A sulphated triterpenoid saponin from Schefflera octophylla.

Dried leaves of Schefflera octophylla afforded a new sulphated triterpene glycoside. From spectroscopic data and chemical transformations the structure of the new constituent was determined as 3-epi-betulinic acid 3-O-sulphate 28-O-[alpha-L-rhamnopyranosyl(1----4)-O-beta-D-glucopyranosyl (1----6)]-beta-D-glucopyranoside.     

Molecular organization of heparan sulphate from human skin fibroblasts.

The molecular structure of human skin fibroblast heparan sulphate was examined by specific chemical or enzymic depolymerization and high-resolution separation of the resulting oligosaccharides and disaccharides. Important features of the molecular organization, disaccharide composition and O-sulphate disposition of this heparan sulphate were identified. Analysis of the products of HNO2 hydrolysis revealed a polymer in which 53% of disaccharide units were N-acetylated and 47% N-sulphated, with an N-/O-sulphate ratio of 1.8:1. These two types of disaccharide unit were mainly located in separate domains. Heparitinase and heparinase scission indicated that the iduronate residues (37% of total hexuronate) were largely present in contiguous disaccharide sequences of variable size that also contained the majority of the N-sulphate groups. Most of the iduronate residues (approx. 70%) were non-sulphated. About 8-10% of disaccharide units were cleaved by heparinase, but only a minority of these originated from contiguous sequences in the intact polymer. Trisulphated disaccharide units [alpha-N-sulpho-6-sulphoglucosaminyl-(1----4)-iduronate 2-sulphate], which are the major structural units in heparin, made up only 3% of the disaccharide units in heparan sulphate. O-Sulphate groups (approx. 26 per 100 disaccharide units) were distributed almost evenly among C-6 of N-acetylglucosamine, C-2 of iduronate and C-6 of N-sulphated glucosamine residues. The results indicate that the sulphated regions of heparan sulphate have distinctive and potentially variable structural characteristics. The high content of non-sulphated iduronate in this heparan sulphate species suggests a conformational versatility that could have important implications for the biological properties of the polymer.     

An anticoagulant fucoidan from the brown seaweed Ecklonia kurome.

The structure of a alpha-L-fucose-rich, sulphated polysaccharide (C-I) with a potent anticoagulant activity, which was isolated from the brown seaweed Ecklonia kurome, has been studied. Methylation analysis showed that C-I consisted mainly of 3-linked and 3,4-disubstituted fucopyranosyl residues in addition to non-reducing terminal fucofuranosyl and fucopyranosyl residues, 2,3-di- and 2,3,4-tri-substituted fucopyranosyl residues and galactopyranosyl residues with various glycosidic linkages. Methanolysis of C-I gave neutral di-, tri-, tetra- and highly polymerized-oligosaccharide fractions. GC-MS and methylation analysis indicated that di- and trisaccharide fractions consisted mainly of Fuc-(1----3)-Fuc and Fuc-(1----3)-Fuc-(1----3)-Fuc, respectively, in addition to small amounts of Fuc-(1----4)-Fuc, Fuc-(1----4)-Gal and Fuc-(1----3)-[Fuc-(1----2)-]Fuc. When methylated C-I was subjected to methanolysis for desulphation followed by remethylation with deuterated methyl iodide, most of deuteriomethyl groups substituted to position 4 of 3-linked Fuc.     

Rapid catabolism of tyrosine-O-sulphated proteins and the formation of free tyrosine O-sulphate as an end product in rat embryo fibroblasts.

Rat embryo fibroblasts, line 3Y1, were prelabelled for 24 h with [35S]sulphate and incubated in fresh medium without [35S]sulphate. A rapid efflux of the overall 35S-labelled compounds from the cells into the medium was observed. After 9 h of incubation, about 50% of the total 35S radioactivity appeared in the medium and up to 84.3% did so at the end of a 48 h incubation. Determination of [35S]sulphated macromolecules present in both the cell-associated and the incubation-medium fractions at different time points during incubation indicated that the majority of the 35S-labelled compounds released from the cells were low-Mr products derived from digestion of the [35S]sulphated macromolecules. Further analysis for tyrosine-O-[35S]sulphated proteins, which constituted only a small fraction of the overall [35S]sulphated macromolecules, showed that, after 9 h of incubation, there was a 65% decrease in the cell-associated fraction, and only 16.4% remained after 48 h. During that time, an amount equivalent to 20.7% of the cell-associated tyrosine-O-[35S]sulphated proteins originally present was released into the medium. Free tyrosine O-[35S]sulphate was generated in the cells and excreted into the incubation medium. Its rate of increase with time, however, was slow, and could account for only 12.4% of the tyrosine-O-[35S]sulphated proteins catabolized at the end of the 48 h incubation.     

