The hydration of proteoglycans of bovine cornea.

The water sorptive and retentive capacities of three corneal proteoglycans with different keratan sulfate/chondroitin-4-sulfate compositions were investigated. The calcium salt of a predominantly keratan sulfate containing proteoglycan had hydration properties similar to that of calcium keratan sulfate. The proteoglycan containing predominantly calcium chondroitin-4-sulfate side chains sorbed water to a greater extent than pure calcium chondroitin-4-sulfate but its retentive power was somewhat less. The proteoglycan containing about twice as much keratan sulfate as chondroitin-4-sulfate, on a dissaccharidic molar basis and had hydration properties which were closer to the behavior of chondroitin-4-sulfate than keratan sulfate. The results are discussed in terms of structure and polymer interaction in the proteoglycan matrices.     

Isolation and characterization of developmentally regulated chondroitin sulfate and chondroitin/keratan sulfate proteoglycans of brain identified with monoclonal antibodies

A panel of monoclonal antibodies prepared to the chondroitin sulfate proteoglycans of rat brain was used for their immunocytochemical localization and isolation of individual proteoglycan species by immunoaffinity chromatography. One of these proteoglycans (designated 1D1) consists of a major component with an average molecular size of 300 kDa in 7-day brain, containing a 245-kDa core glycoprotein and an average of three 22-kDa chondroitin sulfate chains. A 1D1 proteoglycan of approximately 180 kDa with a 150-kDa core glycoprotein is also present at 7 days, and by 2-3 weeks postnatal this becomes the major species, containing a single 32-kDa chondroitin 4-sulfate chain. The concentration of 1D1 decreases during development, from 20% of the total chondroitin sulfate proteoglycan protein (0.1 mg/g brain) at 7 days postnatal to 6% in adult brain. A 45-kDa protein which is recognized by the 8A4 monoclonal antibody to rat chondrosarcoma link protein copurifies with the 1D1 proteoglycan, which aggregates to a significant extent with hyaluronic acid. A chondroitin/keratan sulfate proteoglycan (designated 3H1) with a size of approximately 500 kDa was isolated from rat brain using monoclonal antibodies to the keratan sulfate chains. The core glycoprotein obtained after treatment of the 3H1 proteoglycan with chondroitinase ABC and endo-beta-galactosidase decreases in size from approximately 360 kDa at 7 days to approximately 280 kDa in adult brain. In 7-day brain, the proteoglycan contains three to five 25-kDa chondroitin 4-sulfate chains and three to six 8.4-kDa keratan sulfate chains, whereas the adult brain proteoglycan contains two to four chondroitin 4-sulfate chains and eight to nine keratan sulfate chains, with an average size of 10 kDa. The concentration of 3H1 increases during development from 3% of the total soluble proteoglycan protein at 7 days to 11% in adult brain, and there is a developmental decrease in the branching and/or sulfation of the keratan sulfate chains. A third monoclonal antibody (3F8) was used to isolate a approximately 500-kDa chondroitin sulfate proteoglycan comprising a 400-kDa core glycoprotein and an average of four 28-kDa chondroitin sulfate chains. In the 1D1 and 3F8 proteoglycans of 7-day brain, 20 and 33%, respectively, of the chondroitin sulfate is 6-sulfated, whereas chondroitin 4-sulfate accounts for greater than 96% of the glycosaminoglycan chains in the adult brain proteoglycans.(ABSTRACT TRUNCATED AT 400 WORDS)     

Keratan sulfate proteoglycan isolated from the estrogen-induced medullary bone in Japanese quail

1. Two proteoglycans isolated from the femurs of quail actively producing medullary bone were separated using DEAE Bio-Gel A. 2. The first to elute in the gradient was a keratan sulfate proteoglycan with an average buoyant density of 1.53 g/ml and a Kav = 0.57 on Sepharose CL-4B. 3. The second proteoglycan to elute contained chondroitin 4-sulfate. 4. Apparently only the keratan sulfate proteoglycan is associated with the new medullary bone matrix.     

