Kaeko Hayashi

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.

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.

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.

Desulfation of Sulfated Carbohydrates Mediated by Silylating Reagents

Abstract Efficient methods of desulfation are often required in carbohydrate chemistry and biochemistry. In addition to conventional desulfation methods,1,2 we recently reported a novel desulfation method employing a silylating reagent, N,O-bis(trimethylsilyl)-acetamide.3,4 With this reagent, the 6-O-sulfoxyl groups of the sugar moiety are regioselectively removed and newly formed hydroxyl groups are further converted by silylation into trimethylsilyloxyl groups. The desulfated carbohydrates are easily recovered after desilylation with water or aqueous methanol. Although the mechanism for this reaction remains unclear, silylating reagents can be considered as potential reagents for desulfation reaction. In the present paper, we examined various silylating reagents to find effective and new desulfation reagents for carbohydrate sulfates.

Highly methylated agars with a high gel-melting point from the red seaweed, Gracilaria eucheumoides☆

Highly methylated agars were isolated from the red seaweed, Gracilaria eucheumoides, harvested in Japan. One of the obtained agars formed a thermo-reversible gel with a high melting point up to 121 degrees and was indicated to consist of a regularly repeating structure,-->3 6-O-methyl-beta-D-Gal(1-->4) 3,6-anhydro-2-O-methyl-alpha-L-Gal 1-->.

Enzymic digestibility of reduced-pressurized, heat-moisture treated starch

The digestibility of the reduced-pressurized heat-moisture treated corn starches by α-amylase and glucoamylase was studied. By the treatment, regular and waxy corn starch granules were well digested by α-amylase without gelatinization, while the digestibility of the high amylose corn starch was reduced. Both regular and waxy corn starches, regardless of the treatment, were digested well by enzymes under the gelatinized condition. However, a drastic increase of indigestible portion was observed in the high amylose corn starch. Methylation analysis of the enzyme resistant moiety of the high amylose starch indicated that most of the indigestible moiety was composed of amylose. These findings suggested that the arrangement of regular or waxy corn starch molecules was made more random by the treatment while, in the high amylose corn starch molecule, crammed amylose formed the highly associated structure.