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     

Antibacterial activity of a sulfated galactan extracted from the marine alga <Emphasis Type="Italic">Chaetomorpha aerea</Emphasis> against <Emphasis Type="Italic">Staphylococcus aureus</Emphasis>

The in vitro antimicrobial activity of the marine green algae Chaetomorpha aerea was investigated against gram-positive bacteria, gram-negative bacteria, and a fungus. The water-soluble extract of algae was composed of a sulfated (6.3%) galactan with a molecular weight of 1.160 × 10⁶ Da and a global composition close to commercial polysaccharides, such as dextran sulfate or fucoidan. The polysaccharide was composed of 18% arabinose, 24% glucose, and 58% galactose. The re-suspended extracts (methanol, water) exhibited selective antibacterial activities against 3 gram-positive bacteria including Staphylococcus aureus (ATCC 25923). Minimum inhibitory concentration and minimum bactericidal concentration tests showed that the sulfated galactan could be a bactericidal agent for this strain (40 mg/mL). The results of this study confirmed the potential use of the green algae Chaetomorpha aerea as a source of antibacterial compounds.     

Antiangiogenic properties of a sulfated galactan isolated from a marine green alga, Codium cylindricum

An anticoagulant sulfated galactan isolated from the marine green alga, Codium cylindricum, was shown to have antiangiogeinic activity. This galactan suppressed microvessel formation in an ex vivo serum-free matrix culture model using rat aortic ring. It also inhibited human umbilical vein endothelial cells (HUVEC) tube formation on reconstituted basement membrane gel. These results show the value of algal sulfated galactans in the design of antiangiogenic agents.     

Antioxidant, cytotoxic and hemolytic effects of sulfated galactans from edible red alga Hypnea musciformis

Polysaccharides, galactans, obtained from edible red seaweed Hypnea musciformis were characterized by molecular weight and infrared spectroscopy analysis and were evaluated for antioxidant activity in vitro and for their effects on cell viability. The main components were galactose and sulfate presenting low protein contamination. These sulfated galactans (F1.0) showed a polydisperse profile, and signs in infrared analysis were attributed to a sulfate ester S?=?O bond, the presence of a 3,6-anhydrogalactose C–O bond, nonsulfated β-d-galactose, and a C–O–SO₄ bond in galactose C4. The NMR analysis showed signals at about 95 and 92 attributed to anomeric carbon of 4-linked 3,6-anhydro-α-d-galactopyranose residue of κ-carrageenans and 4-linked 3,6-anhydro-α-d-galactopyranose2-sulfate of ι-carrageenans. Sulfated galactan F1.0 showed strong antioxidant activity under lipid peroxidation assay where F1.0 at 8 mg mL^?1 promoted 57.92% peroxidation inhibition and displayed the scavenging activity on hydroxyl radicals in a dose-dependent manner leading to 32.5% scavenging of these radicals when 5 mg mL^?1 of sulfated galactan F1.0 was used. The sulfated galactan fraction also exhibited strong inhibition on the H₂O₂-induced hemolysis model. Sulfated galactan F1.0 displayed low cytotoxic action in 3 T3 cells and moderate antitumoral action in HeLa cells. These results suggest that sulfated galactan F1.0 from H. musciformis has antioxidant potential, which is a great effect for a compound used as food and in the food industry.     

Polar Constituents of the Tunicate Botryllus schlosseri

Eighteen compounds were identified by GC–MS of their trimethylsilyl derivatives in n-butanolic extract from the biomass of Botryllus schlosseri. Three of them, 5-oxoproline, 5-hydroxyhydantoin, and kinurenic acid, were found in marine invertebrates for the first time. In addition to cellulose, the biomass was also shown to contain complex water-soluble sulfated polysaccharides. These were extracted and fractionated, and sulfate content and monosaccharide composition were determined in the fractions; fucose, xylose, galactose, mannose, glucose, glucosamine, galactosamine, and uronic acids were found. Unlike several other tunicate species, Botryllus schlosseri does not seem to contain any simple galactan sulfate.     

The galactan sulfate from the edible,red alga Porphyra columbina

A galactan sulfate has been isolated from the seaweed Porphyra columbina, and its structure established by a combination of methylation, methanolysis, treatment with alkali followed by methylation, and ¹³C-n.m.r. spectroscopy. The polysaccharide belongs to the porphyran class, and consists of 3-linked β-d-galactosyl residues and 4-linked α-l-galactosyl residues. 3,6-Anhydro-l-galactose and l-galactose 6-sulfate residues total approximately half of the sugar units, the other half being made up of d-galactose and 6-O-methyl-d-galactose residues. Some evidence is presented that suggests that the galactan sulfate does not have a completely alternating structure.     