Extracellular matrix and the maintenance of the differentiated state: proteoglycans synthesized by replated chondrocytes and nonchondrocytes

It has been previously shown that undifferentiated stage 23 to 24 chick limb bud mesenchymal cells can be maintained in culture under conditions which promote chondrogenesis. As the chondrocytes mature in vitro, their proteoglycan synthesis progresses through a specific and reproducible biosynthetic program. By the eighth day of culture, the chondrocytes are making proteoglycans that are similar to proteoglycans isolated from adult animal tissues. Relative to the Day 8 proteoglycans, the proteoglycans synthesized by chick limb bud chondrocytes earlier in culture have a smaller monomer size, longer chondroitin sulfate chains, shorter keratan sulfate chains, a higher ratio of chondroitin-6-sulfate to chondroitin-4-sulfate, and a decreased ability to interact with hyaluronic acid. We have reported a procedure to remove the cells from Day 8 cultures and strip away most, if not all, of the extracellular matrix. In addition, the chondrocytes can be separated from the 40-50% nonchondrocytic cells normally found in Day 8 cultures, and the two cell populations replated separately. This report describes the analysis of the proteoglycans synthesized by replated cells; this analysis demonstrates quantitative and qualitative differences between chondrocyte and nonchondrocyte proteoglycans. The overall rate of proteoglycan synthesis is fourfold higher and the rate of synthesis of high buoyant density proteoglycans 30-fold higher for replated chondrocytes relative to nonchondrocytes. Qualitatively, more newly synthesized nonchondrocyte proteoglycans partition at lower buoyant density on CsCl equilibrium density gradients than do chondrocyte proteoglycans. Nonchondrocyte proteoglycans are of two major classes: One has a monomer size slightly smaller than that of Day 8 chondrocyte proteoglycan, but has much longer glycosaminoglycan chains. The other is considerably smaller than Day 8 chondrocyte proteoglycans, but has glycosaminoglycans of slightly larger size. In contrast, replated chondrocytes synthesize, even as soon as 4.5 hr after replating, proteoglycans that are identical to Day 8 chondrocyte proteoglycan in monomer size, in glycosaminoglycan chain size, in aggregability, and in the ratio of 6-sulfated to 4-sulfated chondroitin. Since denuding mature Day 8 chondrocytes of their extracellular matrix does not cause them to recapitulate their developmentally regulated program for the biosynthesis of proteoglycans, it is concluded that the quality of mature chondrocyte proteoglycan is not altered by the absence of extracellular matrix.     

Organization of glycosaminoglycan chains in a chondroitin sulfate - dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate - dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by β-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

The dynamics of compartmentalization of embryonic muscle by extracellular matrix molecules

In order to delineate the role of proteoglycans in muscle development, the immunohistological localization of glycosaminoglycans and proteoglycan core proteins was studied in embryonic chick leg at Hamburger-Hamilton stages (St.) 36, 39, 43, and 46, and at 2 weeks posthatching. A specific anatomical landmark was chosen (the junction between the pars pelvica and the pars accessoria of the flexor cruris lateralis muscle) in order to ensure the study of anatomically equivalent sites. Frozen cross sections were immunostained with monoclonal antibodies to chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans; to the core proteins of muscle/mesenchymal chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, and basement membrane heparan sulfate proteoglycan; and to laminin and tenascin. Extracellular matrix zones corresponding to the endomysium, perimysium, epimysium, basement membrane, and myotendinous junction each show characteristic immunostaining patterns from St. 36 to St. 46 and have unique matrix compositions by St. 46. In some cases, there is a sequential or coordinate expression of epitopes, first in the epimysium, then the perimysium, and last in the endomysium. Dermatan sulfate proteoglycan is detected in the epimysium at St. 36, in the perimysium at St. 39 (there is no perimysium structure at St. 36), and is not detected in the endomysium until St. 43. A putative mesenchymal proteoglycan core protein (reactive to the monoclonal antibody MY-174) is detected at St. 39 in both epimysium and perimysium, but is not detected in the endomysium until St. 43. Keratan sulfate antibody immunostains epimysium at St. 39 and perimysium at St. 46, but is never detected in the endomysium. Some epitopes are expressed independently in each of the extracellular matrix zones: antibody to tenascin stains only a subset of the epimysium, at the myotendinous junction; and heparan sulfate proteoglycan and laminin are detected only in the endomysium. Between St. 36 and St. 39, the muscle/MY-174-reactive proteoglycan core protein staining decreases in intensity in the endomysium and becomes positive in the epimysium and perimysium. An inverse relationship is found between (1) the disappearance of muscle/MY-174-reactive proteoglycan core protein staining at the surface of myotubes from St. 36 to St. 39 and (2) the infiltration of laminin and heparan sulfate proteoglycan staining encompassing groups of myotubes (St. 36) to circumferential staining of all myotubes (St. 39).(ABSTRACT TRUNCATED AT 400 WORDS)     

