Leishmania donovani: cell-surface heparin receptors of promastigotes are recruited from an internal pool after trypsinization

With the use of [3H]heparin, we recently demonstrated that Leishmania donovani promastigotes express a cell-surface receptor that is specific for the glycosaminoglycan heparin (Mukhopadhyay et al. 1989, The Biochemical Journal, 264, 517-525.). Treatment of the parasite with trypsin abolishes 75-90% of this [3H]heparin-binding activity. When trypsinized promastigotes were resuspended in fresh culture medium in the absence and presence of cycloheximide (10 micrograms/ml), approximately 25-30% of the original heparin-binding capacity was restored within 1 hr, indicating that recruitment of receptors from an internal pool occurred without de novo protein synthesis. Scatchard analysis of the regenerated receptor revealed that the number of regenerated binding sites per cell was 2.3 x 10(5); these sites have a binding affinity of 6.7 x 10(-7) M. Like the native heparin receptors on the surface of freshly isolated cells, the receptors recruited after trypsinization are also highly specific for heparin, as a 25-fold excess of four other glycosaminoglycans displaced less than 10% of bound [3H]heparin from the trypsinized cells. The structural requirements of the ligand heparin, namely the number of monosaccharide units and degree of sulfation, were compared for both the native and regenerated receptor: for both receptors, oversulfated polysaccharide heparin fragments of at least six to eight sugar residues were most efficient at displacing [3H]heparin. The concentrations of oligosaccharide fragments required to displace 50% of [3H]heparin were 0.32 and 0.035 microM for the hexa- and octasaccharides, respectively. Colloidal gold-labeled heparin was bound to promastigotes and visualized by electron microscopy. This analysis revealed that the heparin bound almost exclusively to the flagella of control cells (not subjected to trypsin) and those which had regenerated receptor after trypsinization. The physiological significance of this heparin-binding activity on the surface of promastigotes is discussed.     

Biosynthesis of glycosaminoglycans by cultured mastocytoma cells

Biosynthesis of glycosaminoglycans by several lines of cultured neoplastic mouse mast cells was studied by incorporation of [³⁵S]sulphate (and in some cases [6-³H]glucosamine) into macromolecular materials found in both the cells and their growth media. Such intracellular and extracellular radioactively labelled materials (shown to be glycosaminoglycans by susceptibility to digestion with heparinase) were further characterized by ion-exchange chromatography and by digestion with testicular hyaluronidase and chondroitinase. All but one cell line produced chondroitin sulphate as the major sulphated glycosaminoglycan; the remainder of the glycosaminoglycan was heparin-like material. No [³H]hyaluronic acid was synthesized. Cells of a newly derived line, termed P815S, synthesized more glycosaminoglycan than the other lines. This glycosaminoglycan, found in both cells and growth medium, was almost entirely chondroitin 4-sulphate. No chondroitin 6-sulphate was found. The chondroitin 4-sulphate from the cells was shown by gel filtration to be smaller than the chondroitin 4-sulphate in the media of these cultures. This discovery of relatively high proportions of chondroitin 4-sulphate in these mastocytoma-derived cells is noteworthy, since mast cells have generally been considered to produce heparin as their major glycosaminoglycan.     

Degradation of [3H]chondroitin 4-sulphate and re-utilization of the [3H]hexosamine component by the isolated perfused rat liver.

Radiolabelled chondroitin 4-sulphate was isolated after incubation of rat rib cartilage with N-acetyl-D-[6-3H]galactosamine. After proteolytic digestion of the tissue with either papain or trypsin the released [3H]chondroitin 4-sulphate was added to an isolated perfused rat liver system. Analysis of perfusate after several hours perfusion showed that radiolabelled amino sugars were secreted by the liver in a low-molecular-weight form and as components of glycoproteins.     

Diagnosis of Maroteaux-Lamy syndrome by the use of radiolabelled oligosaccharides as substrates for the determination of arylsulphatase B activity.

