Structural studies of heparitin sulfates.

Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain. The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.     

On the structure of heparitin sulfates. Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum.

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavorbacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylucosamine and an unsaturated uronic, joined by alpha(1 lead to 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by alpha(1 lead to 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by alpha-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.     

Structural studies of heparitin sulfates

Heparitin sulfate fractions with a large range in sulfate content were subjected to degradation by Flavobacterium heparinase and by nitrous acid. The products obtained were fractionated by chromatography, characterized, and used to arrive at tentative structures for these complex polysaccharides. The heparitin sulfate chains examined appear to be composed of: 1. uninterrupted blocks of N-acetylglucosamine containing disaccharides; 2. larger blocks with a molecular weight range of 5000 to 6000 which include the N-acetyl block but do not contain heparinase sensitive linkages; 3. segments containing mainly areas where N-acetyl, N-sulfate and some disulfated units alternate in the chain.The size and arrangement of these polymer segments seem to vary with the sulfate content of a particular heparitin sulfate. For instance, the polysaccharides with the highest degree of sulfation do not appear to contain N-acetyl blocks of significant size.     

On the structure of heparitin sulfates Analyses of the products formed from heparitin sulfates by two heparitinases and a heparinase from Flavobacterium heparinum

The analyses of the products formed from heparitin sulfates by the action of two heparitinases and a heparinase from Flavobacterium heparinum is reported. Heparitin sulfates A and B are degraded by heparitinase I yielding two disaccharides, one of them composed of N-acetylglucosamine and an unsaturated uronic, joined by α(1 → 4) linkage, and the other, with the same composition but with an O-sulfate at the hexosamine moiety. A third disaccharide is also formed from heparitin sulfate B, by the action of the same enzyme, composed of glucosamine N-sulfate and an unsaturated uronic acid joined probably by α(1 → 4) linkage. Besides these three disaccharides, heparitin sulfate B yields, by the action of heparitinase I, an oligosaccharide (with an average molecular weight of 6000) which is completely degraded by the heparitinase II yielding a disaccharide composed of glucosamine 2,6-disulfate and unsaturated uronic acid. All the disaccharides are further degraded by α-glycuronidase from Flavobacterium heparinum yielding the respective monosaccharides. Based on these and other analyses the possible structures of the heparitin sulfates are proposed.     

Chemistry of heparitin sulfate and heparin from normal tissues and from patients with Hunter syndrome.

Some structural features of heparitin sulfate excreted by patients with Hunter syndrome are described. It is shown, with the aid of heparitinases and heparinase from Flavobacterium heparinum, that the Hunter heparitin sulfate is a very complex structure composed of nine different disaccharide units containing regions akin to normal heparitin sulfate and regions akin the heparin. Two-thirds of the iduronic acid residues of Hunter heparitin sulfate are devoid of sulfate, contrasting with heparin in which most of the iduronic acid residues are sulfated. The isolation and characterization of the non-reducing ends of heparin and of the heparitin sulfates is also described. Based on these results the specificity of the heparinase and heparitinases as well as the biosynthesis of iduronic acid-containing heparin-like compounds is discussed.     

Interactions between mucopolysaccharides and cationic polypeptides in aqueous solution: hyaluronic acid, heparitin sulfate, and keratan sulfate

Circular dichroism spectroscopy has been used to study the interactions of hyaluronic acid, heparitin sulfate, and keratan sulfate with cationic polypeptides. The results indicate that the presence of these mucopolysaccharides has an effect in the conformation of poly(L -lysine) and poly(L -arginine), such that the former adopts the “random” form and the latter takes up the α-helical conformation, rather than the “charged coil” form expected at neutral pH. The relative strengths of the interactions can be judged from the melting temperatures above which they are disrupted. Both the stoichiometry and the strength of the interactions depend on the position, number, and type of anionic groups attached to the polysaccharide backbone. Such considerations place the six common mucopolysaccharides in order of increasing strength of interaction: hyaluronic acid < chondroitin 4-sulfate < heparitin sulfate < chondroitin 6-sulfate < keratan sulfate ? dermatan sulfate. These differences should be paralleled by differences in the interaction of the mucopolysaccharides with collagen and fibrous proteins.     

Surface sulfated mucopolysaccharides of primary and permanent mammalian cell lines.

The sulfated mucopolysaccharide composition of the mammalian cell lines: HeLa, H.Ep.2, AV₃, WI-38, BHK and a cell culture of rabbit lung tissue is reported. It is shown that chondroitin sulfate AC and heparitin sulfate are the main mucopolysaccharides of the permanent cell lines whereas chondroitin sulfate B and heparitin sulfate are the major ones in the primary cultures, with no significant change in their relative concentrations up to seven generations. It is also shown that besides heparitin sulfate, chondroitin sulfate AC and chondroitin sulfate B are located at the surface of the cells. These results are in agreement with the earlier proposals that heparitin sulfate and chondroitin sulfate B might play a role in cell recognition and adhesiveness and that chondroitin sulfate AC might act as a stimulant of cell division.     