Organization of glycosaminoglycan chains in a chondroitin sulfate-dermatan sulfate proteoglycan from bovine aorta

A chondroitin sulfate-dermatan sulfate proteoglycan was isolated from bovine aorta intima by extraction of the tissue by 4 M guanidine hydrochloride. The proteoglycan was purified by CsCl isopycnic centrifugation followed by gel filtration and ion-exchange chromatography. The proteoglycan had 21.9% protein, 22.1% uronate, 21.4% hexosamine and 10.8% sulfate. Glycosaminoglycan chains obtained from the proteoglycan by beta-elimination were resolved by gel filtration into two fractions, one containing chondroitin 6-sulfate with an approximate molecular weight of 49 000 and the other containing chondroitin 4-sulfate and dermatan sulfate in a proportion of 2:1 with an approximate molecular weight of 37 000. Digestion of the proteoglycan by chondroitinase ABC or AC yielded a protein core with similar composition and behavior in gel filtration and SDS-polyacrylamide gel electrophoresis. An approximate molecular weight of 180 000 was estimated for the core protein. Dermatan sulfate chains with an approximate molecular weight of 10 000 were observed only in the digest of chondroitinase AC. Limited trypsin hydrolysis of the proteoglycan yielded three peptide fragments containing chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in varied proportions. A tentative structure for the proteoglycan was suggested.     

Highly sulfated glycosaminoglycans inhibit aggrecanase degradation of aggrecan by bovine articular cartilage explant cultures.

The catabolism of 35S-labeled aggrecan and loss of tissue glycosaminoglycans was investigated using bovine articular cartilage explant cultures maintained in medium containing 10(-6) M retinoic acid or 40 ng/ml recombinant human interleukin-1alpha (rHuIL-1alpha) and varying concentrations (1-1000 microg/ml) of sulfated glycosaminoglycans (heparin, heparan sulfate, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate) and calcium pentosan polysulfate (10 microg/ml). In addition, the effect of the sulfated glycosaminoglycans and calcium pentosan polysulfate on the degradation of aggrecan by soluble aggrecanase activity present in conditioned medium was investigated. The degradation of 35S-labeled aggrecan and reduction in tissue levels of aggrecan by articular cartilage explant cultures stimulated with retinoic acid or rHuIL-1alpha was inhibited by heparin and heparan sulfate in a dose-dependent manner and by calcium pentosan polysulfate. In contrast, chondroitin 4-sulfate, chondroitin 6-sulfate, dermatan sulfate and keratan sulfate did not inhibit the degradation of 35S-labeled aggrecan nor suppress the reduction in tissue levels of aggrecan by explant cultures of articular cartilage. Heparin, heparan sulfate and calcium pentosan polysulfate did not adversely affect chondrocyte metabolism as measured by lactate production, incorporation of [35S]-sulfate or [3H]-serine into macromolecules by articular cartilage explant cultures. Furthermore, heparin, heparan sulfate and calcium pentosan polysulfate inhibited the proteolytic degradation of aggrecan by soluble aggrecanase activity. These results suggest that highly sulfated glycosaminoglycans have the potential to influence aggrecan catabolism in articular cartilage and this effect occurs in part through direct inhibition of aggrecanase activity.     

Mucopolysaccharidases from Pseudomonas sp. Isolation and partial characterization of constitutive enzymes involved in the degradation of keratan sulfate and chondroitin sulfate

