Heparitin Sulfate Research Articles

Hormonal effect on glycosaminoglycans and glycoproteins in uteri of ovariectomized rats

1. 1. Glycosaminoglycans such as chondroitin sulfate A (or C), chondroitin sulfate B (dermatan sulfate), heparitin sulfate (hepara sulfate) and hyaluronic acd 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 teh luminal epithelia. 2. 2. Treatment of ovariectomized rats with estradiol-17β makedly 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. 3. Administration of progesterone to overiectomized rats slightly increased the uterine glycosaminoglycans content without appreciable alteration of the uterine glycosaminoglycan pattern.

Aortic glycosaminoglycans in atherosclerosis-susceptible and -resistant pigeons

Quantitative electrophoretic profiles of glycosaminoglycans (GAGs) in the upper thoracic aorta and at the celiac bifurcation were obtained from atherosclerosis-susceptible White Carneau (WC) and atherosclerosis-resistant Show Racer (SR) pigeons at four ages ranging from 1 day to 3 years. While thoracic aortas of WC and SR pigeons contained similar total amounts and similar amounts and profiles of the five individual GAGs (hyaluronic acid, heparitin sulfate, dermatan sulfate, chondroitin 4-sulfate, and chondroitin 6-sulfate) at each age, celiac sites did not show these similarities. Celiac sites in WC pigeons contained more total GAG than corresponding SR sites at all four ages, and, after 6 months of age, WC foci showed an increased amount of chondroitin 6-sulfate, decreased amounts of heparitin sulfate and chondroitin 4-sulfate, and a lower chondroitin 4-sulfate/chondroitin 6-sulfate ratio when compared with corresponding SR celiac foci. In addition, heparitin sulfate in preatherosclerotic and moderately atherosclerotic WC celiac foci was more heterogeneous than in the thoracic aorta of either breed or in SR celiac foci. Differences in GAG content between sites also existed, since the thoracic aorta contained more total GAGs than the celiac foci at each age examined. These findings indicate that changes in aortic GAG content occurring with age are different than those related to atherogenesis and suggests a role for specific GAGs in the pathogenesis of atherosclerosis.

Heparitin sulfates (heparan sulfates) of atherosclerosis-susceptible and atherosclerosis-resistant pigeon aortas

Electrophoretic mobilities of heparitin sulfates (HS) from the thoracic aorta and the celiac bifurcation of atherosclerosis-susceptible White Carneau and atherosclerosis-resistant Show Racer pigeons at four ages were examined. Atherosclerosis-susceptible White Carneau aortas contain, in addition to their normal HS component, a variant type of HS (HS') in pre, early- and moderately-atherosclerotic celiac foci which is not found in the thoracic regions of either breed or in Show Racer celiac foci at any age. These findings are interpreted to suggest a role of HS' in the pathogenesis of atherosclerosis and to propose an explanation for atherosclerotic susceptibility and resistance in pigeons.

Quantitative microanalysis of aortic glycosaminoglycans

Glycosaminoglycans (GAGs) are isolated from approximately 250 mg of aortic tissue by enzymatic degradation of tissue, followed by potassium acetate precipitation of GAGs. Hyaluronic acid, heparitin sulfate, and dermatan sulfate are quantitated by densitometric scanning of cellulose acetate electrophoretograms. Chondroitin 4-sulfate and chondroitin 6-sulfate are quantitated in separate aliquots of the aortic extract by the Morgan-Elson determination of N-acetyl hexosamines, following selective enzymatic digestions of the chondroitin sulfate isomers. In both analyses, absolute amounts of GAGs are determined by comparison with standard curves prepared simultaneously. The accuracy for this micromethod was ±0.03–±0.07 μg at the 99% confidence level for determination of the five individual GAGs in mixtures of GAGs whereas the precision was ±0.03–±0.09 μg at the 99% confidence level over the range of 2.5–50.0 μg of GAG.

Distribution of sulfated mucopolysaccharides in invertebrates.

The sulfated mucopolysaccharide composition of 22 species of invertebrates belonging to the phyla Arthropoda, Mollusca, Annelida, Tunicata, Echinodermata, Coelenterata, and Porifera was analyzed. It is shown that all the species contain variable amounts of one or more types of sulfated mocopolysaccharides, most of which similar to the ones found in vertebrates. It is shown also that each species has a characteristic composition, differing from each other regarding the relative amount and type of chondroitin sulfates A, B, and C, heparitin sulfate, and heparin. The possible biological role of the sulfated mucopolysaccharides in cell recognition or aggregation or both is discussed in view of the present findings.