The kinetic parameters (Km and V) of human arylsulphatase B (4-sulpho-N-acetylgalactosamine sulphatase) activity in cultured skin fibroblasts were determined with a variety of substrates matching structural aspects of the physiological substrates in vivo chondroitin 4-sulphate and dermatan sulphate. More structurally complex substrates, in which several aspects of the aglycone structure of the natural substrate were maintained, were desulphated up to 4400 times faster than the minimum arylsulphatase-B-specific substrate, namely the monosaccharide N-acetylgalactosamine 4-sulphate. Aglycone structures that influence substrate binding and/or enzyme activity were an adjacent-residue C-6 carboxy group and a second but internal N-acetylgalactosamine 4-sulphate residue. Arylsulphatase B activity in fibroblast homogenates assayed with O-(beta-N-acetylgalactosamine 4-sulphate)-(1----4)-O-D-(beta-glucuronic acid)-(1----3)-O-D-N-acetyl[1-3H] galactosaminitol 4-sulphate derived from chondroitin 4-sulphate as substrate clearly distinguished Maroteaux-Lamy-syndrome patients from normal controls and other mucopolysaccharidosis patients. We recommend the use of the above trisaccharide substrate for both postnatal and prenatal diagnosis of Maroteaux-Lamy syndrome.     

Action pattern of polysaccharide lyases on glycosaminoglycans

The action pattern of polysaccharide lyases on glycosaminoglycansubstrates was examined using viscosimetric measurements andgradient polyacrylamide gel electrophoresis (PAGE). Heparinlyase I (heparinase, EC 4.2.2.7 [EC] ) and heparin lyase II (no ECnumber) both acted on heparin in a random endolytic fashion.Heparin lyase II showed an ideal endolytic action pattern onheparan sulphate, while heparin lyase I decreased the molecularweight of heparan sulphate more slowly. Heparin lyase III (heparitinase,EC 4.2.2.8 [EC] ) acted endolytically only on heparan sulphate anddid not cleave heparin. Chondroitin ABC lyase (chondroitinaseABC, EC 4.2.2.4 [EC] ) from Proteus vulgaris acted endolytically onchondroitin-6-sulphate (chondroitin sulphate C) and dermatansulphate at nearly identical initial rates, but acted on chondroitin-4-sulphate(chondroitin sulphate A) at a reduced rate, decreasing its molecularweight much more slowly. Two chondroitin AC lyases (chondroitinaseAC, both EC 4.2.2.5 [EC] ) were examined towards chondroitin-4- and-6-sulphates. The exolytic action of chondroitin AC lyase Afrom Arthrobacter aurescens on both chondroitin-4- and -6-sulphateswas demonstrated viscosimetrically and confirmed using bothgradient PAGE and gel permeation chromatography. ChondroitinAC lyase F from Flavobacterium heparinum (Cytophagia heparinia)acted endolytically on the same substrates. Chondroitin B lyase(chondroitinase B, no EC number) from F.heparinum acted endolyticallyon dermatan sulphate giving a nearly identical action patternas observed for chondroitin ABC lyase acting on dermatan sulphate. action pattern chondroitin lyase glycosaminoglycan heparin lyase.     

Human liver glucuronate 2-sulphatase. Purification, characterization and catalytic properties.

Human glucuronate 2-sulphatase (GAS), which is involved in the degradation of the glycosaminoglycans heparan sulphate and chondroitin 6-sulphate, was purified almost 2,000,000-fold to homogeneity in 8% yield from liver with a four-step six-column procedure, which consists of a concanavalin A-Sepharose/Blue A-agarose coupled step, a DEAE-Sephacel/octyl-Sepharose coupled step, CM-Sepharose chromatography and gel-permeation chromatography. Although more than 90% of GAS activity had a pI of greater than 7.5, other forms with pI values of 5.8, 5.3, 4.7 and less than 4.0 were also present. The pI greater than 7.5 form of GAS had a native molecular mass of 63 kDa. SDS/polyacrylamide-gel-electrophoretic analysis resulted in two polypeptide subunits of molecular mass 47 and 19.5 kDa. GAS was active towards disaccharide substrates derived from heparin [O-(beta-glucuronic acid 2-sulphate)-(1----4)-O-(2,5)-anhydro[1-3H]mannitol 6-sulphate (GSMS)] and chondroitin 6-sulphate [O-(beta-glucuronic acid 2-sulphate-(1----3)-O-(2,5)-anhydro[1-3H]talitol 6-sulphate (GSTS)]. GAS activity towards GSMS and GSTS was at pH optima of 3.2 and 3.0 respectively with apparent Km values of 0.3 and 0.6 microM respectively and corresponding Vmax values of 12.8 and 13.7 mumol/min per mg of protein respectively. Sulphate and phosphate ions are potent inhibitors of enzyme activity. Cu2+ ions stimulated, whereas EDTA inhibited enzyme activity. It was concluded that GAS is required together with a series of other exoenzyme activities in the lysosomal degradation of glycosaminoglycans containing glucuronic acid 2-sulphate residues.     

