Isolation of dermatan sulfate with high heparin cofactor II-mediated thrombin-inhibitory activity from porcine spleen.

We prepared dermatan sulfate specimens from various porcine tissues, and compared their heparin cofactor II-mediated thrombin-inhibitory activities and chemical natures, including disaccharide composition. Electrophoresis of the specimens on cellulose acetate membrane indicated that spleen dermatan sulfate was the most acidic of the dermatan sulfates prepared from the various porcine tissues. Analysis of the disaccharide units of the dermatan sulfate specimens by high-performance liquid chromatography revealed that spleen dermatan sulfate was rich in 4,6-di-O-sulfated N-acetylgalactosamine residues as compared with those of the other tissues.

Inhibitory activities for thrombin and factor Xa of whale heparin: comparative studies on two preparations from different species.

Whale intestinal heparin preparations from Balaenoptera physalus L. (Bp) and Balaenoptera borealis L. (Bb) were fractionated by affinity chromatography on a column of antithrombin III (AT)-Sepharose 4B.

Isolation and characterization of chondroitin sulfate proteoglycans from porcine thoracic aorta.

A chondroitin sulfate proteoglycan fraction was prepared from the 3 M MgCl2 extract of porcine aortas by DEAE-cellulose chromatography, followed by gel filtration through Sepharose CL-4B. Affinity chromatography of the fraction with antithrombin III-agarose yielded two chondroitin sulfate proteoglycans of a non-binding (proteoglycan IA) and binding (proteoglycan IB) nature. Proteoglycans IA and IB were different from each other in molecular size, in proportion of the protein relative to the polysaccharide portion, and in size of the chondroitin sulfate chain.

Liquid-chromatographic determination of urinary glycosaminoglycans for differential diagnosis of genetic mucopolysaccharidoses.

Glycosaminoglycans in urine from patients with various mucopolysaccharidoses were digested with chondroitin ABC lyase (EC 4.2.2.

Thrombin-inhibitory activity of whale heparin oligosaccharides.

Whale heparin was partially digested with a purified heparinase and the oligosaccharide fractions with 8-20 monosaccharide units were isolated from the digest by gel filtration on Sephadex G-50, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. A marked difference in the inhibitory activity for thrombin in the presence of antithrombin III was observed between the high-affinity fractions for antithrombin III of octasaccharide approximately hexadecasaccharide and those of octadecasaccharide approximately eicosasaccharide. The disaccharide compositions of these hexadeca-, octadeca-, and eicosasaccharides were analyzed by high-performance liquid chromatography after digestion with a mixture of purified heparitinases 1 and 2 and heparinase.

High-performance liquid chromatography of pyridylamino derivatives of unsaturated disaccharides produced from chondroitin sulfate isomers by chondroitinases.

A sensitive method was developed for the separation and quantitation of four unsaturated disaccharides (delta Di-0S, delta Di-4S, delta Di-6S, and delta Di-diS) by high performance liquid chromatography. The unsaturated disaccharides were coupled with a fluorescent compound, 2-aminopyridine. Complete separation of the resulting pyridylamino derivatives was achieved on a column of muBondapak-C18 with 8 mM KH2PO4-Na2HPO4 (pH 6.

Comparison of glycosaminoglycans from thoracic aortas of several mammals.

Intima-media of porcine, canine, rabbit, and human thoracic aortas were separately digested with pronase, after extraction of fat. Glycosaminoglycan fractions were then separated from the resulting glycoconjugate fractions by precipitation with cetylpyridinium chloride. Glycosaminoglycan compositions were determined by sequential digestion with Streptomyces hyaluronidase, chondroitinase AC, chondroitinase ABC and heparitinase 1.

Hormonal effects on glycosaminoglycans in thoracic aortas of rabbits.

Intima-media of thoracic aortas obtained from estrogen-treated, estrogen-progesterone-treated and sham-treated ovariectomized rabbits were separately digested with pronase after extraction of fat. Glycosaminoglycan fractions were then separated from the resulting glycoconjugate fractions by precipitation with cetylpyridinium chloride. The glycosaminoglycan levels increased after treatments with estrogen and estrogen-progesterone.

