Nobuko Seno

A modified method for chondrosulfatase assay

The turbidimetric method of Dodgson and Price has been modified for chondrosulfatase assay. The method involves removal of undegraded chondroitin sulfates with cetylpyridinium chloride, modification of the barium chloride-gelatin reagent, and substitution of 0.2N hydrochloric acid for the 3% trichloroacetic acid of the original method. By using this modified method, the presence of chondrosulfatase activity was demonstrated in the liver of the squid, Ommastrephes sloani pacificus.

A new dermatan polysulfate, chondroitin sulfate H, from hagfish notochord

Abstract A novel oversulfated mucopolysaccharide was isolated from the notochord of hagfish, which belongs to the more primitive branch of cyclostomes. It is concluded that the mucopolysaccharide is a new type of dermatan polysulfate consisting mainly of (1 → 4)-α-L-idopyranuronosyl-(1 → 3)-2-acetamido-2-deoxy-β-D-galactopyranosyl 4,6-disulfate unit. The name of chondroitin sulfate H is proposed for this dermatan polysulfate.

Diversities in animal vitronectins differences in molecular weight immunoreactivity and carbohydrate chains

Six animal plasma vitronectins, human, horse, porcine, bovine, rabbit and chicken vitronectins purified by a novel method using two successive heparin affinity columns, showed marked diversity in molecular weight, immunoreactivity and carbohydrate composition. Chicken vitronectin had a distinctly different amino acid composition from the mammalian vitronectins; and bovine vitronectin was the only one to contain N-glycolylneuraminic acid as well as N-acetylneuraminic acid. Binding studies with horseradish peroxidase-labelled lectins indicated that all the vitronectins contained complex-type, sialylated N-linked sugar chains and that only porcine vitronectin had a fucosylated sugar chain. D-Galactosamine determinations and binding studies with horseradish peroxidase-peanut lectin on native and asialovitronectins revealed that the mammalian vitronectins other than human vitronectin contained O-linked sugar chains with sialic acid, chicken vitronectin contained unsialylated chains, and human vitronectin contained neither. The results indicate that diversities in vitronectins are apparent in their molecular weights and glycosylations, especially in the number and structure of O-linked sugar chains.

Derivatization of epoxy-activated agarose with various carbohydrates for the preparation of stable and high-capacity affinity adsorbents: Their use for affinity chromatography of carbohydrate-binding proteins

Abstract Two types of affinity adsorbents for lectins were prepared by new simple procedures. Both types of adsorbents had high ligand concentration and chemically stable linkage between ligand and Sepharose 4B. Oligosaccharide ligands were coupled by reductive amination with sodium cyanoborohydride to amino-Sepharose 4B prepared by amination of epoxy-activated Sepharose 4B. The glycamyl-Sepharose 4B thus obtained had particularly high adsorption capacities for lectins; lactamyl-Sepharose 4B, 58 mg/l ml of gel for peanut lectin; maltamyl-Sepharose 4B, 146 mg/ml for concanavalin A; and tetra- N -acetylchitotetraamyl-Sepharose 4B, 36 mg/ml for wheat germ agglutinin. Hexosamine was coupled by the aid of carbodiimide to carboxyl-Sepharose 4B prepared by succinylation of amino-Sepharose 4B. Galactosamine-Sepharose 4B adsorbed 145 mg soybean agglutinin/l ml gel. The columns turned from a semitransparent white to a milky white as they were saturated with lectins.

Mucopolysaccharides from chicken skin of three age groups.

Abstract Mucopolysaccharides were isolated from the skin of chickens of three age groups: newly hatched, 4 weeks old and 23 months old, and determined by electrophoresis on cellulose acetate strips, Dowex I column chromatography and chondroitinase digestion. Hyaluronic acid and dermatan sulfate were the major components in all age groups; the content of hyaluronic acid decreased and dermatan sulfate increased with age. Small amounts of chondroitin sulfate A, dermatan polysulfate and a heparan sulfate-like substance were also found. The mucopolysaccharide pattern of 4-week-old chicken skin was closer to that of 23-month-old skin than that of newly hatched.

Preparation of affinity adsorbents with toyopearl gels

Abstract The optimal conditions for the activation of Toyopearl by epichlorohydrin and subsequent immobilization of ligands were investigated. The optimal conditions for the activation were very different from those for agarose gel. The concentration of epoxy groups introduced was as high as 330 μmole per gram of wet gel for Toyopearl HW-55 and 150 μmole per gram of wet gel for Toyopearl HW-65 (diol type). Epoxy-activated Toyopearl was converted into amino and carboxyl derivatives and was subsequently coupled with various ligands. Glycamyl Toyopearl was prepared in a much shorter time (6 h) than agarose gel (800 h), because a higher reaction temperature could be used. The adsorbents obtained were successfully used for the affinity chromatography of lectin and trypsin. However, their adsorption capacities were lower than those of the agarose adsorbents prepared by the same methods.