Comparative study on glycosaminoglycans synthesized in peripheral and peritoneal polymorphonuclear leucocytes from guinea pigs.

Glycosaminoglycans synthesized in polymorphonuclear (PMN) leucocytes isolated from blood (peripheral PMN leucocytes) and in those induced intraperitoneally by the injection of caseinate (peritoneal PMN leucocytes) were compared. Both peripheral and peritoneal PMN leucocytes were incubated in medium containing [35S]sulphate and [3H]glucosamine. Each sample obtained after incubation was separated into cell, cell-surface and medium fractions by trypsin digestion and centrifugation. The glycosaminoglycans secreted from peripheral and peritoneal PMN leucocytes were decreased in size by alkali treatment, indicating that they existed in the form of proteoglycans. Descending paper chromatography of the unsaturated disaccharides obtained by the digestion of glycosaminoglycans with chondroitinase AC and chondroitinase ABC identified the labelled glycosaminoglycans of both the cell and the medium fractions in peripheral PMN leucocytes as 55-58% chondroitin 4-sulphate, 16-19% chondroitin 6-sulphate, 16-19% dermatan sulphate and 6-8% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found only in the medium fraction. In peritoneal PMN leucocytes there is a difference in the composition of glycosaminoglycans between the cell and the medium fractions; the cell fraction was composed of 60% chondroitin 4-sulphate, 5.5% chondroitin 6-sulphate, 16.8% dermatan sulphate and 13.9% heparan sulphate, whereas the medium fraction consisted of 24.5% chondroitin 4-sulphate, 28.2% chondroitin 6-sulphate, 33.7% dermatan sulphate and 10% heparan sulphate. Oversulphated chondroitin sulphate and oversulphated dermatan sulphate were found in the cell, cell-surface and medium fractions. On the basis of enzymic assays with chondro-4-sulphatase and chondro-6-sulphatase, the positions of sulphation in the disulphated disaccharides were identified as 4- and 6-positions of N-acetylgalactosamine. Most of the 35S-labelled glycosaminoglycans synthesized in peripheral PMN leucocytes were retained within cells, whereas those in peritoneal PMN leucocytes were secreted into the culture medium. Moreover, the amount of glycosaminoglycans in peritoneal PMN leucocytes was significantly less than that in peripheral PMN leucocytes. Assay of lysosomal enzymes showed that these activities in peritoneal PMN leucocytes were 2-fold higher than those in peripheral PMN leucocytes.     

Interaction of rat liver glucocorticoid receptor with heparin

When rat liver cytosol containing [3H]dexamethasone-glucocorticoid receptor complex is exposed to immobilized heparin (Sepharose-heparin; Seph-hep) the steroid receptor complex binds to the substituted Sepharose avidly [Kd = 3.5 (+/- 1.7) X 10(-10) M], and 80-90% of the receptor present is adsorbed to the solid phase after 40 min at 0 degree C. The binding is enhanced by Mn2+ (10 mM) and Mg2+, whereas Ca2+ and Sr2+ are ineffective. Sodium molybdate (10 mM) does not influence the reaction but enhances receptor stability. Moreover, binding of the receptor to Seph-hep is dependent on the ionic strength of the medium, because binding is totally reversed by 300 mM KCl. The bound [3H]dexamethasone-receptor complex can be recovered from Seph-hep with solutions (4 mg/mL) of heparin (95% release), dextran sulfate (88%), and chondroitin sulfate (63%); total calf liver RNA is less effective (9%), whereas dextran, D-glucosamine, N-acetyl-D-glucosamine, D-glucuronic acid, and sheared calf thymus DNA are totally ineffective (less than 3%). Both "native" and temperature "transformed" forms of the glucocorticoid receptor interact with immobilized heparin. These results strongly suggest that the receptor site that binds heparin is distinct from that binding DNA. An immediate application of this newly found ability of the glucocorticoid receptor to interact with heparin is the use of Seph-hep for affinity chromatography purification of the glucocorticoid receptor. A purification of 10-fold, with a recovery of 55-65%, can be achieved by using either 4 mg/mL heparin or 300 mM KCl to elute [3H]dexamethasone-receptor bound to the resin.     