Changes of sulfated mucopolysaccharides and mucopolysaccharidases during fetal development

The changes of sulfated mucopolysaccharides and mucopolysaccharidases during bovine fetal development were analyzed. It is shown that chondroitin sulfate C increases in concentration up to the 50th day of fetal development and then decreases progressively until its complete disappearance in most adult tissues. Likewise, hyaluronidase also reaches a peak on the 50th day and decreases in activity until its disappearance in adult tissues. On the other hand, heparitin sulfate and chondroitin sulfate B as well as beta-glucuronidase and beta-N-acetylglucosaminidase remain without significant changes during the whole period. The fetal chondroitin sulfate C is tissue specific with different molecular weights depending on the tissue of origin. Some properties of fetal muscle and brain hyaluronidase are also described. The possible role of chondroitin sulfate C and hyaluronidase in the processes of differentiation and division is discussed in view of the present findings.     

Production of heparin related glycosaminoglycans by an extablished mammalian cell line

A sulfated glycosaminoglycan has been isolated from the acid-soluble fraction of an established line of Chinese hamster fibroblasts grown in suspension culture. This material has a molecular weight between 5000 and 10,000, contains equimolar amounts of hexosamine and uronic acid (orcinol method), and about 0.6 sulfate groups per hexosamine residue. About 80% of the sulfate groups are N-sulfates on the basis of lability of the sulfate and the formation of equivalent numbers of free amino groups upon mild acid hydrolysis. The material is completely resistant to testicular hyaluronidase but is degraded to reducing monosaccharides and small oligosaccharides upon treatment with lyophilized cells of Flavobacterium heparinum that were grown on heparin. It is thought, therefore, to be related to the known N-sulfated glycosaminoglycans heparin and heparitin sulfate.     

The effect of acid mucopolysaccharides and acid mucopolysaccharide–proteins on fibril formation from collagen solutions

1. The effects of acid mucopolysaccharides and acid mucopolysaccharide-proteins on the size and rate of formation of fibril aggregates from collagen solutions in pH7.6 buffers were studied by turbidimetric and light-scattering methods. 2. Serum albumin, orosomucoid, methylated cellulose, chondroitin sulphate A and chondroitin sulphate C of molecular weight less than 20000, and hyaluronate of molecular weight less than 40000 did not influence rates of fibril formation. Chondroitin sulphate A, chondroitin sulphate C and hyaluronate of high molecular weight retarded the rate of fibril formation. This effect of high-molecular-weight chondroitin sulphate C decreased with increasing ionic strength. Heparin, though of low molecular weight (13000), was highly effective, as was also heparitin sulphate. The chondroitin sulphate-proteins of very high molecular weight were highly effective, despite the fact that for some preparations the component chondroitin sulphate chains had molecular weights much less than 20000. 3. Agents that had delayed fibril formation were also effective in producing an increase in degree of aggregation of fibrillar collagen, as indicated by dissymmetry changes observed in light-scattering experiments at low collagen concentrations. Methylated cellulose and heparin at 2.5mug./ml. were unusual in decreasing aggregation, but heparin at 0.25mug./ml. increased aggregation. Electron microscopy of gels showed fibrils and fibril aggregates with ;normal' collagen spacing and dimensions consistent with the light-scattering results. 4. The rates of electrical transport of agents and of solvent (electro-osmosis) through collagen gels indicated a contribution of molecular entanglement that increased with increase in molecular size of the agents. Electrostatic binding of heparin to collagen was noted. Binding to collagen during fibril formation was also found for heparitin sulphate and a chondroitin sulphate with extra sulphate groups. 5. Electrostatic binding of acid mucopolysaccharide-proteins to collagen may be an important factor in the organization and functioning of connective tissues at all stages of growth and development. Excluded-volume (molecular-entanglement) effects may also be important. These factors operate simultaneously and interact mutually so that precise assessment of their relative importance is difficult.     