Synthesis of glycosaminoglycans by human skin fibroblasts cultured on collagen gels.

A comparison has been made of the synthesis of glycosaminoglycans by human skin fibroblasts cultured on plastic or collagen gel substrata. Confluent cultures were incubated with [3H]glucosamine and Na235SO4 for 48h. Radiolabelled glycosaminoglycans were then analysed in the spent media and trypsin extracts from cells on plastic and in the medium, trypsin and collagenase extracts from cells on collagen gels. All enzyme extracts and spent media contained hyaluronic acid, heparan sulphate and dermatan sulphate. Hyaluronic acid was the main 3H-labelled component in media and enzyme extracts from cells on both substrata, although it was distributed mainly to the media fractions. Heparan sulphate was the major [35S]sulphated glycosaminoglycan in trypsin extracts of cells on plastic, and dermatan sulphate was the minor component. In contrast, dermatan sulphate was the principal [35S]sulphated glycosaminoglycan in trypsin and collagenase extracts of cells on collagen gels. The culture substratum also influenced the amounts of [35S]sulphated glycosaminoglycans in media and enzyme extracts. With cells on plastic, the medium contained most of the heparan sulphate (75%) and dermatan sulphate (> 90%), whereas the collagenase extract was the main source of heparan sulphate (60%) and dermatan sulphate (80%) from cells on collagen gels; when cells were grown on collagen, the medium contained only 5-20% of the total [35S]sulphated glycosaminoglycans. Depletion of the medium pool was probably caused by binding of [35S]sulphated glycosaminoglycans to the network of native collagen fibres that formed the insoluble fraction of the collagen gel. Furthermore, cells on collagen showed a 3-fold increase in dermatan sulphate synthesis, which could be due to a positive-feedback mechanism activated by the accumulation of dermatan sulphate in the microenvironment of the cultured cells. For comparative structural analyses of glycosaminoglycans synthesized on different substrata labelling experiments were carried out by incubating cells on plastic with [3H]glucosamine, and cells on collagen gels with [14C]glucosamine. Co-chromatography on DEAE-cellulose of mixed media and enzyme extracts showed that heparan sulphate from cells on collagen gels eluted at a lower salt concentration than did heparan sulphate from cells on plastic, whereas with dermatan sulphate the opposite result was obtained, with dermatan sulphate from cells on collagen eluting at a higher salt concentration than dermatan sulphate from cells on plastic. These differences did not correspond to changes in the molecular size of the glycosaminoglycan chains, but they may be caused by alterations in polymer sulphation.     

Regulation of heparan sulphate metabolism by adenosine 3′:5′-cyclic monophosphate in hepatocytes in culture

Freshly isolated rat hepatocytes maintained as monolayers in a serum-free medium synthesize sulphated glycosaminoglycans, most of which behave as heparan sulphate and are mainly distributed into intracellular compartments. Cyclic AMP, dibutyryl cyclic AMP, glucagon, noradrenaline, prostaglandin E(1), and theophylline, all drugs and hormones known to increase intracellular cyclic AMP concentrations, decreased the incorporation of (35)SO(4) (2-) into heparan sulphate of intra-, extra- and peri-cellular pools. The inhibition mediated by dibutyryl cyclic AMP was dose-dependent and observed as early as 2h after exposure to the drug. In the presence of 1mm-dibutyryl cyclic AMP, incorporation of (35)SO(4) (2-) or [(14)C]glucosamine into heparan sulphate was decreased to 40-50%, suggesting that dibutyryl cyclic AMP interfered with the synthesis of heparan sulphate. This was further supported by pulse-chase experiments, where dibutyryl cyclic AMP had no effect on the degradation of sulphated glycosaminoglycans. Heparan sulphates synthesized and secreted into the extracellular pool in the presence of dibutyryl cyclic AMP were smaller in size, whereas the degree of sulphation and molecular size of the heparan sulphate chains released by beta-elimination from these proteoglycans were not different from control values. In the presence of 1mm-cycloheximide, (35)SO(4) (2-) incorporation was decreased to 5%. Addition of p-nitrophenyl beta-d-xyloside, an artificial acceptor of glycosaminoglycan chain synthesis, enhanced this incorporation to 18%. Dibutyryl cyclic AMP did not have any inhibitory effect on the synthesis of chains initiated on p-nitrophenyl beta-d-xylosides. Incorporation of [(3)H]serine into heparan sulphate was not affected by dibutyryl cyclic AMP, whereas the degree of substitution of serine residues with heparan sulphate chains was less in heparan sulphate synthesized in the presence of dibutyryl cyclic AMP, suggesting that cyclic AMP exerts its effect on the metabolism of sulphated glycosaminoglycans by affecting the transfer of xylose on to the protein core.     