Selective binding of zinc ions to heparin rather than to other glycosaminoglycans.

The relative binding affinity of Zn2+ to several glycosaminoglycans was determined by gel-filtration chromatography. Binding was observed only between Zn2+ and heparin. No binding was observed between Zn2+ and chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate of hyaluronic acid. All of the glycosaminoglycans contained carboxy groups, but only heparin bound Zn2+. This observation suggests that, contrary to a previously proposed hypothesis, simple electrostatic interactions between the negatively charged carboxy groups of the glycosaminoglycans and the positively charged Zn2+ cannot explain the observed binding.     

A sulfatase specific for glucuronic acid 2-sulfate residues in glycosaminoglycans

Although 2-O-sulfated L-iduronic acid (IdoA) residues have been known to occur in heparin, 2-O-sulfated D-glucuronic acid (GlcA) residues have been reported only recently (Bienkowski, M. J., and Conrad, H. E. (1985) J. Biol. Chem. 250, 356-365). Disaccharides prepared by cleavage of heparin and N-deacetylated chondroitin 6-sulfate with nitrous acid were used to demonstrate a new sulfatase that catalyzed the removal of the 2-O-sulfate substituents from GlcA but not IdoA residues. The deamination products were labeled by NaB3H4 reduction to give disaccharides from heparin and chondroitin sulfate which had reducing terminal 2,5-anhydro-D-mannitol ([3H]AManR) and 2,5-anhydro-D-talitol ([3H]ATalR) residues, respectively. IdoA(2-SO4)-[3H]AManR(6-SO4) from heparin and GlcA(2-SO4)-[3H]ATalR(6-SO4) from chondroitin sulfate were purified for use as substrates. GlcA(2-SO4)-[3H]AManR(6-SO4) was prepared by epimerization of IdoA(2-SO4)-[3H]AManR(6-SO4) with hydrazine at 100 degrees C. Lysosomal enzyme preparations from chick embryo chondrocytes and from two normal human fibroblast cell lines catalyzed the removal of the 2-O-SO4 substituent from the uronic acid residues of IdoA(2-SO4)-[3H]AManR(6-SO4), GlcA(2-SO4)-[3H] AManR(6-SO4), and GlcA(2-SO4)-[3H]ATalR(6-SO4). In contrast, a lysosomal enzyme preparation from a human fibroblast cell line deficient in idurono-2-sulfatase (Hunter's-syndrome), which had no activity on the IdoA(2-SO4)-[3H]AManR(6-SO4), converted GlcA(2-SO4)-[3H]AManR(6-SO4) to a mixture of GlcA-[3H] AManR(6-SO4) and [3H]AManR(6-SO4). This enzyme also converted GlcA(2-SO4)-[3H]ATalR(6-SO4) to a mixture of GlcA-[3H]ATalR(6-SO4) and [3H]ATalR(6-SO4). Digestion of both GlcA(2-SO4)-[3H]AManR(6-SO4) and GlcA(2-SO4)-[3H]ATalR(6-SO4) was inhibited by 35SO2-4 and was arrested at the monosulfated disaccharide stage by 1,4-saccharolactone. The glucurono-2-sulfatase exhibited a pH optimum of 4. The results indicate that there exists a separate sulfatase for the removal of sulfate substituents from C-2 of GlcA residues in glycosaminoglycans.     

