Binding-site Analysis of the Ether Linkages between Lignin and Hemicelluloses in Lignin–Carbohydrate Complexes by DDQ-Oxidation

Lignin-carbohydrate complexes (LCC; nor-C-1-M, com-C-1-A) isolated from normal and compression woods of Pinus densiflora were hydrolyzed with two types of cellulase preparations, and the hydrolyzates formed were fractionated by adsorption chromatography on polyvinyl gel into water-soluble materials and LCC fragments. To elucidate the binding sites between the lignin and carbohydrate, the cellulase-degraded LCC fragments were subjected to acetylation, and then oxidation with 2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ), which was confirmed to oxidatively cleave the benzyl ether linkages between the lignin and carbohydrate. The DDQ-oxidized fraction was then methylated by the method of Prehm, hydrolyzed, reduced and acetylated. A GC-MS analysis of the methylated sugar revealed that alditol acetates from 6-O-methyl mannose, 6-O-methyl galactose, 6-O-methyl glucose and a small amount of their 2-O- or 3-O-methyl isomers existed in both methylated fractions. 2-O-Methyl xylose and 3-O-methyl xylose were also identified in the fraction from the acidic LCC (com-C-1-A). These results led to the conclusion that acetylglucomannan and β-1,4-galactan were preferably bound to the lignin at C-6 position of the hexoses, and that arabinoglucuronoxylan did likewise at the C-2 and C-3 positions of xylose units.     

Lectin-binding studies of a human skin mastocytoma

Summary A solitary mastocytoma of the skin was investigated to assess the lectin-binding pattern of human mast cells. Of 18 Fluorescein-labelled lectins tested, nine reacted with mast cell granules. While lectins recognizingN-acetylgalactosamine or fucose residues did not stain mast cells, lectins with binding sites forN-acetylglucosamine, -methyl mannopyranoside, galactose, complex carbohydrates ofN-acetyl-lactosamine type and sialic acid gave a positive reaction.     

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (M_r 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.Abbreviations A₂pm diaminopimelic acid - ATCC American Type Culture Collection - CE cell envelope - CM cytoplasmic membrane - CW cell wall - dOcla 3-deoxy-d-manno-2-octulosonic acid - GalN galactosamine - GlcN glucosamine - GlcUA glucuronic acid - HF hydrofluoric acid - LPS lipopolysaccharide - ManN mannosamine - M relative molecular mass - MurN muramic acid - MurN-6-P muramic acid-6-phosphate - OMe O-methyl - PAGE polyacrylamide gel electrophoresis - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - SH sheath     

Studies on the metabolic determinants of D-galactose-induced neurotoxicity in the chick

The contents of galactose, galactitol, galactose 1-phosphate, UDP-galactose and UDP-glucose in the brains of chicks fed a diet containing 40 % (w/w) D-galactose were determined at regular intervals during a 48 h period which terminated in convulsive activity and death of the animals. Although levels of galactose and galactitol were markedly elevated, UDP-galactose and UDP-glucose levels were not significantly increased. The level of galactose 1-phosphate rose to 1-3 μg/g of fresh tissue by 14 h but gradually diminished until, at 48 h, the content was 0-25 μg/g. The metabolic turnover of these compounds, as shown by labelling experiments with inorganic [³²P]phosphate and [U-¹⁴C]galactose, indicated that galactose 1-phosphate and UDP-galactose were rapidly metabolized, yet relatively little galactose was utilized by the brain as a source of energy. These observations have prompted us to propose a mechanism for the turnover of galactose 1-phosphate that involves cyclical phosphorylation and dephosphorylation reactions in the brains of galactose-fed chicks. In support of this hypothesis, we have identified phosphatase activity which has a relatively low K_m value for galactose 1-phosphate (0-06-0-07 mM) in virtually all subcellular fractions of homogenates of chick brain. Maximum activity of the phosphatase is several-fold greater than that recorded for galactokinase (EC 2.7.1.6) and galactose 1-phosphate uridyltransferase (EC 2.7.710) from chicken brain.     