Biochemical studies on the sulphated glycosaminoglycan fraction of skin fibroblasts cultured from a patient with the Hurler syndrome

1. The metabolism of the sulphated glycosaminoglycan fraction in cultured skin fibroblasts derived from a patient with the Hurler syndrome and from a normal subject was studied. Two labelled precursors, Na(2) (35)SO(4) and d-[2-(3)H]glucose, were used and their intracellular fates during uptake and ;chase' periods were assessed after separation of sulphated glycosaminoglycans from hyaluronic acid. After 4 or 8h of exposure to culture medium containing both labels, [(35)S]sulphate incorporation into the sulphated glycosaminoglycan fraction was twofold greater in Hurler-syndrome cells than in normal cells. At the same time, the rate of incorporation of [(3)H]glucose into the sulphated glycosaminoglycan fraction was approximately the same for both cell types. Consequently, an increased (35)S/(3)H ratio (nmol of [(35)S]sulphate incorporated/nmol of [(3)H]glucose incorporated) was observed for Hurler-syndrome cells compared with normal cells. 2. The results of ;chase' experiments revealed that although the expected loss and relative retention of labelled sulphate occurred in the sulphated glycosaminoglycan fraction of normal and Hurler-syndrome cells, both cell types retained all of their radioactivity derived from [(3)H]glucose. 3. After 34h exposure to a ;corrective-factor' preparation from urine, the sulphated glycosaminoglycan content (as hexosamine and [(35)S]sulphate) of the Hurler-syndrome cells approached normal values. At the same time, there was an increase in specific radioactivity of ;corrected' Hurler-syndrome cells.     

The effect of growth factors on the cytotoxicity of sulphated polysaccharides

The cytotoxicity of sulphated polysaccharides on 3T3-L1 fibroblasts was investigated. Dextran sulphate with a molecular weight of more than 100000 and with a number of sulphate groups per sugar unit (degree of sulphation) of 2.4 showed strong cytotoxicity to 3T3-L1 fibroblasts. The synthetic (1→6)-- -mannopyranan sulphate having a degree of sulphation of more than 1.0 and with a molecular weight of approx. 100000 also exhibited cytotoxicity. The results indicated that the cytotoxicity of sulphated polysaccharides strongly depended on their molecular weight and degree of sulphation. FGF-1 (aFGF) and FGF-2 (bFGF) protected the cell from the damage caused by the sulphated polysaccharide, while PDGF, EGF, and HGF had no such effect. Therefore, it is suggested that the rescuing effect is one of the special biological activities, and is shown only by FGFs.     

Host plant-dependent metabolism of 4-hydroxybenzylglucosinolate in Pieris rapae: substrate specificity and effects of genetic modification and plant nitrile hydratase