Hormonal effect on glycosaminoglycans and glycoproteins in uteri of ovariectomized rats

1. Glycosaminoglycans such as chondroitin sulfate A (or C), chondroitin sulfate B (dermatan sulfate), heparitin sulfate (heparan sulfate) and hyaluronic acid were identified as major glycosaminoglycan components in whole uteri as well as in uterine stroma of rats. Two types of sialoglycoproteins with different electrophoretic mobilities (fast- and slow-migrating) were detected in the glycosaminoglycan fraction from the luminal epithelia. 2. Treatment of ovariectomized rats with estradiol-17beta markedly increased the uterine contents of glycosaminoglycans. Chondroitin sulfate A (or C) was found to increase more than chondroitin sulfate B. Furthermore, it was found that the estrogen treatment specifically increases the fast-migrating sialoglycoprotein level in the luminal epithelia and results in the appearance of it in the uterine fluid. 3. Administration of progesterone to ovariectomized rats slightly increased the uterine glycosaminoglycan content without appreciable alteration of the uterine glycosaminoglycan pattern.     

Electrofocusing of heparin: fractionation of heparin into 21 components distinguishable from other acidic mucopolysaccharides.

Twenty-one fractions have been demonstrated in each of 15 different commercially available heparins subjected to electrofocusing. These fractions show a molecular-weight range from 3000 to 37,500 with a constant interval between molecular weights. Degradation of each fraction by purified enzymes of Flavobacterium heparinum yielded identical end products, suggesting chemical identity. Only fractions with a molecular weight of 7000 and up had significant anticoagulant activities. The phenomenon of electrofocusing of mucopolysaccharides is dependent upon pH, molecular weight, and ampholyte availability. Chemical composition of the mucopolysaccharide is also an essential factor since N- and O-desulfation of heparin markedly changed the focalization pattern. The pattern produced when heparin is subjected to electrofocusing is not duplicated by any other naturally occurring acidic mucopolysaccharide tested. Heparitin sulfate D shows some similarities to heparin and it is probable that heparitin sulfate D is a normal contaminant of heparin preparations (this assumption is supported by molecular-weight and anticoagulant activity determinations). The technique is specific and reproducible and unequivocally distinguishes heparin from other acid mucopolysaccharides.     

Characterization of a chondroitin 4-sulfate proteoglycan carrier for heparin neutralizing activity (platelet factor 4) released from human blood platelets

Platelet heparin neutralizing activity (platelet factor 4) is released from human blood platelets by thrombin in the form of a high molecular weight proteoglycan-platelet factor 4 complex. This complex was partially purified by isoelectric precipitation and gel filtration. At high ionic strength (I = 0.75) the complex dissociates into the active component (mol. wt 29000) and the proteoglycan carrier. The components were separated by gel filtration and the proteoglycan further purified by Na₂SO₄ treatment. The molecular weight of the purified carrier was 59000. The carbohydrate moieties of the proteoglycan isolated after papain digestion and ion-echange chromatography were shown to consist of chondroitin 4-sulfate by chemical, physical and electrophoretic analysis. The multichain proteoglycan consists of four chondroitin 4-sulfate chains (mol. wt 12000) in covalent linkage to a single polypeptide. The molecular weight (350000) of the fully saturated proteoglycan carrier suggests that 4 moles of platelet factor 4 are bound per mole of proteoglycan and that the carrier occurs in the form of a dimer consisting of 8 moles of platelet factor 4 and 2 moles of proteoglycan. The isolated chondroitin 4-sulfate moieties combine with platelet factor 4 at a binding ratio of one mole of platelet factor 4 per carbohydrate chain. Heparin completely displaces platelet factor 4 from both the saturated proteoglycan and chondroitin 4-sulfate complexes. Heparitin sulfate, dermatan sulfate and chondroitin 6-sulfate also combine stoichiometrically with platelet factor 4 and are displaced by equimolar amounts of heparin. Hyaluronic acid did not combine with platelet factor 4. The relative binding capacities of glycosaminoglycans for platelet factor 4 were shown to be: heparin (100), heparitin sulfate (75), chondroitin 4-sulfate (50), dermatan sulfate (50), chondroitin 6-sulfate (50), and hyaluronic acid (o). Chondroitin 4-sulfate was identified as the major glycosaminoglycan in all platelet subcellular fractions; in addition, the soluble fraction contains a minor amount of hyaluronic acid. Subcellular distribution studies revealed that 55% of both the proteoglycan carrier and platelet factor 4 activity were localized in the “granule rich” fraction. This data together with the low recovery of both these components in the membrane fraction, suggest that they occur together as a complex within specific granules and are released in this form under physiologic conditions.     

Sulfated mucopolysaccharides from normal Swiss 3T3 cell line and its tumorigenic mutant ST1: possible role of chondroitin sulfates in neoplastic transformation.