Studies on the transport of glucose from disaccharides by hamster small intestine in vitro. I. Evidence for a disaccharidase-related transport system

In the presence of saturating concentrations of free d-glucose, total glucose uptake was enhanced beyond the theoretical V for free glucose uptake when disaccharides were incubated with intestinal rings. This phenomenon was not seen when glucose 1-phosphate was the substrate.Analogs of d-glucose transport system, galactose and β-methyl glucoside, had an inhibitory effect on glucose uptake from sucrose and their uptake was in turn inhibited by the disaccharide. This inhibition was non-competitive.The effect of phlorizin on glucose uptake from sucrose was 2-fold, competitive at low concentrations and non-competitive at high concentrations.Sucrose did not induce counterflow of the preloaded β-methyl glucoside.These observations indicate that with a disaccharide as the substrate, there is a component of glucose transport which is in addition to the monoscaccharide-transport system and that this could arise as a consequence of the association of disaccharidases with the brush border membrane.     

Comparison of Extracellular Polysaccharide Composition, Rhizobitoxine Production, and Hydrogenase Phenotype among Various Strains of Bradyrhizobium japonicum

A survey of 51 strains of Bradyrhizobium japonicum was performedwith respect to the composition of extracellular polysaccharide(EPS), the production of rhizobitoxine and the hydrogenase phenotype.A good correlation was found among these three different characteristics.Thirty-six strains producing an EPS composed of glucose, mannose,galactose, 4-O-methyl galactose and galacturonic acid did notsynthesize rhizobitoxine, whereas 14 strains producing an EPScomposed of rhamnose and 4-O-methyl glucuronic acid were allfound to synthesize rhizobitoxine. Hydrogen-uptake positive(Hup⁺) strains were confined exclusively to the former groupof strains which produced an EPS composed of glucose, mannose,galactose, 4-O-methyl galactose and galacturonic acid. Theseresults suggest that the phenotype with respect to rhizobitoxineproduction and hydrogen uptake is involved in the phylogenyof Bradyrhizobium japonicum as well as in the productivity ofnodulated soybeans. (Received March 24, 1989; Accepted June 16, 1989)     

DNA-dependent in vitro synthesis of enzymes of the galactose operon of Escherichia coli

Summary Two active enzymes of the galactose operon of Escherichia coli, uridyl transferase and galactokinase have been synthesized with high yields in a DNA dependent system for protein synthesis. The unspecific blank values amount to less than two percent of the rate obtained under optimal conditions and permit the accurate determination of even a small fraction of the maximum synthesis rate. Therefore this system provides a sensitive assay for the biological activity of DNA that contains the intact galactose operon of Escherichia coli.The synthesis of these galactose enzymes is to a high extent dependent on the presence of cyclic adenosine-3:5-monophosphate.D-fucose, known as an inducer of the galactose operon in vivo, stimulates the synthesis of galactokinase, indicating that the repressor of the galactose operon in active under these conditions. This stimulation is not observed, if the bacterial extract is prepared from a strain defective for the galactose repressor or if the DNA carries an operator constitutive mutation in the galactose operon. Therefore the stimulation by D-fucose is true derepression.     

Regulation of methyl beta-galactoside permease activity in pts and crr mutants of Salmonella typhimurium