After ingestion of transgenic Arabidopsis thaliana CYP79A1 containing sinalbin (4-hydroxybenzylglucosinolate) due to genetic modification, only one major sinalbin-derived sulphate ester (the sulphate ester of 4-hydroxyphenylacetonitrile) was excreted by Pieris rapae caterpillars (corresponding to 69mol% of ingested sinalbin). An additional sulphate ester (the sulphate ester of 4-hydroxyphenylacetamide) was excreted when the caterpillars were reared on two plant species (Sinapis alba and Sinapis arvensis) that contained sinalbin naturally. Artificial addition of sinalbin to S. arvensis leaves resulted in increased levels of the sulphated amide, and an enzymatic activity (nitrile hydratase) explaining the formation of the sulphated amide from sinalbin was detected in both Sinapis species, but not in A. thaliana. In agreement with the suggested minor metabolic pathway, the caterpillars were able to sulphate 4-hydroxyphenylacetamide offered as part of an artificial diet. In fact, phenol and seven para-substituted phenol derivatives with substituents of moderate size were sulphated and excreted, but all tested phenols devoid of a nitrile functional group were less efficiently sulphated than the primary sinalbin detoxification product, 4-hydroxyphenylacetonitrile. This suggests that the specificity of the sulphation step involved in sinalbin metabolism may be adapted to nitriles formed as metabolites of phenolic glucosinolates. On the contrary, there was no specificity for products (4-hydroxybenzylascorbigen and 4-hydroxybenzylalcohol) derived from the semistable isothiocyanate produced from sinalbin in the absence of nitrile specifier protein.     

D(+)catechin enhances heparan sulphate content in rat liver

Administration of (D+) catechin (100 mg/kg body wt) to rats resulted in an increase in the amount of total sulphated glycosaminoglycans (GAG) in liver. The increase was more pronounced in the case of heparan sulphate than chondroitin sulphate and dermatan sulphate. The liver slices prepared from catechin-treated rats showed a significant increase in the rate of incorporation of 35S-sulphate into GAG. Similarly there was a concentration-dependent increase in the rate of 35S-sulphate incorporation into GAG by normal liver slices in presence of catechin in vitro. Susceptibility to nitrous acid degradation and chondroitinase ABC digestion showed that more than 80% of the GAG labelled in vivo with 35S-sulphate, was heparan sulphate and about 10% chondroitin sulphate and dermatan sulphate. Gel filtration of the 35S-labelled material isolated from livers of normal and catechin-treated animals over sephacryl S-300 did not show any difference probably excluding the possibility of free GAG chains initiated on catechin or any of its metabolites in vivo. These results indicate that catechin stimulates the synthesis of sulphated GAG, particularly heparan sulphate in liver.     

Studies on the incorporation of [U-14C]glucose and [35S]sulphate into the acid glycosaminoglycans of neonatal rat skin

1. The incorporation of [(35)S]sulphate in vivo into the acid-soluble intermediates extracted from young rat skin showed three sulphated hexosamine-containing components. 2. The rates of synthesis of these components were determined in vivo by measuring the incorporation of radioactivity from [U-(14)C]glucose into their isolated hexosamine moieties. 3. The incorporation of radioactivity from [U-(14)C]glucose into the isolated hexosamine and uronic acid moieties of the acid glycosaminoglycans was also measured. These results, combined with those obtained on the intermediary pathways of hexosamine and uronic acid biosynthesis previously determined in this tissue, indicated that the acid-soluble sulphated hexosamine-containing components were not precursors of the sulphated hexosamine found in the acid glycosaminoglycans. 4. The rates of synthesis of the acid glycosaminoglycan fractions were calculated from the incorporation of radioactivity from [U-(14)C]glucose into the hexosamine moiety. The sulphated components containing principally dermatan sulphate, chondroitin 6-sulphate and in smaller amounts, chondroitin 4-sulphate, heparan sulphate and heparin appeared to be turning over about twice as rapidly as hyaluronic acid and about four times as rapidly as the small keratan sulphate fraction. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from the incorporation of [(35)S]sulphate and were in agreement with those from (14)C-labelling studies.     

