Ryo Takano

Concurrence of agaroid and carrageenan chains in funoran from the red seaweed Gloiopeltis furcata Post. et Ruprecht (Cryptonemiales, Rhodophyta)

Abstract Funoran extracted from the red seaweed Gloiopeltis furcata Post. et Ruprecht was fractionated into four polysaccharide fractions in terms of differences in solubility of their cetylpyridinium salt in potassium chloride solution. Besides the main fraction constructed by an agarose sulphate structure with negative optical rotation, minor polysaccharide fractions with positive optical rotation were obtained. One of the minor polysaccharide fractions contained d - and l -galactose (Gal), 6-O-methyl- d -Gal, 3,6-anhydro- l -Gal and sulphate. Partial hydrolysis and partial methanolysis studies of this fraction led to identification of oligosaccharides attributable to both agaroid and carrageenan backbones, i.e. [ → 3) d -Gal(1 → 4) l -Gal(1 → ][→ 3) d -Gal(1 → 4)3,6-and [ → 3)6- O -methyl- d -Gal(1 → 4)D-Gal(l → ]. Methylation analysis and alkaline treatment study of this fraction revealed that the sulphate groups were located at O-6 of all the (1 → 4)-linked l - and a part of the (1 → 4)-linked d -Gal residues, O-6 of a part of the (1 → 3)-linked d -Gal residue, and both O-2 and O-4 of the (1 → 3)-linked 6-O-methyl- d -Gal residue and the other (1 → 3)-linked d -Gal residue.

A novel regioselective desulfation of polysaccharide sulfates: specific 6-O-desulfation with N,O-bis(trimethylsilyl)acetamide

Abstract Treatment of the pyridinium salts of glycosaminoglycans and galactan sulfates with N,O -bis(trimethylsilyl)acetamide (BTSA) in pyridine for 2 h at 60 °C caused specific 6- O -desulfation without depolymerisation or other chemical changes.

Molecular characteristics and gelling properties of the carrageenan family, 1: Preparation of novel carrageenans and their dilute solution properties

The carrageenans are a family of sulfated polysaccharides extracted from red seaweed, and are conventionally classified into three major groups as κ-, ι- and λ-carrageenan according to the maximum number (1, 2 and 3, respectively) of sulfate groups per disaccharide repeating unit in corresponding ideal structures. β- and θ- carrageenan (not available in nature) possessing none and two sulfate groups per repeating unit, respectively, were prepared from κ- and λ-carrageenan by desulfonation. Here the aqueous solutions of κ-, ι-, β- and θ-carrageenans undergo the thermoreversible gelation by lowering temperature, whereas the aqueous solution of λ-carrageenan forms no gel.Light scattering and small-angle X-ray scattering were observed from the aqueous solution of κ-, ι-, λ-, β- and θ-carrageenans, and the results were analyzed conventionally and by adapting the molecular model in order to elucidate the effect of sulfate groups and anhydrous residue on the conformational characteristics of carrageenans.

Pectinolytic enzymes from Pseudomonas marginalis MAFF 03-01173.

Two pectinolytic enzymes were purified from the culture broth of Pseudomonas marginalis pv. marginalis MAFF 03-01173 with total 33% recovery of the initial activity. From the substrate specificities against pectin and polygalacturonic acid, the requirement of calcium ion for the enzymatic activity, and the N-terminal sequences, the enzymes were identified as pectin lyase and pectate lyase. The M,s of pectin lyase and pectate lyase were estimated to be 34,000 and 43,000, respectively, by SDS polyacrylamide gel electrophoresis. Both enzymes showed almost the same pH dependent activity curves with the highest activity at pH 8.3

Funoran from the red seaweed, Gloiopeltis complanata : polysaccharides with sulphated agarose structure and their precursor structure

Abstract Funoran extracted from the red seaweed, Gloiopeltis complanata, was fractionated based on the solubility of its cetylpyridinium salt in KCl solution to give the fractions PSl and PS2. PS2 was further fractionated into PS2G that formed a gel in 0.5 n KCl, and PS2S that did not form a gel in saturated KCl. PSl, PS2G and PS2S were different in composition and properties showing that the funoran was heterogeneous. On the basis of the results from composition analyses, partial methanolysis studies, methylation studies, 13C-NMR spectrum measurements, and regioselective desulphation studies employing a silylating reagent, N,O-bis(trimethylsilyl)acetamide, PS2G was shown to consist chiefly of 6-O-sulphated agarose, repeats of →3)6-SO3-β- d -Gal(1→4)3,6-anhydro-α- l -Gal(1→; and PS2S to contain repeats of →3)6-SO3-β- d -Gal(1→4)6-SO3-α- l -Gal(1→ as well as smaller amounts of →3)2,6-di-SO3-β- d -Gal(1→ and →3)2-SO3-β- d -Gal(1→ residues.