Four constitutive enzymes, capable of degrading keratan sulfate, were isolated from Pseudomonas sp.: a particulate endoglycosidase, a soluble endoglycosidase, a soluble exo-beta-D-galactosidase and a soluble exo-beta-D-N-acetylglucosaminidase. The endoglycosidases were shown to act only upon keratan sulfate forming beta-D-2-acetamido-2-deoxy-6-O-sulfoglucosyl-(1----3)-D-galactose, as the main product. This results indicates that the enzyme catalyses the hydrolysis of beta-D-galactose-(1----4)-N-acetylglucosamine linkages. It was also shown that this monosulfated disaccharide inhibits the particulate keratan sulfate endoglycosidase. The bovine nucleus pulposus keratan sulfate is depolymerized at a lower rate and extent when compared to the corneal keratan sulfate. The soluble endoglycosidase is very labile, in contrast to the particulate enzyme, which has been stored at -20 degrees C or at 4 degrees C for at least 12 months with no loss in activity. The particulate endoglycosidase and the soluble exo-beta-D-galactosidase and exo-beta-D-N-acetylglucosaminidase are induced when the bacteria is grown in adaptative media containing either 0.1% keratan sulfate or 0.1% chondroitin sulfate. Furthermore, particulate forms of the exoenzymes were detected. The soluble endoglycosidase specific activity, in contrast, is approximately the same in extracts of cells grown in glucose, keratan sulfate or chondroitin sulfate. A chondroitin sulfate lyase was also identified in the soluble extracts of Pseudomonas sp. cells. This enzyme depolymerizes chondroitin 4-sulfate, chondroitin 6-sulfate and hyaluronic acid forming unsaturated disaccharides as main products. It is also active upon the glucuronic-acid-containing regions of the dermatan sulfate molecules. The properties of the soluble enzymes, further purified by ion-exchange chromatography, and of the particulate keratan sulfate endoglycosidase are presented.     

Proteoglycans of guinea-pig coastal cartilage. Fractionation and characterization.

Proteoglycans were extracted, in a yield of about 90%, from costal cartilage of young, growing guinea-pigs. Three solvents were used in sequence: 0.4 M guanidine - HCl, pH 5.8, 4 M guanidine - HCl, pH 5.8, and 4 M guanidine - HCl/0.1 M EDTA, pH 5.8. The proteoglycans were purified and fractionated by cesium chloride density gradient ultracentrifugation under associative and dissociative conditions. Gel chromatography on Sepharose 2 B of proteoglycan fractions from associative centrifugations showed the presence of both aggregated and monomer proteoglycans. The ratio of aggregates to monomers was higher in the second extract than in the other two extracts. Dissociative gradient centrifugation gave a similar distribution for proteoglycans from all three extracts. Thus, with decreasing buoyant density there were decreasing ratios of polysaccharide to protein, and of chondroitin sulfate to keratan sulfate. In addition, there was with decreasing density an increasing ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. Amino acid analyses of dissociative fractions were inaccordance with previously published results. On comparing proteoglycan monomers of the three extracts, significant differences were found. Proteoglycans, extracted at low ionic strength, contained lower proportions of protein, keratan sulfate, chondroitin 6-sulfate and basic amino acids than those of the second extract. The proteoglycans of the third extract also differed from those of the other extracts. The results indicate that the proteoglycans of guinea-pig costal cartilage exist as a very polydisperse and heterogenous population of molecules, exhibiting variations in aggregation capacity, molecular size, composition of protein core, degree of substitution of the protein core, as well as variability in the type of polysaccharides substituted.     

Immunolocation analysis of glycosaminoglycans in the human growth plate.

Monoclonal antibodies were used in this study to immunolocate glycosaminoglycans throughout the human growth plate. Chondroitin-4-sulfate, chondroitin-6-sulfate, and keratan sulfate were observed in the extracellular matrix of all zones of the growth plate and persisted into the cartilage trabeculae of newly formed metaphyseal bone. Also present in the extracellular matrix was an oversulfated chondroitin/dermatan sulfate glycosaminoglycan which appeared to be specific to the proliferative and hypertrophic zones of the growth plate. As with the other extracellular matrix molecules, this epitope persisted into the cartilage trabeculae of the metaphyseal bone. Zonal differences between the extracellular and pericellular or lacunae matrix were also observed. The hypertrophic chondrocytes appeared to synthesize chondroitin sulfate chains containing a non-reducing terminal 6-sulfated disaccharide, which were located in areas immediately adjacent to the cells. This epitope was not found to any significant extent in the other zones. The pericellular region around hypertrophic chondrocytes also contained a keratan sulfate epitope which was also observed in the resting zone but not in the proliferative zone. These cell-associated glycosaminoglycans were not found in the cartilage trabeculae of metaphyseal bone, indicating their removal as the terminal hypertrophic chondrocytes and their lacunae are removed by invading blood vessels. These changes in matrix glycosaminoglycan content, both in the different zones and within zones, indicate constant subtle alterations in chondrocyte metabolic products as they proceed through their life cycle of proliferation, maturation, and hypertrophy.     