The sulfated mucopolysaccharide composition of normal Swiss 3T3 cell line and its tumorigenic mutant ST1 is reported. It is shown that chondroitin sulfate B and heparitin sulfate are the sulfated mucopolysaccharides of the normal 3T3 line whereas chondroitin sulfate A and heparitin sulfate are the major ones of the ST1 variant. Degradation of the chondroitin sulfates derived from both cell lines with chondroitinases B and ABC have shown that they contain only 4-sulfated disaccharides differing from each other by the type of uronic acid residue. It is also shown that the chondroitin sulfate A from the tumorigenic variant is mostly located at the cell surface whereas the chondroitin sulfate B from the normal line is less accessible to trypsinization. A relative increase of chondroitin sulfate A was also observed in 3T3 that had lost contact inhibition after successive subcultures, and in the 3T6 cell line. These combined results are in agreement with the earlier proposal that glucuronic acid-containing chondroitin sulfate plays a role in the stimulation of cell division in neoplastic and embryonic tissues.     

Purification and characterization of glutathione reductase from bovine erythrocytes

Glutathione reductase (E.C.1.8.1.7; GR) was purified from bovine erythrocytes and some characteristics properties of the enzyme were investigated. The purification procedure was composed of preparation of the hemolysate, ammonium sulfate fractionation, affinity chromatography on 2',5'-ADP Sepharose 4B, and gel filtration chromatography on Sephadex G-200. As a result of four consecutive procedures, the enzyme was purified 31,250-fold with a yield of 11.39%. Specific activity at the final step was 62.5 U (mg proteins)(-1). For the enzyme, optimum pH, optimum temperature, optimum ionic strength, and stable pH were found to be 7.3, 55 degrees C, 435 mM, 7.3, respectively. The molecular weight of the enzyme was found to be 118 kDa by Sephadex G-200 gel filtration chromatography and the subunit molecular weight was found to be 58 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). In addition, Km and Vmax values were determined for glutathione disulfide (GSSG) and NADPH. Ki constants and inhibition types were established for glutathione (GSH) and NADP+. Also, effects of NADPH and GSSG were investigated on the enzyme activities.     

Sulfated glycosaminoglycans of cultured fibroblasts from guinea-pig embryo kidney

The sulfated glycosaminoglycan content of primary cultures of fibroblasts from guinea-pig embryo kidney is reported. A hybrid chondroitin sulfate comprises approx. 90% of these glycosaminoglycans from the cell coat. Changes in the proportion of labelled heparitin sulfate were also observed after successive subcultures. We postulate a possible correlation between the pattern of glycosaminoglycans and processes of cell selection and cell dedifferentiation in these cultures.     

Isolation and characterization of argininosuccinate synthetase from human liver

This communication describes the purification and characterization of argininosuccinate synthetase from human liver. By numerous criteria including electrophoresis in sodium dodecyl sulfate containing gels, electrophoresis in nondissociating gels, and analytical ultracentrifugation, the protein is homogeneous at a specific activity of 4.2 mumol/(min mg) assayed at 37 degrees C in the direction of argininosuccinate synthesis. The enzyme has a molecular weight of 183,000, as determined by gel filtration. Electrophoresis in the presence of sodium dodecyl sulfate yielded a single band migrating with an Rf corresponding to 43,000 daltons. Thus, the enzyme is considered to contain four subunits of identical molecular weight. The s20,w of the enzyme is 8.2 S. Antibodies were prepared in rabbits directed against the purified protein. These antibodies react specifically with argininosuccinate synthetase, as determined by electrophoretic analysis of the immunoadsorbed product from crude extracts of human liver. The human enzyme has very similar properties to those published for the beef and rat liver enzymes.     

Correlation of specific sulfated glycosaminoglycans with collagen types I,II, and III

Summary A qualitative and quantitative biochemical study of the glycosaminoglycans was performed in tissues constituted predominantly by one type of collagen, or in tissues containing mixtures of different types of collagen. The results obtained show the presence of dermatan sulfate, chondroitin sulfate, and heparitin sulfate in tissues containing collagen types I, II, or III, respectively, suggesting a specific correlation of different glycosaminoglycans with these three types of collagen.This work was aided by grant N° 79/306 from the Fundação de Amparo à Pesquisa do Estado de São PauloSupported by CNPq (Conselho Nacional de Desenvolvimento Cientifico e Tecnológico)     

Cell recognition and adhesiveness: a possible biological role for the sulfated mucopolysaccharides.

The sulfated mucopolysaccharide composition of different neonate, adult and tumoral tissues is reported. It is shown that each tissue has a characteristic composition with respect to the relative amount, type and molecular size of chondroitin sulfate $\text{A}\text{C}$, chondroitin sulfate B and heparitin sulfate. Neonate and tumor tissues contain large amounts of chondrotin sulfate $\text{A}\text{C}$ which is nearly absent in most adult and normal tissues respectively. Based on these and other results a possible role for the sulfated mucopolysaccharides in cell recognition and adhesiveness is proposed.