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 A C , chondroitin sulfate B and heparitin sulfate. Neonate and tumor tissues contain large amounts of chondrotin sulfate A 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.

Susceptibility to experimental atherosclerosis: Relation to mast cells and heparin

Fifteen chicken tissues were examined for mast cells. Numerous mast cells were found in peritoneum and spleen. The sulfated mucopolysaccharides extracted from these two tissues, corresponding in amount to that in mast cells, were found to be dermatan sulfate, chondroitin sulfate and heparitin sulfate but no evidence of heparin. We have shown a similar situation occurs in the rabbit which is also highly susceptible to the production of atherosclerosis by diet. These observations provide further evidence of a role for heparin and mast cells in limiting atherogenesis.

Triolein emboli clearance from the mouse lung: Effect of heparin and heparitin sulfate

We have designed a model system in mice in which one can study the localization, accumulation, and clearance of experimentally induced pulmonary fat emboli. When 125I-labeled triolein emulsions are injected via the caudal vein, the lipid is trapped predominantly in the lung vasculature. Both heparin and heparitin sulfate significantly lowered the level of trapped 125I-labeled triolein in the lungs compared to control mice and caused the release of significant amounts of lipoproptein lipase into the plasma. We suggest that a possible mechanism for clearance of triglycerides from the lung vasculature may involve the release of plasma lipoprotein lipase. This in turn hydrolyzes the localized glycerides into more water-soluble components, so they can be removed from the site of embolization.

Effect of hydrocortisone on composition of acid mucopolysaccharide fractions of the aorta

Five fractions of acid mucopolysaccharides were identified in the rabbit aorta: hyaluronic acid, heparitin sulfate, and chondroitin sulfates, A, C, and B. During prolonged administration of hydrocortisone the concentration of hyaluronic acid rose but that of heparitin sulfate fell. The relative percentages of chondroitin sulfate A, C, and B were lowered 15 days after administration of the hormone ceased.

Characteristic distribution of sulfated mucopolysaccharides in different tissues and in their respective mitochondria

The distribution of sulfated mucopolysaccharides in different rat tissues and in their respective mitochondria is reported. It is shown that each tissue has a characteristic composition, differing from each other regarding the relative amount, type and molecular size of chondroitin sulfate A C , chondroitin sulfate B, and heparitin sulfate. It is also shown that the mucopolysaccharide composition of the mitochondria is similar to the composition of the tissue of origin. The possible biological functions of these compounds are discussed in view of this characteristic distribution.

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.

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

AbstractTwenty‐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.

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.

The uronidic linkages in heparitin sulfate.

Beta-glucuronidase preparations obtained from various sources were shown to liberate free D-glucuronic acid from heparitin sulfate oligosaccharides. Some free L-iduronic acid could also be detected in the hydrolyzates indicating the presence of an iduronidase in the preparations. The data presented indicate that heparitin sulfate contains a substantial amount of beta-D-glucuronidic and some alpha-L-iduronidic linkages.

Stimulation of transcription of mouse kidney chromatin by sulfated polysaccharides

The sulfated polysaccharides polydextran sulfate (PDS) and heparin stimulate in vitro transcription of mouse kidney chromatin by E. coli RNA polymerase by about 100 and 40 fold respectively. Heparin which has been N-desulfated and N-acetylated stimulates only 13 fold. Chondroitin sulfate B and heparitin sulfate do not stimulate transcription under similar conditions. PDS inhibits transcription of deproteinized chromatin. Therefore, the stimulation with chromatin is due to interaction with the chromatin and not the polymerase. Polydextran sulfate has no effect on the size of the RNA that is made either under conditions in which the enzyme can reinitiate or under conditions in which reinitiation is blocked. If reinitiation of the enzyme is blocked, the time required to complete the synthesis of the RNA is the same whether or not the enzyme is stimulated by PDS. These observations indicate that sulfated polysaccharides stimulate transcription by making available new RNA polymerase binding sites on the chromatin.

Heparin and heparin-like substances of cells.