Expression of externally-disposed heparin/heparan sulfate binding sites by uterine epithelial cells

A class of high-affinity binding sites that preferentially bind heparin/heparan sulfate have been identified on the external surfaces of mouse uterine epithelial cells cultured in vitro. [3H]Heparin binding to these surfaces was time-dependent, saturable, and was blocked specifically by the inclusion of unlabeled heparin or endogenous heparan sulfate in the incubation medium. A variety of other glycosaminoglycans did not compete for these binding sites. The presence of sulfate on heparin influenced, but was not essential for, recognition of the polysaccharide by the cell surface binding sites. [3H]-Heparin bound to the cell surface was displaceable by unlabeled heparin, but not chondroitin sulfate. Treatment of intact cells on ice with trypsin markedly reduced [3H]heparin binding, indicating that a large fraction of the surface binding sites were associated with proteins. Scatchard analyses revealed a class of externally disposed binding sites for heparin/heparan sulfate exhibiting an apparent Kd of approximately 50 nM and present at a level of 1.3 x 10(6) sites per cell. Approximately 9-14% of the binding sites were detectable at the apical surface of cells cultured under polarized conditions in vitro. Detachment of cells from the substratum with EDTA stimulated [3H]heparin binding to cell surfaces. These observations suggested that most of the binding sites were basally distributed and were not primarily associated with the extracellular matrix. Collectively, these observations indicate that specific interactions with heparin/heparan sulfate containing molecules can take place at both the apical and basal cell surfaces of uterine epithelial cells. This may have important consequences with regard to embryo-uterine and epithelial-basal lamina interactions.     

Complexes of heparin proteoglycans, chondroitin sulfate E proteoglycans, and [3H]diisopropyl fluorophosphate-binding proteins are exocytosed from activated mouse bone marrow-derived mast cells

The predominant [3H]diisopropyl fluorophosphate (DFP)-binding proteins that are released from the secretory granules of activated mouse bone marrow-derived mast cells (BMMC) are demonstrated to have an isoelectric point of approximately 9.1 and to be complexed to proteoglycans. Upon Sepharose CL-2B chromatography of the supernatants of calcium ionophore-activated BMMC, 67-78% of the total exocytosed [3H]DFP-binding proteins co-eluted in the excluded volume of the column as a greater than 1 X 10(7) Mr complex bound to 4-7% of the total exocytosed proteoglycans. The remainder of the exocytosed proteoglycans, which filtered in the included volume of the gel filtration column with a Kav of 0.66, contained chondroitin sulfate E glycosaminoglycans. After dissociation of the large Mr complexes of [3H]DFP-binding proteins-proteoglycans with 5 M NaCl and removal of the proteins via phenyl-Sepharose chromatography, the proteoglycans filtered from the Sepharose CL-2B column as a single peak with a Kav of 0.66. The susceptibility of 24-59% and 36-76% of the glycosaminoglycans in the large Mr complex to degradation by nitrous acid and chondroitinase ABC, respectively, indicated the presence of proteoglycans that contained heparin and chondroitin sulfate glycosaminoglycans. Disaccharide analysis revealed that the chondroitin sulfate in the high Mr complex was chondroitin sulfate E. Following chondroitinase ABC treatment of the large Mr complex, the residual heparin proteoglycans filtered on Sepharose CL-4B under dissociative conditions with the same Kav as the original, untreated proteoglycans. Thus, the protein-proteoglycan complexes that are exocytosed from activated mouse BMMC contain approximately equal amounts of proteoglycans of comparable size that bear either predominantly heparin or predominantly chondroitin sulfate E glycosaminoglycans. The demonstration of these secreted complexes indicates that the intragranular protease-resistant heparin and chondroitin sulfate E proteoglycans in the T cell factor-dependent BMMC bind serine proteases throughout the activation-secretion response.     

Isolation and characterization of sulphated mucopolysaccharides from rat leukaemic (RBL-1) basophils.