Improved method for the immobilization of heparin

The optimal conditions for immobilizing heparin through its terminal formyl group were investigated. When Amino Sepharose (1 g) was suspended in 1 ml of phosphate buffer (pH 7) containing 30 mg of heparin and 3 mg of sodium cyanoborohydride, with shaking at room temperature, the maximum immobilization of heparin (10 mg of heparin per gram of wet gel) was reached within 2 days. The Heparin Sepharose thus obtained was stable: no significant loss of the heparin content was observed after storage for 4 months at 4 degrees C. Heparin was also immobilized by the same method with Amino TSK gel G5000PW instead of Amino Sepharose 4B and was successfully applied to the high-performance liquid affinity chromatography of fibronectin and thrombin.

Vitronectin diversity in evolution but uniformity in ligand binding and size of the core polypeptide

We isolated vitronectins from the plasma or sera of 14 animal species including mouse and rat by heparin affinity chromatography. They cross-reacted with anti-vitronectin antibody and their amino terminal sequences showed strong homology. They also promoted spreading of BHK cells and were bound to heparin and collagen in the same way. Therefore, these properties appear to be essential for vitronectin function. However, the apparent molecular weights of these vitronectins varied considerable from 59 to 78 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). In addition, the number of bands also varied from 1 to 3. To search for the uniformity of vitronectin polypeptide, vitronectins were deglycosylated and examined by Ferguson plot analysis. The size of the polypeptide portion of vitronectins was estimated to range from 40 to 57 kDa which was 19-26 kDa smaller than original values. Supposing a possible cleavage site at 5-13 kDa far from the carboxyl terminus, all vitronectin polypeptides were speculated to be synthesized de novo in the size range of 50-57 kDa. Proteins reacting with anti-vitronectin antibody were also detected on the immunoblot of 13 more species including Drosophila and Physarum. Almost all of these vitronectin-like proteins showed marked species-specific variations in their apparent molecular weights from 51 to 96 kDa in SDS-PAGE.

Structural determination of novel tetra- and hexasaccharide sequences isolated from chondroitin sulfate H (oversulfated dermatan sulfate) of hagfish notochord

Oversulfated chondroitin sulfate H (CS-H) isolated from hagfish notochord is a unique dermatan sulfate consisting mainly of IdoAα1-3GalNAc(4S,6S), where IdoA, GalNAc, 4S and 6S represent L-iduronic acid, N-acetyl-D-galactosamine, 4-O-sulfate and 6-O-sulfate, respectively. Several tetra- and hexasaccharide fractions were isolated from CS-H after partial digestion with bacterial chondroitinase B to investigate the sequential arrangement of the IdoAα1-3GalNAc(4S,6S) unit in the CS-H polysaccharide. A structural analysis of the isolated oligosaccharides by enzymatic digestions, mass spectrometry and 1H NMR spectroscopy demostrated that the major tetrasaccharides shared the common disulfated core structure Δ4,5HexAα1-3GalNAc(4S)β1-4IdoAα1-3GalNAc(4S) with 0 ∼ 3 additional O-sulfate groups, where Δ4,5HexA represents 4-deoxy-α-L-threo-hex-4-enepyranosyluronic acid. The major hexasaccharides shared the common trisulfated core structure Δ4,5HexAα1-3GalNAc(4S)β1-4IdoAα1-3GalNAc(4S)β1-4IdoAα1-3GalNAc(4S) with 1 ∼ 4 additional O-sulfate groups. Some extra sulfate groups in both tetra- and hexasaccharides were located at the C-2 position of a Δ4,5HexA or an internal IdoA residue, or C-6 position of 4-O-sulfated GalNAc residues, forming the unique disulfated or trisulfated disaccharide units, IdoA (2S)-GalNAc(4S), IdoA-GalNAc(4S,6S) and IdoA (2S)-GalNAc(4S,6S), where 2S represents 2-O-sulfate. Of the demonstrated sequences, five tetra- and four hexasaccharide sequences containing these units were novel.

Preparation of three types of heparin-Sepharose and their binding activities to thrombin and antithrombin III

Abstract Three types of immobilized heparin, heparin-Sepharoses I, II, and III, were prepared to study the effect of the mode of the heparin attachment on the binding activities to thrombin and antithrombin III (AT-III). Heparin-Sepharose I was prepared by the coupling of the carboxyl groups of heparin with amino-Sepharose, obtained by the epoxy activation of Sepharose 4B with epichlorohydrin and subsequent amination, with the aid of water-soluble carbodiimide. Heparin-Sepharose II was prepared similarly by the coupling of the amino groups of heparin with the carboxyl groups of carboxyl-Sepharose obtained by the succinylation of amino-Sepharose. Heparin-Sepharose III was prepared by the reductive amination of heparin and amino-Sepharose with sodium cyanoborohydride. Heparin-Sepharoses I, II, and III contained heparin in the ratio of 16:1:3. However, their binding activities to thrombin and AT-III were not proportional to the heparin content, and heparin-Sepharose III was found to be the most efficient affinity adsorbent for both proteins.