic acid for this molecule. Here, also, all carbohydrate appears on the heavy chain of the plasmin, which is prepared by activation of this particular plasminogen. Thus, the carbohydrate differences which we reported earlier in rabbit plasminogen isozymes are confirmed and extended.     

The major human urinary trypsin inhibitor is a proteoglycan

The major urinary trypsin inhibitor (Mr 44 000), isolated from human urine, contains 35% carbohydrate. In addition to N-acetylglucosamine and neutral sugars (primarily mannose and galactose), the carbohydrate moiety contains hexuronic acid and N-acetylgalactosamine and corresponds to a glycosaminoglycan. This carbohydrate chain is an integral component of the inhibitor: it does not dissociate from the inhibitor when using dissociative conditions such as sodium dodecyl sulfate, guanidinium chloride, or by increasing ionic strength or mixing with cetylpyridinium chloride. This glycosaminoglycan chain is sensitive to chondroitinase ABC or testicular hyaluronidase digestion and corresponds to slightly sulfated chondroitin 4-sulfate or 6-sulfate. After treatment by these enzymes, the urinary inhibitor has a lower molecular mass (Mr 26 000) but still inhibits trypsin.     

Subunit glycosylation of Lupinus angustifolius seed globulins

Reduced polypeptide subunits of α-, β- and γ-conglutins from Lupinus angustifolius seeds were resolved by preparative SDS gel electrophoresis of the fluorescent labelled proteins, into four, six and two major components, respectively. All subunits were glycosylated, to varying degrees, containing mannose, galactose and glucosamine. The major glycopeptides released by pronase digestion of each conglutin had similar galactose/mannose ratios; the MW of the glycopeptide released from α- and β-conglutin was ca 5000. Although on average, each molecule of α-conglutin contains one main oligosaccharide chain, and β-conglutin two, the presence of carbohydrate in all polypeptide subunits suggests that some subunits may arise by proteolytic cleavage of a larger polypeptide after glycosylation. The presence of minor glycopeptide components indicates that modification of carbohydrate chains during seed development may also occur.     

Dark and Photometabolism of Sugars by a Blue Green Alga: Tolypothrix tenuis

The carbohydrate metabolism of the autotrophically grown blue-green alga, Tolypothrix tenuis, was studied. The alga respires glucose, fructose, galactose, and ribose.     

The oligosaccharide units of rabbit immunoglobulin G. Multiple carbohydrate attachment sites

The carbohydrate structure of rabbit immunoglobulin G isolated from pooled sera was investigated. Amino sugar analysis of fragments of the molecule allowed three oligosaccharides to be located at separate sites on the H-chain. The corresponding glycopeptides were isolated. The average composition of the C1-oligosaccharide was 2 glucosamine, 1 mannose and 2 galactose residues. It appeared to be present in approx. 15% of the H-chains; the carbohydrate was coupled through the amide group of asparagine in a peptide containing asparagine, glycine and threonine within the Fd fragment of the molecule. The average composition of the C2-oligosaccharide was 1 galactosamine, 1 galactose and either 1 or 2 sialic acid residues. It was present on approx. 40% of the H-chains and was attached glycosidically to the OH group of threonine in a peptide Ser-Lys-Pro-Thr-Cys-Pro-Pro-Glu-Leu in the hinge region of the molecule. The average composition of the C3-oligosaccharide was 5 glucosamine, 2 galactose, 5 mannose, 1 fucose and 1 sialic acid residue. It appeared to be present in all the H-chains and was linked through the amide group of asparagine in a peptide Gln-Gln-Phe-Asn-Ser-Thr-Ile-Arg within the Fc fragment of the molecule.     

On the carbohydrate composition of bovine colostrum trypsin inhibitor.

The trypsin inhibitor from bovine colostrum has been separated into several forms by CM-Sephadex and DEAE-cellulose chromatography. These forms differ in the amount and composition of the carbohydrate they contain, which has been quantitated for four components by gas-liquid chromatography and standrad colorimetric procedures. The monosaccharides fucose, mannose, galactose, galactosamine, glucosamine and sialic acids have been determined. A microheterogeneity was establish ed in the carbohydrate moiety, which amounts to about 40% of the total molecular weight (Mr 11 000 - 14 000) of bovine colostrum inhibitor.     

Arabinogalactans in a purified allergen preparation from pollen of timothy (Phleum pratense)

The purified allergen preparation representing a certain fraction of an aqueous timothy pollen extractcontained ca. 20% carbohydrate, mainly as arabinose (7%) and galactose (13%). The protein content was 63%. Fractionation on DEAE-Sephadex and Sephadex G-100 gave one neutral and two acidic fractions, all containing protein, arabinose and galactose. The structure of the carbohydrate moiety was investigated by methylation analysis, periodate oxidation and enzyme incubation. The acidic fraction contained (1→6)-linked galactose residues, some being substituted on O-3 with arabinose. The neutral fraction consisted of a more extensively branched arabinogalactan with longer side chains of (1→3)- and (1→5)-linked arabinose. The arabinose was present mainly as α-l-arabinofuranosyl residues. Alkaline degradation and subsequent fractionation indicated the presence of a covalent linkage between hydroxyproline and arabinose. Periodate oxidation or incubation with α-l-arabinofuranosidase did not affect the allergenic activity of the extract.     