Proteoglycans of 300 000 mol.wt. were isolated from dispersed rat basophil tumour cells after labelling of the sulphated mucopolysaccharides with 35S in vitro:90% of the 35S-labelled mucopolysaccharides were extracted at high salt concentration. Alkali degradation of the 35S-labelled proteoglycans yielded glycosaminoglycan chains of 40 000 mol.wt. The composition of the salt-extracted 35S-labelled mucopolysaccharides, as defined by parallel or sequential degradation with chondroitinase AC, chondroitinase ABC and heparinase and resolution of the disaccharide-digestion products obtained with chondroitinase AC, was 48--61% chondroitin 4-sulphate, 20--30% dermatan sulphate, 10--15% heparin and 7--9% chondroitin 6-sulphate. Most of the salt-extracted 35S-labelled mucopolysaccharides were highly charged, with heparin and chondroitin 6-sulphate being relatively uniform in this regard, whereas chondroitin 4-sulphate and dematan sulphate exhibited a range of charge characteristics. The diversity of sulphated mucopolysaccharides present in the rat leukaemic basophil is in contrast with the predominance of heparin in the rat mast cell.     

Biosynthesis of glycosaminoglycans and proteoglycans by the lymph node

Previous studies of hyaluronan uptake and catabolism by lymph nodes indicated that the nodes might also add some HA of low molecular weight to the unabsorbed fraction that passes through from afferent to efferent lymph vessels.The ability of lymph nodes to synthesise HA and proteoglycans was therefore examined (i) by perfusion of [³H] acetate through an afferent lymph vessel in vivo, and recovery of labeled products from the efferent lymph vessel and from the node after perfusion; and (ii) by tissue culture of lymph nodes with [³H] acetate.Perfusion of lymph nodes with [³H] acetate in situ yielded: (a), in outflowing lymph, small amounts of chondroitin/dermatan sulfate within the first hour which continued to be produced for up to 24[emsp4 ]h; heparin in the second hour and HA in the third. In the nodes removed 17 to 19[emsp4 ]h later, equal amounts of hyaluronan and chondroitin/dermatan sulfate and heparan sulfate proteoglycans were detected. In the tissue culture of lymph nodes: (1) HA, heparin and proteoglycans of heparan sulfate and chondroitin/dermatan sulfate were released into the medium but in the cell extract only heparan sulfate proteoglycan was detected; and (ii) molecular weight of the released hyaluronan ranged widely but was mostly less than 4–5×10⁵[emsp4 ]D; heparan sulfate proteoglycan was 2.8×10⁴ to 9.4×10⁵[emsp4 ]D; heparin 7.9×10⁴[emsp4 ]D and chondroitin sulfate 1.3×10⁴[emsp4 ]D, suggesting that the chondrotin sulfate were released from their proteoglycans core by enzymic degradation.It is concluded that lymph nodes can release HA, heparin, heparan sulfate and chondroitin/dermatan sulfate proteoglycans into efferent lymph but the amount of hyaluronan is likely to be small without immune or other stimulation and its molecular weight is lower than in other tissues.     

Autoactivation of prolegumain is accelerated by glycosaminoglycans

The cysteine protease legumain participates in several biological and pathological processes including tumour invasion and metastasis. Legumain is synthesized as a zymogen and undergoes pH-dependent autoactivation of the proform in order to reach an enzymatically active form. Here we demonstrate that the naturally occurring polyanionic glycosaminoglycans (GAGs) chondroitin 4-sulphate (C4S), chondroitin 6-sulphate (C6S), chondroitin 4,6-sulphate (C4,6S), heparin, heparan sulphate (HS) as well as chondroitin sulphate (CS)-derived decasaccharides accelerated the autocatalytic activation of prolegumain through ionic interactions in a concentration-, size- and time-dependent manner at pH 4.0. In contrast, at pH 5.0 only C4S and C4,6S were able to promote prolegumain activation, while CS-derived decasaccharides, C6S, heparin and HS lost their effect at this pH.     

Renal plasma membrane receptors for certain modified serum albumins. Evidence for participation of a heparin receptor.