The correlation between heparin and mast cells was first recognized by Jorpes (7) in 1937, and the history of heparin has since generally been considered to be the history of mast cells. After this correlation was made, however, heparin-like material with lower anticoagulant activity was found by several investigators as a by-product of heparin isolation from various tissues, such as lung and liver (6, 16). This material, called heparitin sulfate, contained less sulfate per disaccharide unit than heparin and contained a mixture of N-sulfated and N-acetylated glucosamine units (13). Both heparin and heparitin sulfate have been shown to be covalently linked to protein and can therefore by called proteoglycans.KeywordsMast CellHyaluronic AcidChondroitin SulfateSugar NucleotideCommercial HeparinThese keywords were added by machine and not by the authors. This process is experimental and the keywords may be updated as the learning algorithm improves.

Characterization of the mucopolysaccharides associated with fractions of guanidine-denatured amyloid fibrils

An association between intact amyloid fibrils and mucopolysaccharides has long been recognized. Since portions of some amyloid fibrils have been shown recently to exhibit homology to immunoglobulin, a study was undertaken to determine whether mucopolysaccharides were present also in fractions of fibrils homologous to a portion of a Bence Jones protein. Typical fibrils were obtained from the liver of one patient and the spleen of another with primary amyloidosis. Two fractions, both of which showed homology to the V k segment of Bence—Jones protein, type kappa, were obtained from the hepatic fibrils after gel chromatography in 4 M guanidine hydrochloride. A major component (80% by weight) of the splenic fibrils was obtained in identical fashion and contained the same N-terminal amino acid, Asp, as the fractions of hepatic amyloid. Each fraction was metachromatic and contained approximately 0.7% uronic acid and a similar amount of hexosamine. Based upon fractionation of cetylpyridinium chloride complexes of the mucopolysaccharides and upon the hexosamine lost on dialysis after digestion with hyaluronidase or chondroitinase ABC, heparitin sulfate was the predominant polysaccharide, with lesser amounts of chondroitin sulfate and dermatan sulfate also present in each fraction. Thus, mucopolysaccharides remain closely associated with highly purified fractions of amyloid fibrils which resemble immunoglobulin.

Fractionation and properties of four heparitin sulfates from beef lung tissue: Isolation and partial characterization of a homogeneous species of heparitin sulfate

Several commerical batches of heparitin sulfate extracted from beef lung tissue were fractionated into at least four distinct mucopolysaccharides by a combination of polyacrylamide and agarose gel electrophoresis. The four heparitin sulfates (A, B, C and D) were distinguished from each other and from heparin by several physical and chemical properties such as electrophoretic migration, molecular weight, presence of N-acetyl, N- and )-sulfate residues, optical rotation and enzymatic degradation. Of particular significance was the isolation of a heparitin sulfate (heparitin sulfate C) with a homogeneous molecular weight.

Some observations on a protein-mucopolysaccharide complex found in sea urchin embryos

A complex of protein and mucopolysaccharide was isolated chromatographically from developing sea urchin embryos. The protein moiety of the complex was found to vary electrophoretically from one developmental stage to another. On the other hand, the mucopolysaccharide moiety was invariable throughout all the stages tested, and was very probably heparitin sulfate. The results of pulse-labeling and chase experiments showed that the complex was synthesized in the cytoplasm and subsequently transferred into the nucleus. Data were obtained which indicate the possibility that the mucopolysaccharide in the nucleus is localized preferentially in the template-active part of the chromatin. An augmentation of RNA synthesis in isolated nuclei was observed when the complex was added to the incubation mixture. A possible role of the complex molecule in the mechanisms of nucleo-cytoplasmic interactions is discussed.

Isolation and partial characterization of three induced enzymes from flavobacterium heparinum involved in the degradation of heparin and heparitin sulfates

Summary The isolation and partial characterization of two heparitinases and a heparinase from induced extracts from F. heparinum are reported. The heparinase acts upon heparin, heparitin sulfates C and D producing as the main degradation product a trisulfated disaccharide. The heparitinase I degrades only heparitin sulfate A and B producing as main products two N-acetylated disaccharides, one of them O-sulfated. The heparitinase II degrades heparitin sulfate B producing an N-O-sulfated disaccharide as the main product. Based on these results, the mode of action of the three enzymes as well as the structure of heparin and heparitin sulfates are proposed.