Assimilatory sulfur metabolism in marine microorganisms: Sulfur metabolism,growth, and protein synthesis of Pseudomonas halodurans and Alteromonas luteo-violaceus during sulfate limitation

Sulfate concentration in the growth medium exerted a strong influence on the sulfur content of protein in two marine bacteria, Pseudomonas halodurans and Alteromonasluteo-violaceus, but the distribution of sulfur in major biochemical fractions was not affected. 90% of the total cellular sulfur was contained in low molecular weight organic compounds and protein; inorganic sulfate was not an important component. The sulfur content of isolated protein and total cellular sulfur increased in proportion to the external sulfate concentration for both bacteria, reaching a maximum at about 100–250 M. The growth rate of P. halodurans only was dependent on the sulfate concentration.Sulfur starvation of cells labeled to equilibrium with ³⁵S-sulfate resulted in a rapid decrease in low molecular weight organic S with a concommitant increase in alcohol soluble (P. halodurans) or residue protein (A. luteo-violaceus). Although cell division was prevented, total protein increased in both bacteria, resulting in synthesis of sulfur-deficient protein. This effect was most pronounced in P. halodurans.Addition of ³⁵S-sulfate to sulfur-starved A. luteo-violaceus further demonstrated that sulfur metabolism was restricted primarily to the synthesis and utilization of sulfurcontaining protein precursors. The low molecular weight organic S pool was replenished rapidly, and the pool size per cell reached twice the normal value before cell division resumed. Incorporation into protein was very rapid.Abbreviations L.M.W. low molecular weight - TCA trichloroacetic acid     

Expression, purification and characterisation of soluble GlfT and the identification of a novel galactofuranosyltransferase Rv3782 involved in priming GlfT-mediated galactan polymerisation in Mycobacterium tuberculosis

The arabinogalactan (AG) component of the mycobacterial cell wall is an essential branched polysaccharide which tethers mycolic acids (m) to peptidoglycan (P), forming the mAGP complex. Much interest has been focused on the biosynthetic machinery involved in the production of this highly impermeable shield, which is the target for numerous anti-tuberculosis agents. The galactan domain of AG is synthesised via a bifunctional galactofuranosyltransferase (GlfT), which utilises UDP-Galf as its high-energy substrate. However, it has proven difficult to study the protein in its recombinant form due to difficulties in recovering pure soluble protein using standard expression systems. Herein, we describe the effects of glfT co-induction with a range of chaperone proteins, which resulted in an appreciable yield of soluble protein at 5 mg/L after a one-step purification procedure. We have shown that this purified enzyme transfers [¹⁴C]Galf to a range of both β(1 → 5) and β(1 → 6) linked digalactofuranosyl neoglycolipid acceptors with a distinct preference for the latter. Ligand binding studies using intrinsic tryptophan fluorescence have provided supporting evidence for the apparent preference of this enzyme to bind the β(1 → 6) disaccharide acceptor. However, we could not detect binding or galactofuranosyltransferase activity with an n-octyl β-d-Gal-(1 → 4)-α-l-Rha acceptor, which mimics the reducing terminus of galactan in the mycobacterial cell wall. Conversely, after an extensive bioinformatics analysis of the H37Rv genome, further cloning, expression and functional analysis of the Rv3792 open reading frame indicates that this protein affords galactofuranosyltransferase activity against such an acceptor and paves the way for a better understanding of galactan biosynthesis in Mycobacterium tuberculosis.     

Species specific release of sulfate from adenylyl sulfate by ATP sulfurylase or ADP sulfurylase in the green sulfur bacteria Chlorobium limicola and Chlorobium vibrioforme

High activities of ATP sulfurylase were found in the soluble protein fraction of two Chlorobium limicola strains, whereas ADP sulfurylase was absent. ATP sulfurylase was partially purified and characterized. It was a stable soluble enzyme with a molecular weight of 230,000, buffer-dependent pH optima at 8.6 and 7.2 and an isoelectric point at pH 4.8. No physiological inhibitor was found. Inhibition was observed with p-CMB and heavy metals. Sulfur compounds had no effect on enzyme activity. The stoichiometry of the reaction was proven. In contrast, an ADP sulfurylase, but no ATP sulfurylase, was found in Chlorobium vibrioforme. This enzyme was very labile with a molecular weight of about 120,000 and buffer-dependent pH optima at 9.0 and 8.5. Under test conditions the apparent K _m value was determined to be 0.28 mM for adenylyl sulfate and 8.0 mM for phosphate.Abbreviations APS adenylyl sulfate - p-CMB parachloromercuribenzoate - PP_i inorganic pyrophosphate     