Differential inhibition of polymorphonuclear leukocyte recruitment in vivo by dextran sulphate and fucoidan

The selectin-mediated rolling of leukocytes along the endothelial cells is a prerequisite step followed by firm adhesion and extravasation into the inflamed tissue. This initial contact can be suppressed by sulphated polysaccharides. We have studied the effect of sulphated polysaccharides on the ultimate polymorphonuclear leukocyte (PMN) recruitment and plasma leakage in rabbit skin in response to intradermal injection of various inflammatory mediators. PMN infiltration evoked by various PMN chemoattractants (FMLP, C5a desArg, LTB(4) and IL-8) was significantly inhibited after intravenous injection of dextran sulphate (25 mg/kg), heparin (2 x 90 mg/kg) or fucoidan (1 mg/kg). PMN-dependent plasma leakage was equally well reduced by the different sulphated polymers. Vascular permeability induced by histamine or thrombin acting via a PMN-independent mechanism was not reduced. Fucoidan was the only polysaccharide able to suppress IL-1-induced PMN infiltration for 60-70%. Local administration of dextran sulphate had no effect on PMN-dependent plasma leakage. Differential inhibition of PMN recruitment was determined after injection of dextran sulphate or fucoidan depending on the type of insult. Therefore, these results suggest that different adhesion pathways are utilized during PMN recruitment in vivo in response to chemoattractants and IL-1.     

The synthesis of glycosaminoglycans by cultures of rabbit corneal endothelial and stromal cells.

Confluent monolayer cultures of rabbit corneal endothelial and stromal cells were incubated independently with [35S]sulphate and [3H]glucosamine for 3 days. AFter incubation, labelled glycosaminoglycans were isolated from the growth medium and from a cellular fraction. These glycosaminoglycans were further characterized by DEAE-cellulose column chromatography and by sequential treatment with various glycosamino-glycan-degrading enzymes. Both endothelial and stromal cultures synthesized hyaluronic acid as the principal product. The cell fraction from the stromal cultures, however, had significantly less hyaluronic acid than that from the endothelial cultures. In addition, both types of cells synthesized a variety of sulphated glycosaminoglycans. The relative amounts of each sulphated glycosaminoglycan in the two cell lines were similar, with chondroitin 4-sulphate, chondroitin 6-sulphate and dermatan sulphate as the major components. Heparan sulphate was present in smaller amounts. Keratan sulphate was also identified, but only in very small amounts (1-3%). The presence of dermatan sulphate and the high content of hyaluronic acid are similar to the pattern of glycosaminoglycans seen in regenerating or developing tissues, including cornea.     

Isolation and characterization of sulphated oligosaccharides released from bovine corneal keratan sulphate by the action of endo-beta-galactosidase

A series of oligosaccharides has been isolated from the keratan sulphate peptidoglycan (3 M NaCl fraction) of bovine cornea after digestion with the endo-beta-galactosidase of Bacteroides fragilis. Structural information on the major oligosaccharides was obtained from (a) their susceptibilities to endo-beta-galactosidase before and after desulphation, (b) their elution positions on a column of Bio-Gel P-4 and retention times on a high-performance anion-exchange column and (c) negative-ion fast-atom-bombardment mass spectrometry. More than 75% of the oligosaccharides were sulphated unbranched poly(N-acetyllactosamine) sequences, (-3/4GlcNAc beta 1-3Gal beta 1-)n, and approximately 3% was the neutral disaccharide, GlcNAc beta 1-3Gal. The sulphated disaccharide, GlcNAc-SO-3 beta 1-3Gal, accounted for almost 35% of the oligosaccharide material while 40% consisted of four oligosaccharides, unbranched tetra-, hexa-, octa- and decasaccharides of poly(N-acetyllactosamine) type, having 3, 5, 7 and 9 sulphate residues respectively. Proton nuclear magnetic resonance studies at 500 MHz (Hounsell, E. F., et al. following paper in this journal) have shown that a sulphate residue is attached to the C-6 position of each N-acetylglucosamine and each internal galactose residue of these four oligosaccharides which express to varying degrees the antigenic determinants recognised by three monoclonal antibodies to keratan sulphate (Mehmet, H. et al., paper which follows the next paper in this journal).     