Agarose-carrageenan hybrid polysaccharides fromLomentaria catenata

Two polysaccharide fractions, PS1 and PS2 from Lomentaria catenata consisted of D-Gal, L-Gal, D-Glc, D-Xyl, D-GlcA and sulphate. Partial hydrolysis led to the isolation and identification of oligosaccharides indicating the co-existence of an agarose and carrageenan backbone structure, in which D-Glc and D-GlcA residues occur as single units branching at O-3 of -->4)alpha-D-Gal(1--> and O-4 of -->3)beta-D-Gal(1-->, respectively.

Trypsin inhibitors from bottle gourd (Lagenaria leucantha Rusby var Depressa Makino) seeds. Purification and amino acid sequences

Two almost identical trypsin isoinhibitors, LLDTI-I and LLDTI-II, from bottle gourd (Lagenaria leucantha Rusby var. Depressa Makino) seeds were purified by acetone precipitation, gel filtration and reversed phase chromatography. LLDTI-I and LLDTI-II consist of 30 and 29 amino acid residues, respectively, and have identical sequences, except that LLDTI-I has one additional pyroglutamic acid residue at N-terminus. Both proteins are strong inhibitors of bovine trypsin, with Ki values of 2.4.10(-10) M (LLDTI-I) and 9.6.10(-11) M (LLDTI-II). Amino acid sequences are as follows: [sequence: see text]

Sulfation of Polysaccharides with Sulfuric Acid Mediated by Dicyclohexylcarbodiimide

Abstract Starch, agarose, κ-carrageenan and porphyran were sulfated by sulfuric acid with dicyclohexylcarbodiimide as a condensation reagent. In all the cases, the sulfation at O-6 appeared to be predominant on the basis of results from methylation analyses. Although the reactivities of the other hydroxyl groups towards the present sulfation reaction were less than that at C-6, they varied depending on the position in the sugar residue of the polysaccharides. The sulfated agarose was further fractionated in terms of the difference in the solubility of its cetylpyridinium salt to KCl solution. The major fraction resembled the main polysaccharide of funoran (agarose 6-sulfate) on the basis of the behavior of its cetylpyridinium salt, 13C NMR measurements and methylation analyses.

Structural Requirements of Human Tissue Factor Pathway Inhibitor (TFPI) and Heparin for TFPI-Heparin Interaction

Heparin affinity chromatography of synthetic peptide fragments mimicking tissue factor pathway inhibitor (TFPI) indicated that the minimal heparin binding sequence consists of 12 amino acid residues located at the C-terminal tail. Within this minimal sequence, Arg-257 and Arg-259 appeared to contribute most significantly to interaction with heparin. Affinity chromatography of TFPI using immobilized heparin derivatives regiospecifically desulfated at O-6 of the glucosamine residue, N-2 of the glucosamine residue, and/or O-2 of the iduronic acid residue indicated that all the sulfate groups in heparin appeared to be required for TFPI-heparin interaction. Among them, however, the 6-O-sulfate groups appeared to make the largest contribution to the interaction, while the 2-O-sulfate groups contributed the least. In vitro experiments on the inhibition of factor Xa by TFPI enhanced with native and chemically modified heparins afforded similar results.

Effect of heparin chain length on the interaction with tissue factor pathway inhibitor (TFPI)

Tissue factor pathway inhibitor (TFPI) is a heparin-binding protein involved in the extrinsic blood coagulation system. In order to elucidate the minimal size of heparin chain required for the interaction with TFPI, we prepared a series of heparin-derived oligosaccharides with tailored chain length ranged from disaccharide to eicosasaccharide after the successive treatments of heparin, including partial N-desulphation, deaminative cleavage with nitrous acid and gel-filtration. Affinity chromatography study of each oligosaccharide fraction using TFPI as the ligand indicated that increasing the degree of polymerisation causes increased affinity, and that a remarkable change in the affinity occurs between the decamers and dodecamers. Measurement of factor Xa inhibitory activity of TFPI in the presence of each oligosaccharide fraction indicated that the fractions shorter than dodecamers only slightly enhanced the TFPI activity for factor Xa inhibition, while the fractions larger than octadecamers had an effect comparable to full-length heparin. These were compatible to the results from the kinetic analyses of the interaction between TFPI and heparin-derived oligosaccharide with an evanescent wave-based biosensor system, IAsys, using a TFPI C-terminal peptide as the ligand.