Biosynthesis of the storage polysaccharide from the snail Biomphalaria glabrata,identification and specificity of a branching β1→6 galactosyltransferase

Adult snails synthesize in their albumen glands a storage polysaccharide called galactan which is utilized by the developing embryos. With [6-³H]-uridine 5diphosphogalactose the incorporation of labelled d-galactose into the polysaccharide can be traeed in freshly removed glands maintained in a bathing buffer. After centrifugation of homogenized glands, galactosyltrasferase activity is only found in the insoluble fraction. Chaps extracts of this material retain almost all of their activity and can be used for comparison of the incorporation rates into different native galactans or in various oligosaccharides. A highly efficient -(16) galactosyltransferase was detected when methyl 3-O-(-d-galactopyranosyl)--d-galactopyranoside was offered as acceptor. The substitution at the penultimate residue resulted in a branched trisaccharide as demonstrated by ¹H-NMR-spectroscopy and permethylation analysis of the reaction product. Comparable results were obtained with various oligosaccharides containing an internal galactose unit glycosidically linked 13. Attempts to separate and purify the various enzymes involved resulted in the isolation of a fraction which is able to transfer d-Gal exclusively to native galactan, but not to oligosaccharides. A further fraction was obtained from a different resin with activity for native galactan and 6-O-(-d-galactopyranosyl)-d-galactopyranose. but without any for methyl-3-O-(-d-galactopyranosyl)--d-galactopyranose. It is thus concluded that at least three different enzymes are involved in the biosynthesis of this snail galactan.Abbreviation Gal galactose - glc gas-liquid chromatography - Gro glycerol - tlc thin layer chromatography     

Structure of a β-galactan isolated from the nuclei of Physarum polycephalum

A sulfated and phosphorylated β-D-galactan ([α]_D + 8°) was isolated from the nuclei of the acellular slime mould Physarum polycephalum. The polysaccharide was isolated from cesium chloride gradients during the preparation of ribosomal DNA and purified. The purified galactan contained 89% galactose, 2.5% phosphate and 9.6% sulfate groups and had an average degree of polymerisation of 560. Periodate degradation and permethylation studies indicated the presence of mainly (1 → 4)-, but also of (1 → 3)-, and (1 → 6)-linked galactose units with one branch every 13 units. These results suggested that the intranuclear galactan, apart from its higher sulfate content, is similar to the extra-cellular polysaccharide produced by P. polycephalum.     

Biosynthesis of an acidic galactan and sulfated glycosaminoglycans during embryonic development of the mollusc Pomacea sp.

The synthesis of acidic polysaccharides by eggs of Pomacea sp. at different stages of development was studied. By day 3 after oviposition an acidic galactan became apparent. This compound reaches its maximum concentration by day 6 and then slowly decreases in concentration, no longer being detectable by day 12. Among the sulfated glycosaminoglycans, chondroitin sulfate is the first to appear (day 10), followed by heparan sulfate and other sulfate glycosaminoglycans, which were synthesized in large amounts until hatching (around day 15). Each one of the compounds was purified and characterized by chemical analysis and enzymatic degradation. The synthesis and characterization of the sulfated glycosaminoglycans was confirmed by the use of radioactive sulfate applied to intact eggs at different stages of development. These studies suggest that the main difference between vertebrate and mollusc development regarding the acidic polysaccharides is that hyaluronic acid seems to be absent during early mollusc development. It is proposed that the acidic galactan may be synthesized from the neutral galactan, already present in the eggs in high amounts, and may replace this glycosaminoglycan in the mollusc embryogenesis.     