Summary We have studied the regulation of the synthesis and activity of a major galactose transport system, that of methyl -galactoside (MglP), in mutants of Salmonella typhimurium. Two classes of mutation that result in a (partially) defective phosphoenolpyruvate: sugar phosphotransferase system (PTS) interfere with MglP synthesis. pts mutations, which eliminate the general proteins of the PTS Enzyme I and/or HPr and crr mutations, which result in a defective glucose-specific factor III^Gle of the PTS, lead to a low MglP activity, as measured by methyl -galactoside transport. In both ptsH,I, and crr mutants the amount of galactose binding protein, one of the components of MglP, is only 5%–20% of that in wild-type cells, as measured with a specific antibody. We conclude that synthesis of MglP is inhibited in pts and crr mutants. Once the transport system is synthesized, its transport activity is not sensitive to PTS sugars (i.e., no inducer exclusion occurs). The defect in pts and crr mutants with respect to MglP synthesis can be relieved in two ways: by externally added cyclic adenosine 3, 5-monophosphate (cAMP) or by a mutation in the cAMP binding protein. The conclusion that MglP synthesis is dependent on cAMP is supported by the finding that its synthesis is also defective in mutants that lack adenylate cyclase. pts and crr mutations do not affect growth of S. typhimurium on galactose, however, since the synthesis and activity of the other major galactose transport system, the galactose permease (GalP), is not sensitive to these mutations. If the galactose permease is eliminated by mutation, growth of pts and crr mutants on low concentrations of galactose becomes very slow due to inhibited MglP synthesis. Residual growth observed at high galactose concentrations is the result of yet another transport system with low affinity for galactose.     

UPTAKE OF ORGANIC,COMPOUNDS BY TWO FACULTATIVELY HETEROTROPHIC MARINE CENTRIC DIATOMS1,2

Glucose and galactose uptake ability in Cyclotella cryptica (clone WT-1-8) and glucose uptake ability in Coscinodiscus sp. develop rapidly in the dark. Induction of sugar uptake ability in the dark does not require the presence of sugar in the medium. The sugars are taken up by carrier-mediated systems. In C. cryptica glucose and galactose are probably taken up by the same system. The capacity of glucose uptake in this recently isolated clone of C. cryptica is nearly 5 times that of a previously studied clone (0-3A). Other organic compounds, which by themselves do not support heterotrophic growth, can be taken up and respired by both diatoms at considerable rates compared, to glucose and galactose. Therefore, in nature, these diatoms may be able to utilize a variety of dissolved organic compounds as sources of intermediary metabolites and as respiratory substrates.     

Energetics of galactose,proline, and glutamine transport in a cytochrome-deficient mutant of salmonella typhimurium

The effect of inhibitors and uncouplers on the osmotic shock-sensitive transport systems for glutamine and galactose (by the β-methyl galactoside permease) was compared to their effect on the osmotic shock-resistant proline and galactose permease systems in cytochrome-deficient cells of Salmonella typhimurium SASY28. Both osmotic shock-sensitive and -resistant systems were sensitive to uncouplers and to inhibitors of the membrane-bound Ca²⁺, Mg²⁺-activated adenosine triphosphatase. This suggests that uptake by both types of systems is energized in these cells by an electrochemical gradient of protons formed by ATP hydrolysis through the ATPase.     

Studies on 1-methyl adenine transfer RNA methyltransferase of Mycobacterium smegmatis

The presence of 1-methyl adenine in transfer RNA is a feature that Mycobacterium smegmatis shares with only a few other prokaryotes. The enzyme 1-methyl adenine tRNA methyl transferase from this source has been purified and the preliminary results show the presence of two activity peaks with different substrate specificity.List of abbreviations tRNA Transfer Ribonucleic acid - EDC 1-ethyl-3(3-dimethyl)amino Propyl Carbodiimide - PCA Perchloric acid - SAH S-adenosyl-homocystine - SIBA 5-deoxy-5-S-isobutylthioadenosine - SAM S-adenosyl methionine     

Utilization of a wood-based syrup as a fermentation carbon source

Summary An industrial by-product stream consisting primarily of - and -methyl xylosides supported microbial growth provided the treated syrup was diluted such that the osmotic pressure was within the range tolerated by the particular microorganism used. A culture screening study identified two organisms that efficiently utilized both - and -methyl xyloside. Xylose, -, and -methyl xylosides were all utilized simultaneously during batch growth ofArthrobacter sp. in a controlled-pH fermentor, although xylose was consumed faster than either xyloside. In addition,Arthrobacter sp. produced glucose isomerase when grown on the syrup.     