Glycosaminoglycans of human colonic mucosa and colonic adenocarcinoma.

The mucosal scrapings of the normal human colons and the colonic adenocarcinoma tissues were digested with pronase, and the glycosaminoglycan fractions were prepared by fractionation with cetylpyridinium chloride after treatment with deoxyribonuclease. Chondroitin sulfate A/C, heparan sulfate, and hyaluronic acid were contained in the glycosaminoglycan fraction of the normal tissue, but dermatan sulfate was undetectable. In contrast, the glycosaminoglycan fractions derived from the tumors contained substantial amounts of dermatan sulfate together with the foregoing three glycosaminoglycans.

Unsaturated disaccharides in the enzymatic digests of heparan sulfates.

High performance liquid chromatography was performed by an ion-pair reversed-phase method of six standard unsaturated disaccharides derived from heparan sulfate and heparin. Separation of delta Di-GlcNAc, delta Di-GlcN(2S), delta Di-GlcNAc(6S), delta Di-GlcN(2,6- or 2,2'-diS) and delta Di-GlcN(2,6,2'-triS) was achieved on a column of Jasco SC-02 with 10 mM tetrabutylammonium phosphate (pH 7.0) containing 30 or 47% methanol as a mobile phase.

Sialoglycopeptides obtained from a transplantable rat colorectal adenocarcinoma: a comparison with those from normal colonic mucosa.

A transplantable colorectal adenocarcinoma and the normal colonic mucosa derived from rats of ACI/N strain were digested successively with pronase, deoxyribonuclease, chondroitinase ABC, and heparitinase to obtain the corresponding glycopeptide fractions. The amino acid compositions of these two fractions suggested that the polypeptide backbones were quite similar. However, the electrostatic net charges of these fractions were shown to be different by cellulose acetate membrane electrophoresis, ion exchange chromatography, and measurement of sialic acid contents.

Enzymatic determination of urinary glycosaminoglycans from orthopedic patients.

Crude glycosaminoglycan (GAG) fraction was directly precipitated with cetylpyridinium chloride without prior dialysis of urine of orthopedic patients. The crude GAG fraction was then fractionated with trichloroacetic acid (TCA). The TCA-insoluble peptide-bound GAG fraction thus obtained was treated with alkali to eliminate the peptide moiety for enzymatic analysis.

A simple method for the quantitation of glycuronic acid-containing glycosaminoglycans with mucopolysaccharidases.

A simple method for the quantitative determination of glycuronic acid-containing glycosaminoglycans (UA-GAG) is described. Sample solutions of glycosaminoglycans were digested with chondroitinase AC, chondroitinase C, chondroitinase B, heparitinases, and Streptomyces hyaluronidase, respectively, and the absorbance was read at 232 nm after digestion. The contents of 4-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-4S), 6-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-6S) plus N-acetylgalactosaminyl beta (1 leads to 4)D-glucosiduronyl units (Ch-OS), 4-O-sulfated N-acetylgalactosaminyl beta (1 leads to 4)L-idosiduronyl units (D-4S) plus N-acetylgalactosaminyl beta (1 leads to 4)L-idosiduronyl units (D-OS), heparan sulfate, and hyaluronic acid in the sample solutions were calculated from the absorbance with reference to that of the digestion products of known amounts of standard UA-GAG.

Glycosaminoglycans of rat colorectal adenocarcinoma.

A transplantable colorectal adenocarcinoma and the normal colonic mucosa derived from rats of ACI/N strain were digested with pronase, and the glycosaminoglycan fractions were obtained by fractionation with cetylpyridinium chloride. The glycosaminoglycan fraction derived from the adenocarcinoma contained substantial amounts of chondroitin sulfate A/C, dermatan sulfate, heparan sulfate, and hyaluronic acid, whereas chondroitin sulfate A/C and dermatan sulfate were undetectable in that derived from the normal colonic mucosa. An increment in the heparan sulfate content was also apparent in the adenocarcinoma, while the level of hyaluronic acid appeared to be unchanged.