Binding of formaldehyde-treated (f-alb), reduced-carboxymethylated (ac-alb) or reduced-acetamidated (am-alb) bovine serum albumins to purified rat renal plasma membranes was studied. Radioiodinated f-alb or ac-alb bound to kidney membranes while am-alb neither bound significantly nor competed with f-alb binding to kidney membranes. The binding was specific, saturable and heat- and proteinase-sensitive. Competition studies showed that f-alb and ac-alb sites may be the same on these membranes. To determine the role played by charge in binding, competition experiments with polyanions were performed. Polyanions such as nucleic acid or glycosaminoglycans were effective competitors of f-alb binding to cell membranes. Heparin was especially inhibitory, being several-fold more so than chondroitin sulphate. Completely reduced and carboxymethylated albumin was a better competitor than its partially modified counterpart. Furthermore, f-alb was a significant competitor of [35S]heparin binding to kidney membranes. Also, partially purified heparin receptor demonstrated specific binding of 125I-f-alb. These data suggest that a heparin receptor is responsible for binding and internalization of intravenously injected f-alb. A Scatchard plot revealed two classes of receptors with dissociation constants of 3.2 X 10(-6) M and 4.7 X 10(-5) M.     

Inhibition by heparin-modulated antithrombin III of amidolysis catalysed by m beta-acrosin.

Purified m beta-acrosin catalysed amidolysis of several p-nitroanilides with C-terminal arginine residues. Antithrombin III inhibited amidolysis catalysed by the enzyme. This effect of antithrombin III was potentiated by heparin, and to a modest extent by heparan sulphate, cellulose sulphate, dextran sulphate and xylan sulphate. De-N-sulphated heparin, de-N-sulphated N-acetylated heparin, heparin of low relative molecular mass, chondroitin 4-sulphate, chondroitin 6-sulphate, dermatan sulphate and hyaluronic acid were ineffective.     

Identification of N-sulphated disaccharide units in heparin-like polysaccharides.

1. Preparations of heparin and heparan sulphate were degraded with HNO2. The resulting disaccharides were isolated by gel chromatography, reduced with either NaBH4 or NaB3H4 and were then fractionated into non-sulphated, monosulphated and disulphated species by ion-exchange chromatography or by paper electrophoresis. The non-sulphated disaccharides were separated into two, and the monosulphated disaccharides into three, components by paper chromatography. 2. The uronic acid moieties of the various non- and mono-sulphated disaccharides were identified by means of radioactive labels selectively introduced into uronic acid residues (3H and 14C in D-glucuronic acid, 14C only in L-iduronic acid units) during biosynthesis of the polysaccharide starting material. Labelled uronic acids were also identified by paper chromatography, after liberation from disaccharides by acid hydrolysis or by glucuronidase digestion. Similar procedures, applied to disaccharides treated with NaB3H4, indicated 2,5-anhydro-D-mannitol as reducing terminal unit. On the basis of these results, and the known positions and configurations of the glycosidic linkages in heparin, the two non-sulphated disaccharides were identified as 4-O-(beta-D-glucopyranosyluronic acid)-2,5-anhydro-D-mannitol and 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol. 3. The three monosulphated [1-3H]anhydromannitol-labelled disaccharides were subjected to Smith degradation or to digestion with homogenates of human skin fibroblasts, and the products were analysed by paper electrophoresis. The results, along with the 1H n.m.r. spectra of the corresponding unlabelled disaccharides, permitted the allocation of O-sulphate groups to various positions in the disaccharides. These were thus identified as 4-O-(beta-D-glucopyranosyl-uronic acid)-2,5-anhydro-D-mannitol 6-sulphate, 4-O-(alpha-L-idopyranosyluronic acid)-2,5-anhydro-D-mannitol 6-sulphate and 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol. The last-mentioned disaccharide was found to be a poor substrate for the iduronate sulphatase of human skin fibroblasts, as compared with the disulphated species, 4-O-(alpha-L-idopyranosyluronic acid 2-sulphate)-2,5-anhydro-D-mannitol 6-sulphate. 4. The identified [1-3H]anhydromannitol-labelled disaccharides were used as reference standards in a study of the disaccharide composition of heparins and heparan sulphates. Low N-sulphate contents, most pronounced in the heparin sulphates, were associated with high ratios of mono-O-sulphated/di-O-sulphated (N-sulphated) disaccharide units, and in addition, with relatively large amounts of 2-sulphated L-iduronic acid residues bound to C-4 of N-sulpho-D-glucosamine units lacking O-sulphate substituents.     