Chloroplast sulfate transport in green algae – genes,proteins and effects

This review summarizes evidence at the molecular genetic, protein and regulatory levels concerning the existence and function of a putative ABC-type chloroplast envelope-localized sulfate transporter in the model unicellular green alga Chlamydomonas reinhardtii. From the four nuclear genes encoding this sulfate permease holocomplex, two are coding for chloroplast envelope-targeted transmembrane proteins (SulP and SulP2), a chloroplast stroma-targeted ATP-binding protein (Sabc) and a substrate (sulfate)-binding protein (Sbp) that is localized on the cytosolic side of the chloroplast envelope. The sulfate permease holocomplex is postulated to consist of a SulP–SulP2 chloroplast envelope transmembrane heterodimer, flanked by the Sabc and the Sbp proteins on the stroma side and the cytosolic side of the inner envelope, respectively. The mature SulP and SulP2 proteins contain seven transmembrane domains and one or two large hydrophilic loops, which are oriented toward the cytosol. The corresponding prokaryotic-origin genes (SulP and SulP2) probably migrated from the chloroplast to the nuclear genome during the evolution of Chlamydomonas reinhardtii. These genes, or any of its homologues, have not been retained in vascular plants, e.g. Arabidopsis thaliana, although they are encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the Photosystem II D1 reaction center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in Chlamydomonas reinhardtii is discussed along with its impact on the repair of Photosystem II from a frequently occurring photo-oxidative damage and H₂-evolution related metabolism in this green alga.     

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.     

Structure and properties of agar from two unexploited agarophytes from Venezuela

The red seaweeds Gelidiella acerosa and Gracilaria mammillaris growing along the coast of Venezuela were investigated as potential economic sources of agar. Agar extracted from Gracilaria mammillaris accounted for 27% of the algal dry weight but had quite a poor gelling ability because of the presence of alkali-stable sulphate groups on the D-galactose residue. However, the gel strength of its aqueous solutions was considerably enhanced by the addition of potassium, sodium and calcium ions (up to 1N). On the contrary, the galactan from Gelidiella acerosa (yield of about 20% w/w) exhibited quite good properties. In particular, the gel strength was comparable to that of commercial agaroses. Sulphate esters were not detectable by chemical methods and NMR spectroscopy revealed an agarose backbone with a high degree of methylation on both D and L-galactose residues.     

Bifunctional glycosyltransferases catalyze both extension and termination of pectic galactan oligosaccharides

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side‐chains of rhamnogalacturonan‐I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP‐Gal to growing β‐1,4‐galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP‐α‐d ‐Gal and the transfer of an arabinopyranose from UDP‐β‐l ‐Ara_p to galactan chains. The two substrates share a similar structure, but UDP‐α‐d ‐Gal is the preferred substrate, with a 10‐fold higher affinity. Transfer of Ara_p to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage.     

Siroheme sulfite reductase isolated from Chromatium vinosum. Purification and investigation of some of its molecular and catalytic properties

Cells of the phototrophic bacterium Chromatium vinosum strain D were shown to contain a siroheme sulfite reductase after autotrophic growth in a sulfide/bicarbonate medium. The enzyme could not be detected in cells grown heterotrophically in a malate/sulfate medium. Siroheme sulfite reductase was isolated from autotrophic cells and obtained in an about 80% pure preparation which was used to investigate some molecular and catalytic properties of the enzyme. It was shown to consist of two different types of subunits with molecular weights of 37,000 and 42,000, most probably arranged in an ₄₄-structure. The molecular weight of the native enzyme was determined to 280,000, 51 atoms of iron and 47 atoms of acid-labile sulfur were found per enzyme molecule. The absorption spectrum indicated siroheme as prosthetic group; it had maxima at 280 nm, 392 nm, 595 nm, and 724 nm. The molar extinction coefficients were determined as 302×10³ cm²xmmol^-1 at 392 nm, 98×10³ cm² xmmol^-1 at 595 nm and 22×10³ cm²x-mmol^-1 at 724 nm. With reduced viologen dyes as electron donor the enzyme reduced sulfite to sulfide, thiosulfate, and trithionate. The turnover number with 59 (2 e^-/enzyme moleculexmin) was low. The pH-optimum was at 6.0. C. vinosum sulfite reductase closely resembled the corresponding enzyme from Thiobacillus denitrificans and also desulfoviridin, the dismilatory sulfite reductase from Desulfovibrio species. It is proposed that C. vinosum catalyses anaerobic oxidation of sulfide and/or elemental sulfur to sulfite in the course of dissimilatory oxidation of reduced sulfur compounds to sulfate.Non-common abbreviations APS adenylyl sulfate - SDS sodium dodecyl sulfate     

Purification and partial characterization of alanine dehydrogenase from Streptomyces aureofaciens