Subcellular characterization of glycoproteins in the principal cells of human gallbladder

Gallbladder mucus is mainly composed of glycoproteins, which seem to play a critical role in cholesterol nucleation during gallstone formation. The biosynthetic pathway and sequential processing as well as the characterization of the oligosaccharide sidechains of human gallbladder secretory glycoproteins have not been completely defined. The aim of the present study is the subcellular characterization of the glycoproteins in the principal cells of human gallbladder. Principal cells of normal human gallbladder were studied by means of a variety of cytochemical techniques, including lectin histochemistry, enzyme and chemical treatments, immunocytochemistry and lectin-gold technology. Fucose, galactose, N-acetylglucosamine, N-acetylgalactosamine and N-acetylneuraminic acid residues were detected in mucous granules, Golgi apparatus and apical membrane of principal cells. Mannose residues were only observed in dense bodies. Oligosaccharide side-chains of the glycoproteins contained in the biliary mucus are synthesized in the Golgi apparatus of the principal cells of the gallbladder epithelium and are also contained in the mucous granules of these cells. Terminal N-acetylneuraminic acid(2-3)galactose(1-3)N-acetylgalactosamine, N-acetylneuraminic acid(2-3)galactose(1-4)N-acetylglucosamine and galactose(1-4)N-acetylglucosamine sequences are contained in the oligosaccharide chains of gallbladder mucus glycoproteins. The dense bodies detected in the cytoplasm of the principal cells contained N-linked glycoproteins. Mucin-type O-linked glycoproteins were the main components of the mucous granules although some N-linked chains were also detected.     

Regulation of sugar transport in Neurospora crassa

Sugar uptake systems in Neurospora crassa are catabolically repressed by glucose. Synthesis of a low K(m) glucose uptake system (system II) in Neurospora is derepressed during starvation for an externally supplied source of carbon and energy. Fasting also results in the derepression of uptake systems for fructose, galactose, and lactose. In contrast to the repression observed when cells were grown on glucose, sucrose, or fructose, system II was not repressed by growth on tryptone and casein hydrolysate. System II was inactivated in the presence of 0.1 m glucose and glucose plus cycloheximide but not by cycloheximide alone. Inactivation followed first-order kinetics with a half-time of 40 min. The addition of glycerol to the uptake medium had no significant effect on the kinetics of 3-0-methyl glucose uptake, suggesting that the system was not feedback inhibitable by catabolites of glycerol metabolism.     

Replication properties ofARS1 plasmids inSaccharomyces cerevisiae: dependence on the carbon source

The replication behaviour of a number ofARS1-based plasmids was investigated on propagation inSaccharomyces cerevisiae grown with either glucose or galactose as carbon source. Growth on galactose results in reduced plasmid stability, as well as in reduced replication efficiency, when the entire 1.5-kbTRP1-ARS1 fragment is present on a plasmid. The galactose sensitivity is mediated by a 0.13-kb fragment harbouring part of theGAL3 promoter. This fragment exerts its effect when situated either 5 or 3 to the ARS core consensus at distances up to 0.9 kb. The endogenous 2 µm plasmid remained unaffected by the choice of carbon source.     

Synthesis of polyprenol-monophosphate-β-galactose by Acetobacter xylinum

Summary A particulate enzyme preparation fromAcetobacter xylinum synthesizes ficaprenol-monophosphate--galactose from ficaprenol monophosphate (FMP) and UDP-galactose in the presence of detergent. The product has the same properties as those previously reported for the compound formed with the endogenous acceptor. Dolichol-monophosphate (DolMP) is also a good galactose acceptor but the product obtained has different properties. Lipid extracts fromAcetobacter contain galactose acceptor capacity which is lost by mild acid treatment. FMP behaves in a similar manner but DolMP is resistant to this treatment. It is concluded that the endogeneous acceptor is an allylic phosphate ester of a polyprenol.Abbreviations FMP ficaprenol monophosphate - DolMP dolichol monophosphate Dedicated to ProfessorLuis F. Leloir on the occasion of his 70^th birthday.     