The antigenic determinants recognized by three monoclonal antibodies to keratan sulphate involve sulphated hepta- or larger oligosaccharides of the poly(N-acetyllactosamine) series

The carbohydrate determinants of keratan sulphate recognized by three monoclonal antibodies (5-D-4, 1-B-4 and MZ15) have been investigated by solid-phase radioimmunoassay using bovine corneal keratan sulphate as the immobilized reference antigen. The antibodies appeared highly specific for sulphated poly(N-acetyllactosamine) sequences, for their binding was strongly inhibited by preparations of keratan sulphate, but not by glycoproteins with non-sulphated poly(N-acetyllactosamine) sequences of I and i antigen types, a desulphated keratan sulphate hexasaccharide, an array of neutral and sulphated mono- and disaccharides and other glycosaminoglycans. Inhibition of binding assays using a series of structurally characterized sulphated di, tetra-, hexa-, octa- and decasaccharides, and partially characterized larger oligosaccharides, isolated from bovine corneal keratan sulphate after digestion with endo-beta-galactosidase (see preceding two papers in this journal) showed that the smallest oligosaccharide reactive with all three antibodies was the linear pentasulphated hexasaccharide, E-II although antibody 1-B-4 reacted with a tetrasulphated analogue. The heptasulphated octasaccharide, G-III, was more active; among the structurally characterized keratan sulphate oligosaccharides the nonasulphated decasaccharide, I-IV, was the most active. Thus, the hepta- and octasaccharide sequences, indicated by brackets below are proposed as candidate antigenic structures recognized by the three monoclonal antibodies. (Formula: see text). Antibody 5-D-4 differs from the other two antibodies in reacting relatively strongly with a minor oligosaccharide which chromatographs as a hexasulphated octasaccharide, G-I, and most strongly with a minor sulphated, linear dodecasaccharide, J-II, which has been partially characterized [Tang, P.W., Scudder, P., Mehmet, H., Hounsell, E. F. & Feizi, T., unpublished results] and may contain N-sulphated glucosamine residues.     

ISOLATION AND CHARACTERIZATION OF GLYCOSAMINOGLYCANS FROM BRAIN OF CHILDREN WITH PROTEIN-CALORIE MALNUTRITION

Abstract— The uronic acid containing glycosaminoglycans (GAGs) were isolated from the brains of 1-year-old and 4-year-old kwashiorkor children and characterised by constituent analyses. A marked reduction is the total GAG concentration of brain was noticed in both cases of kwashiorkor. In the 1-year-old kwashiorkor brain, hyaluronic acid is the most predominant GAG (73.5 per cent) whereas heparan sulphate, chondroitin sulphates and low sulphated chondroitin sulphate constituted less than 10 per cent. In the 4-year-old kwashiorkor brain, the proportion of hyaluronic acid was 27.5 per cent, low sulphated chondroitin sulphate 31.2 per cent, chondroitin sulphates 28.3 per cent and heparan sulphate 10 per cent. This marked reduction in the concentration as well as qualitative changes in GAG in protein-calorie malnutrition as compared to the normal is discussed in relation to brain function.     

Types of oligosaccharide sulphation, depending on mucus glycoprotein source, corpus or antral, in rat stomach.

Radiolabelled mucus glycoprotein was obtained from tissue and a culture medium each of the corpus and antrum of rat stomach incubated with [35S]sulphate in vitro. Gel-filtration analysis of oligosaccharides liberated by alkaline-borohydride treatment from glycoproteins indicated that 35S-labelled oligosaccharides from the corpus vary considerably with respect to chain length whereas those from antral mucus glycoprotein are composed of small oligosaccharides. Examination of the reduced radiolabelled products obtained by HNO2 cleavage of the hydrazine-treated oligosaccharides indicated sulphate esters of N-acetylglucosamine to be present at three locations on a carbohydrate unit: [35S]sulphated monosaccharide (2,5-anhydromannitol 6-sulphate), [35S]sulphated disaccharide [galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate] and [35S]sulphated trisaccharide [fucosyl(alpha 1-2)-galactosyl(beta 1-4)-2,5-anhydromannitol 6-sulphate]. Sulphated disaccharide and trisaccharide, possibly originating from the N-acetyl-lactosamine and fucosyl-N-acetyl-lactosamine sequences respectively, were detected in the corpus, especially as large oligosaccharides, but were present in the antrum in only very small amounts. The sulphated monosaccharide, however, most probably originating from 6-sulphated N-acetylglucosamine residues at non-reducing termini, was present in all oligosaccharide fractions in both the corpus and antrum.     

Co-purification of galactosyltransferases from chick-embryo liver.