Studies on the structure of a phosphoglycoprotein from the parasitic protozoan Trypanosoma cruzi.

A glycoprotein (GP72) has been isolated from Trypanosoma cruzi and found to contain 41% protein, 49% carbohydrate and 10% phosphate. All phosphate was covalently attached to the carbohydrate which contained the following sugars: ribose, xylose, fucose, galactose, mannose, glucose and glucosamine. The carbohydrate side chains were linked to protein by fucose, xylose and N-acetylglucosamine; 50% of the total N-acetylglucosamine was involved in glycoprotein linkages. Two classes of carbohydrate side chains were detected. One class comprised 15% of the total carbohydrate and contained glucosamine, mannose and galactose; some of these chains were phosphorylated. The other class comprised 85% of the total carbohydrate and contained xylose, ribose, fucose, galactose, mannose, glucosamine and phosphate; these chains were antigenic and reacted with a monoclonal antibody with specificity for the whole glycoprotein.     

Sargassan: A sulphated heteropolysaccharide from Sargassum linifolium

A new sulphated heteropolysaccharide containing glucuronic acid, mannose, galactose, xylose, fucose and a protein moiety has been extracted from Sargassum linifolium. The polysaccharide extracted with HCl was richer in its carbohydrate and protein contents and contained lower amounts of ash than that extracted with oxalic acid.     

The isolation and partial characterization of a glycoprotein isolated from human gastric aspirates and from extracts of gastric mucosae

The isolation and partial characterization of a glycoprotein isolated from individual gastric aspirates and extracts of gastric mucosae solubilized with N-acetylcysteine is described.The isolated glycoproteins and the glycoproteins from proteolysed gastric aspirates showed virtually the same carbohydrate and amino acid composition. The results indicate that they consist of a protein core to which are attached carbohydrate side-chains composed of four sugars: N-acetylgalactosamine N-acetylglucosamine, galactose, fucose showing a ratio of 1 : 3 : 4 : 2. Superimposed on this basic structure were additional sugar residues, the blood-group determinants. The results also suggest that the carbohydrate side-chains are linked by an alkali-labile O-glycosidic linkage to the threonine and serine residues of the protein core, N-acetylgalactosamine forming the link.     

Glycoproteins of the opium poppy

Two carbohydrate-protein fractions were isolated from the water-soluble biopolymer from opium poppy capsules by chromatography on SP-Sephadex. The carbohydrate chains are composed of arabinose, rhamnose, xylose, mannose, glucose, galactose, galacturonic acid, glucuronic acid and 4-O-methyl glucuronic acid. Methylation analysis indicated a high degree of branching suggesting a very complex structure. Treatment of the glycoprotein with NaOH in the presence of NaBH₄ resulted in a significant decrease in the serine and threonine content. The carbohydrate side chains released contained the sugar alcohol, galactitol. These results indicate that polysaccharide chains are linked to protein via serine-O-galactoside linkages.     

Haemagglutinin activity in the haemolymph of individual Acrididae (grasshopper) specimens

Twenty-four individual grasshopper specimens representing four Melanoplus spp. contained similar broad-spectrum haemolymphatic haemagglutinin. The agglutinin activity showed highest titre toward human ABO and rabbit cells among nine types of erythrocytes tested. Titre values differed between individual insects but agglutination specificity toward different erythrocytes was similar. Agglutination of type-O red cells by individual grasshopper haemolymph was inhibited by 34 of 41 tested carbohydrates, carbohydrate derivatives, alcohols and chelating agents. Individual insects showed similar patterns of haemagglutination inhibition. Non-inhibitory compounds were mannose and mannose derivatives (excepting N-acetylneuraminate), several glucose derivatives, amino sugars and ethanol. The observations indicated that haemolymph from an individual grasshopper contained complex heteroagglutinin activity similar to that found in haemolymph pooled from several insects. Determination of minimal effective inhibitor concentrations confirmed the presence of heteroagglutinin activity primarily directed toward galactose and glucose and related α-linked glycosidic derivatives.     