13C-nmr spectroscopy of red algal galactans

¹³C-nmr spectra of red seaweed galactans, belonging to the agar and carrageenan groups or having the “intermediate” type of structure, were interpreted on the basis of ¹³C-nmr spectra of model compounds. Signal assignments have been made for most of the known extreme structures of such galactans. ¹³C-nmr spectroscopy was shown to be a rapid and convenient method of structural analysis, which permits one to determine the type of galactan structure, the absolute configurations of its constituents (galactose and 3,6-anhydrogalactose), and the positions of the sulfate and O-methyl groups in a polysaccharide molecule.     

Specificity of the Glucose Transport System in Leishmania tropica Promastigotes*

SYNOPSIS. The glucose transport system in Leishmania tropica promastigotes was characterized by the use of labeled 2-deoxy-D-glucose (2-DOG), a nonmetabolizable glucose analog. The uptake system has a Q₁₀ of 2 and a heat of activation of 10.2 kcal/mole. The glucose transport system is subject to competitive inhibition by 2-DOG, glucosamine, N-acetyl glucosamine, mannose, galactose, and fructose which suggests that substitutions in the hexose chain at carbons 2 and 4 do not affect carrier specificity. In contrast, changes at carbon 1 (α-methyl-D-glucoside, 1,5-anhydroglucitol) and carbon 3 (3–0-methyl glucose) lead to loss of carrier affinity since these sugars do not compete for the glucose carrier. Sugars that compete with the glucose carrier have one common feature—they all exist in the pyranose form in solution. The carrier for D-glucose does not interact with L-glucose or any of the pentose sugars tested. Uptake of 2-DOG is inhibited by glycerol. This inhibition, however, is noncompetitive; it is evident, therefore, that glucose and glycerol do not compete for the same carrier. Glycerol does not repress the glucose carrier since cells grown in presence of glycerol transport the sugar normally.     

Chemical structure analysis of water-soluble sulfated polysaccharide fromSchizymenia dubyi (Rhodophyta,Gigartinales)

Chemical and spectroscopic methods showed that the water-soluble polysaccharide extracted fromSchizymenia dubyi from Sicily was composed of 1/0.75/1.3 galactose, glucuronic acids and sulfate groups; 45% of total galactose was present as the L-form and no 3,6-anhydrogalactose was detected. The structural characteristics of this galactan of molecular weight 290 000 were close to sulfated polysaccharide with 1,3-, 1,4- and terminal-linked galactose units and secondary ramifications in 1,3,6; 1,4,6; 1,3,4 and 1,6. Permethylation analysis suggested the presence of sulfate groups on positions O-2 and/or O-3 of 1,4-linked galactose and on O-2 and/or O-4 of 1,3-linked residues.Author for correspondence     

Induction and genetics of two alpha-galactosidase activities in Saccharomyces cerevisiae

Summary The induction of -galactosidase of Saccharomyces cerevisiae was investigated. We have demonstrated the existence of inducible internal and external -galactosidase activities and have studied the relationship between the two -galactosidases by examining a mutant strain which lacks both the internal and external activities. The mutant possesses a mutation in a single locus (mel 1-1) which does not affect the synthesis of the other galactose pathway enzymes or the ability of the yeast to grow on media containing only galactose as the carbon source. Genetic studies of the mutant indicate that mel 1-1 is recessive and allelic to the wild type allele for melibiose fermentation Mel 1.This investigation was supported in part by Grant No. AM15325 from the National Institutes of Arthritis, Metabolism and Digestive Diseases, and a grant from the Office of Research Administration, University of Hawaii. R.G. Buckholz was a National Science Foundation Pre-Doctoral Fellow during part of this study