Two galactosyltransferases with nearly identical Mr values were purified 5000-7000-fold from microsomal membranes of chick-embryo livers by using several affinity columns. One enzyme transfers galactose from UDP-galactose to form a beta-(1----4)-linkage to GlcNAc (N-acetylglucosamine) or AsAgAGP [asialo-agalacto-(alpha 1-acid glycoprotein)]. The other enzyme forms a beta-(1----3)-linkage to AsOSM [asialo-(ovine submaxillary mucin)]. Both enzymes were solubilized (85%) from a microsomal pellet by using 1% Triton X-100 in 0.1 M-NaCl. The supernatant activities were subjected to DEAE-Sepharose chromatography and four affinity columns: UDP-hexanolamine-Sepharose, alpha-lactalbumin-Sepharose, GlcNAc-Sepharose and either AsAgAGP-Sepharose or AsOSM-Sepharose. The AsAgAGP enzyme [(1----4)-transferase] and the AsOSM enzyme [(1----3)-transferase] behave identically on the DEAE-Sepharose and UDP-hexanolamine-Sepharose columns, and similarly on the alpha-lactalbumin-Sepharose column. Final separation of the two enzymes, however, could only be achieved on affinity columns of their immobilized respective acceptors. Both purified enzymes migrate as a single band on sodium dodecyl sulphate/polyacrylamide-gel electrophoresis after silver staining, and both have an apparent Mr of 68 000. The enzymes were radioiodinated and again subjected to sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Radioautographic analyses showed only one, intensely radioactive, band. Activity stains performed for both transferases after cellulose acetate electrophoresis indicate that, with this system too, both activities have identical mobilities, and co-migrate, as well, with the major, silver-stained, protein band. Kinetic studies with the purified enzymes show that the Km value for GlcNAc, for the (1----4)-transferase, is 4mM; for the (1----3)-transferase the Km value for AsOSM is 5mM, in terms of GalNAc (N-acetylgalactosamine) equivalents. Both enzymes have a Km value of 25 microM for UDP-galactose.     

Antiangiogenic effect of sulphated and nonsulphated glycosaminoglycans and polysaccharides in the chick embryo chorioallantoic membrane

The inhibiting effect of sulphated and nonsulphated glycosaminoglycans and polysaccharides on the normal outgrowth of capillaries was tested in the chick embryo chorioallantoic membrane (CAM) with and without the presence of hydrocortisone. An antiangiogenic response to 50 µg of heparin and heparan sulphate (without hydrocortisone present) was observed in 38.8% and 23.1% of the CAMS, respectively, while the antiangiogenic response rate for dermatan sulphate, chondroitin sulphate A or C, hyaluronic acid and keratan sulphate was 15.9–0%. All sulphated homopolysaccharides tested were more effective than the naturally occurring glycosaminoglycans. Nonsulphated dextran and (methyl) cellulose had no antiangiogenic effect, while largely desulphated heparin retained such an effect. Hydrocortisone generally improved the antiangiogenic effect, a 100% response was obtained when it was combined with cellulose sulphate or fucoidan (polyfucose sulphate derived from marine algae), but the antiangiogenic effect of the largely desulphated heparin was unaffected by the presence of hydrocortisone. The results show that different polysulphated polysaccharides also have an antiangiogenic effect, without the addition of corticosteroids. The effect was apparently independent of their degree of sulphation, but the glycosidic structure may be of critical importance.     

Initiation of altered heparan sulphate on beta-D-xyloside in rat hepatocytes

Inhibition of protein synthesis by cycloheximide 10(-3)M reduced the incorporation of [35S]sulphate into heparan sulphate to about 5% of untreated hepatocytes. Addition of rho-nitrophenyl beta-D-xyloside could partially revert this inhibitory effect. The sulphated material isolated from the cell layer or secretions of hepatocytes grown in presence of cycloheximide and rho-nitrophenyl beta-D-xyloside were shown to be mostly free heparan sulphate chains not bound to core protein. Covalent association of beta-xylosides to the heparan sulphates was demonstrated for heparan sulphate synthetized in the presence of [35S]sulphate, cycloheximide and the fluorogenic 4-methylumbelliferyl beta-D-xyloside. Beta-Xylosides served as an initiator of heparan sulphate chain synthesis in rat hepatocytes only in the absence of protein synthesis. Heparan sulphates primed on artificial beta-xylosides are slightly smaller in molecular size and are more sulphated than chains linked to core protein.