Carbohydrate composition of cells and plasma membranes of Distyostelium discoideum at selected stages of development

Cells of Distyostelium discoideum representing four developmental stages were atuo-analysed for constituent monosaccharides and their compositions compared. Rhamnose, ribose, fucose, glucose, mannose, galactose, glucosamine, galactosamine and an unidentified sugar were recovered after hydrolysis in 2 M HCl for 2 h at 100°C. The relative proportions of the individual sugars were found to vary as a function of development. The largest variations were in the proportions contributed by galactose (from 2% of vegetative cell carbohydrate to 12% of the carbohydrate of fruiting bodies) and galactosamine (present in measurable quantity only in fruiting bodies).Plasma membrane “ghosts” were found to have the same monosccharide constituents as whole cells, but in different proportions. Mannose contributed over 24% of the total carbohydrate recovered from aggregating cell “ghosts”, but only 13% of carbohydrate recovered from “ghosts” prepared from vegetative cells. Galactose was the most abundant sugar recovered from vegetative “ghosts”, and was second only to mannose in aggregating “ghosts”.     

Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte Isolement et caracterisation de glycopeptides sulfatesSulfated glycoproteins form egg shell membranes and hen oviduct. Isolation and characterization of sulfated glycopeptides

Sulfated glycopeptides were isolated from pronaisc and tryptic digests of egg shell membranes and hen oviduct. They were precipitated by cationic detergents and separated by preparative electrophoresis, after removal of small quantities of glucuronoglycosaminoglycans detected only in the oviduct (isthmus and magnum). The principal isolated sulfated glycopeptides were divided according to increasing electrophoretic mobilities into two groups A and B. The homogeneity of the purified glycopeptides was confirmed by gel filtration and polyacrylamide gel electrophoresis.Glycopeptides from pool preparation of tissue are analysed and carbohydrate and amino acids average values are estimated. Hexosamines (mainly N-acetylglucosamine), hexoses (galactose, glucose, mannose) and fucose were found in Glycopeptides A. The molar ratio of hexose/hexosamine was 0.4. N-Acetylneuraminic acid and sulfate were also present in Glycopeptides A. The molar ratio of sulfate/hexosamine ranged from 0.1 to 0.25. The Glycopeptides A composition indicated the presence of chains with many glycosyl groups and a few of amino acids residues. The carbohydrate components of Glycopeptides B from egg shell membranes and magnum were found to be hexosamines (N-acetylgalactosamine and N-acetylglucosamine in equimolar proportions), hexoses (galactose mainly and glucose), N-acetylneuraminic acid, and fucose. The molar ratio of hexose/hexosamine was 1. Sulfate was also present and the molar ratio of N-acetylneuraminic acid and sulfate to hexosamine was ranged from 0.8 to 1. The main amino acid residues in these glycopeptides were serine and threonine with destruction of these hydroxyamino acids during alkali treatment. Glycopeptides B probably consist of short carbohydrate chains, linked to the polypeptide through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Approximately 40% of the amino acid residues were linked to carbohydrate chains.Glycopeptides B from egg shell membranes magnum and egg white were very similar in their carbohydrate and amino acid composition and in their properties.Gylcopeptides A from egg shell membranes, isthmus and magnum showed similarities and divergences especially in the amino acid composition. These results suggest that magnum and isthmus in oviduct are both concerned with the synthesis of egg shell membrane glycoproteins.     

Immunochemistry of the Cell Walls of Listeria monocytogenes

The antigenic specificity of Listeria monocytogenes types I, II, III, IVa, and IVb was studied by immunochemical techniques. Immunologically active carbohydrates of the various types were extracted from cell walls and were chemically analyzed. Types I and II contained predominantly glucosamine and rhamnose; type III, galactose, rhamnose, and glucosamine; and types IVa and IVb, glucose and galactose. Quantitative precipitin inhibition tests with purified monosaccharides indicated that the major antigenic determinant of types I and II is rhamnose. Precipitin reactions could not be detected with type III carbohydrate and homologous or heterologous antisera. The major determinants of types IVa and IVb were found to be galactose and glucose, respectively. As much as 87% inhibition of the quantitative precipitin test for types I and II was obtained with rhamnose, 72% for type IVa with galactose, and 72% for type IVb with glucose. The immunochemical basis for the antigenic specificity of L. monocytogenes types I, II, IVa, and IVb was further confirmed by using agar gel diffusion. Cross-reactions among the various type-specific carbohydrates and heterologous antisera were also studied. Type II carbohydrate was found to contain galactose and react with type IVa antisera. This reaction could be blocked by galactose. Type I carbohydrate did not contain galactose nor did it react with antiserum prepared from type IVa cells. Therefore, the somatic antigens of type I and type II L. monocytogenes, previously thought to be identical, appeared to differ. The dominant immuno-specific group in the cross-reaction between type IVb carbohydrate and type IVa antisera was found to be galactose. Type IVa absorbed antisera did not produce a significant cross-reaction with type IVb carbohydrate. The results obtained from this investigation indicate a lesser degree of antigenic relationship between type IVa and type IVb L. monocytogenes than was previously believed to exist.