Alanine dehydrogenase was purified to near homogeneity from cell-free extract of Streptomyces aureofaciens, which produces tetracycline. The molecular weight of the enzyme determined by size-exclusion high-performance liquid chromatography was 395 000. The molecular weight determined by sodium dodecyl sulfate gel electrophoresis was 48 000, indicating that the enzyme consists of eight subunits with similar molecular weight. The isoelectric point of alanine dehydrogenase is 6.7. The pH optimum is 10.0 for oxidative deamination of L-alanine and 8.5 for reductive amination of pyruvate. K _M values were 5.0 mM for L-alanine and 0.11 mM for NAD⁺. K _M values for reductive amination were 0.56 mM for pyruvate, 0.029 mM for NADH and 6.67 mM for NH₄Cl.Abbreviation AlaDH alanine dehydrogenase     

Isolation and characterization of membrane-associated proteoglycans from normal and malignant human mammary epithelial cells

The plasma membrane-associated proteoglycans of a malignant human breast cell line (MDA-MB-231) were compared with the corresponding proteoglycans from a normal cell line (HBL-100). The labeled proteoglycans were isolated from the plasma membranes of cells grown in the presence of [³H]glucosamine and [³⁵S]Na₂SO₄ by extraction with guanidine hydrochloride and subsequently purified by DEAE-ion exchange chromatography. Their structural properties were established by treatment with nitrous acid, heparitinase and chondroitinase ABC, and by gel filtration before and after alkaline -elimination. About 18% of the proteoglycans synthesized by these cell lines were associated with the plasma membranes. The HBL plasma membranes contained 80% heparan sulfate and 20% chondroitin sulfate proteoglycans whereas MDA plasma membranes had 50% heparan sulfate and 50% chondroitin sulfate proteoglycans. The MDA plasma membrane contained two heparan sulfate proteoglycans, both having nearly the same molecular size as the two species secreted into the medium by these cells. The HBL plasma membrane also contained two hydrodynamic size heparan sulfate proteoglycans. The larger hydrodynamic size species has a slightly lower molecular size than that secreted into the medium, and the smaller hydrodynamic size species was not detectable in the medium. Even though the major chondroitin sulfate proteoglycans from MDA plasma membranes were smaller in size than those from HBL plasma membrane, a larger proportion of the glycosaminoglycan chains of the former were bigger than those from the latter.Abbreviations CHAPS 3-[(3-cholamidopropyl)dimethylammonio]propane-1-sulfonate - Di-OS 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-d-galactose - Di-4S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-4-O-sulfo-d-galactose - Di-6S 2-acetamido-2-deoxy-3-O-(-d-gluco-4-ene-pyranosyluronic acid)-6-O-sulfo-d-galactose - Gdn-HCl guanidine hydrochloride - WGA wheat germ agglutinin     

A κ-carrageenase from a newly isolated pseudoalteromonas-like bacterium, WZUC10

A bacterial strain able to produce κ-carrageenase, designated WZUC10, was isolated from a live specimen of the red alga Plocamium telfainae collected in the East China Sea. The phylogenetic evidence and phenotypic features indicate that this strain belongs to the genus Pseudoalteromonas. WZUC10 requires NaCl for growth and κ-carrageenan to induce κ-carrageenase synthesis; galactose and lactose do not induce it. The optimal growth temperature is 23∼27°C. The secreted enzyme, which has a molecular mass of 45 kDa, breaks down κ-carrageenan into κ-neocarratetraose sulfate and larger oligosaccharides with a repeating β-D-Galp4S-(1→4)-α-D-AnGalp structure, but cannot degrade κ-neocarratetraose sulfate or κ-neocarrahexaose sulfate into κ-neocarrabiose sulfate. The enzyme retains 90% of its activity after 2 h at 40°C and is completely inactivated after 7.5 min at 70°C. The enzyme’s optimal temperature is 30°C and its optimal pH is 7.5. The enzyme-catalyzed reaction follows Michaelis-Menten kinetics, with the Michaelis constant (K _m) and the turnover number (k) being 0.015 mM and 125 s⁻¹, respectively. WZUC10 produces 50 U/mL κ-carrageenase after cultivation at 25°C for 35 h on a medium containing 80 g/L glucose, 5 g/L corn steep liquor, 3 g/L κ-carrageenan, and 15 g/L NaCl. κ-Neocarratetraose sulfate was prepared simply with precipitation by ethanol:water (5:1, v/v).