Nature and distribution of chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone

Summary The type and distribution of mineral binding and collagenous matrix-associated chondroitin sulphate and dermatan sulphate proteoglycans in rabbit alveolar bone were studied biochemically and immunocytochemically, using three monoclonal antibodies (mAb 2B6, 3B3, and 1B5). The antibodies specifically recognize oligosaccharide stubs that remain attached to the core protein after enzymatic digestion of proteoglycans and identify epitopes in chondroitin 4-sulphate and dermatan sulphate; chondroitin 6-sulphate and unsulphated chondroitin; and unsulphated chondroitin, respectively. In addition, mAb 2B6 detects chondroitin 4-sulphate with chondroitinase ACII pre-treatment, and dermatan sulphate with chondroitinase B pre-treatment. Bone proteins were extracted from fresh specimens with a three-step extraction procedure: 4m guanidine HCl (G-1 extract), 0.4m EDTA (E-extract), followed by guanidine HCl (G-2 extract), to characterize mineral binding and collagenous matrix associated proteoglycans in E- and G2-extracts, respectively. Biochemical results using Western blot analysis of SDS-polyacrylamide gel electrophoresis of E- and G2-extracts demonstrated that mineral binding proteoglycans contain chondroitin 4-sulphate, chondroitin 6-sulphate, and dermatan sulphate, whereas collagenous matrix associated proteoglycans showed a predominance of dermatan sulphate with a trace of chondroitin 4-sulphate and no detectable chondroitin 6-sulphate or unsulphated chondroitin. Immunocytochemistry showed that staining associated with the mineral phase was limited to the walls of osteocytic lacunae and bone canaliculi, whereas staining associated with the matrix phase was seen on and between collagen fibrils in the remainder of the bone matrix. These results indicate that mineral binding proteoglycans having chondroitin 4-sulphate, dermatan sulphate, and chondroitin 6-sulphate were localized preferentially in the walls of the lacunocanalicular system, whereas collagenous associated dermatan sulphate proteoglycans were distributed over the remainder of the bone matrix.     

Binding of [3H]heparin to human plasma low density lipoprotein.

The binding of [³H]heparin to human plasma lipoproteins was measured using a gel filtration assay on columns of Ultrogel AcA 54. [³H]Heparin formed a soluble complex with low density lipoprotein (LDL) as evidenced by the appearance of a new radioactive peak emerging at the void volume where the lipoproteins elute. Free heparin on the other hand was retarded on this column and eluted at a later volume. Heparin binding to LDL could also be demonstrated on columns of Sepharose 4B, in which case two included peaks of ³H were observed to elute in the area of LDL and of heparin. [³H]Heparin did not bind to either high or very low density lipoproteins as determined by the gel filtration assay. The binding of the [³H]heparin to LDL was proportional to both the concentration of LDL and of heparin and both showed saturation kinetics. Cations were not necessary for binding, nor was binding inhibited by EDTA. LDL showed a marked specificity for heparin. Thus, the binding of [³H]heparin to LDL was strongly inhibited by the addition of unlabeled heparin, while other glycosaminoglycans such as chondroitin sulfate, heparan sulfate, keratan sulfate, and dermatan sulfate were not effective inhibitors except at very high concentrations. Salts, especially K₂HPO₄ and (NH₄)₂SO₄, also inhibited binding when added at concentrations of 10 mm or higher suggesting an ionic interaction between heparin and LDL. The pH optimum for binding was between 7.5 and 8.5 but binding fell off markedly above pH 9.0. The [³H]heparin was heterogeneous and could be separated into four fractions on columns of Sephadex G-75. When these fractions were tested for binding to LDL, only the high molecular weight fraction bound to any significant extent. LDL was treated with reagents used to selectively modify basic amino acid residues, and the effect of these treatments on heparin binding was examined. Thus, ethoxyformic anhydride was used for histidine modification, acetic anhydride and succinic anhydride for lysines and cyclohexanedione for arginine residues. In each case there was a significant loss in heparin binding suggesting that various basic amino acids are involved in binding and/or that basic amino acids are necessary to maintain the proper conformation of LDL.