Structure and biological activity of finback-whale (Balaenoptera physalus L.) heparin octasaccharide. Chemical, carbon-13 nuclear-magnetic-resonance, enzymic and biological studies.

Finback-whale (Balaenoptera physalus L.) heparin was partially digested with a purified heparinase and an octasaccharide with high affinity for antithrombin III was isolated from the digest by gel filtration, followed by affinity chromatography on a column of antithrombin III immobilized on Sepharose 4B. This octasaccharide possessed high inhibitory activity for Factor Xa in the presence of antithrombin III, but was essentially inactive for thrombin-antithrombin III reaction.

Screening study on excretion pattern of urinary glycosaminoglycans from orthopedic patients.

To obtain a clue for the metabolic disorder of glycosaminoglycans (GAG) in orthopedic diseases, a screening study on excretion pattern of urinary GAG from orthopedic patients was performed by the procedures of Nagatsuka et al. (1980). All the urines examined gave three regular GAG-bands.

Anticoagulant activity of heparin octasaccharide.

Porcine intestinal heparin was partially digested with a purified heparinase and an octasaccharide with high-affinity for antithrombin III was isolated from the digest by gel filtration, followed by affinity chromatography on a column of Sepharose 4B coupled with antithrombin III. The anticoagulant activity determined by the activated partial thromboplastin time method of the octasaccharide was 240 units/mg. Fifty percent inactivation activities of the octasaccharide for thrombin and factor Xa in the presence of antithrombin III were 2 and 6.

Comparative studies on the structures of highly active and relatively inactive forms of whale heparin.

Whale heparin was separated by affinity chromatography on an antithrombin III-Sepharose column into two distinct fractions. The high-affinity fraction accounted for most the anticoagulant activity of the unfractionated material, while the low-affinity fraction was relatively inactive. The yields of the two fractions were substantially equivalent.

Purification of heparinase and heparitinase by affinity chromatography on glycosaminoglycan-bound AH-Sepharose 4B.

Heparinase and heparitinase were separated from an extract of Flavobacterium heparinum, induced with heparin by using column chromatography on hydroxylapatite. As the heparinase preparation contained chondroitinases B and C, chondroitinase B was removed by rechromatography on a hydroxylapatite column. Chondroitinase C was then eliminated by column chromatography on O-phosphono("phospho")-cellulose.

A method of screening test for excretion pattern of urinary glycosaminoglycans and its application to normal human urine.

To obtain a clue for the metabolic disorder of glycosaminoglycans (GAG) in connective tissue diseases (CTD), a device of screening test for excretion pattern of urinary GAG was achieved. The crude GAG were separated from voluntary 100-20 ml urine by precipitation with cetylpyridinium chloride, followed by digestion with pronase P. The crude GAG were then separated by one-dimensional Separax (cellulose acetate membrane) electrophoresis in 0.

Glycans and glycoproteins in 0.05M LaCl3-soluble fraction from bovine costal cartilage.

The supernatant fraction (Fr. S) obtained by ten-fold dilution of the 0.5 M LaCl3-extract of bovine costal cartilage was fractionated by DEAE-Sephadex A-25 column chromatography, followed by gel filtration on Sepharose 4B.

High performance liquid chromatography of unsaturated disaccharides produced from chondroitin sulfates by chondroitinase.

High performance liquid chromatography was performed by the ion pair method on unsaturated disaccharides produced from chondroitin sulfates by the action of chondroitinase. Completely separated peaks corresponding to deltaDi-0S, deltaDi-4S, and deltaDi-6S were obtained on a column of mu-bondapak-C18 with 0.035 M tetra-butylammonium phosphate (pH 7.

Purification of chondroitinase B and chondroitinase C using glycosaminoglycan-bound AH-Sepharose 4B.

Chondroitinase B and chondroitinase C were separated from an extract of Flavobacterium heparinum induced with chondroitin 6-sulfate by using column chromatography on hydroxylapatite. Chondroitinase C was eluted together with the activities of hyaluronidase, delta4,5glycosiduronase, and sulfatase. The latter two activities were eliminated exclusively by passing the crude chondroitinase C fraction through a phosphono-cellulose column pre-equilibrated with 0.