Chondroitin sulfate synthesis by mouse embryonic, extraembryonic, and teratoma cells in vitro

Sulfated glycosaminoglycan (GAG) synthesis by primary cultures of embryo, yolk sac, and trophoblast was compared with synthesis by the same tissues in utero. In general, the in vivo and in vitro results were in good agreement. As was the case in vivo, the three tissues synthesized chondroitin-4-sulfate and chondroitin-6-sulfate (but no dematan sulfate) at characteristic ratios.Cultured embryos are already capable of synthesizing chondroitin sulfates, primarily chondroitin-4-sulfate, before, or at, the 64-cell stage. During the attachment and initiation of outgrowth stages, blastocysts synthesize more chondroitin-6-sulfate than chondroitin-4-sulfate. Thereafter, progressively more chondroitin-4-sulfate is synthesized so that the 4:6 ratio increases, resembling that of trophoblast cells.Blastocyst-derived cell lines and teratoma cell cultures were also studied. One blastocyst-derived line, MB4, synthesized GAG with a pattern similar to that of yolk sac, which it resembles biochemically in other respects as well. The GAG profile of MB2, a parietal endoderm-like cell line resembled neither that of embryo, yolk sac, nor trophoblast cells. Embryonal carcinoma (undifferentiated teratoma) cells had a chondroitin sulfate pattern different from that of most of the other cultures.     

Composition of glycosaminoglycans in human pancreatic cancer

Five glycosaminoglycans were isolated from tryptic digestion of both cancerous and normal tissues of the human pancreas and were assayed by determining the carbohydrate content of materials. Separation of these five polymers was achieved by Dowex 1-X2 column chromatography and fractionation with Benedict's solution. They were identified as hyaluronic acid, heparan sulfate, dermatan sulfate, chondroitin-4-sulfate, and chondroitin-6-sulfate, respectively. The total amount of glycosaminoglycans in cancer tissue increased in comparison to the controls. The increase in tissue content of glycosaminoglycans was accompanied by increases in chondroitin-4-sulfate and chondroitin-6-sulfate levels.     

The glycosaminoglycans of estrogen-induced medullary bone in Japanese quail

Glycosaminoglycans were isolated from the femurs of estrogen-treated male Japanese quail. During the 72 h after the injection of estrogen the incorporation of a 1-h pulse of H₂³⁵SO₄ into keratan sulfate increased more than 100-fold in a pattern corresponding to the production of the induced medullary bone. The rate of incorporation into chondroitin 4-sulfate, the only other glycosaminoglycan detected, remained constant throughout the same time period. The rate of incorporation of the 1-h pulse of sulfate into chondroitin 4-sulfate and keratan sulfate was the same at 48 h of estrogen treatment. When birds (48 h estrogen) were allowed to live 6 h after the injection of the isotope, chondroitin 4-sulfate accumulated 5-fold over that found for similar animals labeled for only 1 h. Keratan sulfate, into which the isotope was incorporated at the same rate as the chondroitin sulfate in this experiment, did not accumulate much more in 6 h of labeling than in 1 h of labeling. This suggests that the keratan sulfate turns over more rapidly than the chondroitin 4-sulfate in this tissue. Autoradiography showed that the chondroitin 4-sulfate was associated mainly with the marrow cells near the cortical bone and the keratan sulfate with the newly synthesized medullary bone. These results suggest that keratan sulfate is a specific marker for this secondary bone matrix.     

Immunochemical characterization and ultrastructural localization of chondroitin sulfates and keratan sulfate in embryonic chick bone marrow

Summary Monoclonal antibodies directed against specific carbohydrate epitopes on chondroitin 4-/dermatan sulfate, chondroitin 6-sulfate, keratan sulfate, and a monoclonal antibody directed against the hyaluronate binding region were used to characterize proteoglycans extracted from embryonic chick bone marrow. About half of the proteoglycans separate into the high density fraction on a CsCl gradient. Glycosaminoglycan-specific antibodies recognize proteoglycans from all fractions; this includes an antibody directed against keratan sulfate. Some proteoglycans, principally in the high buoyant density fraction, contain sites recognized by the antibody specific for the hyaluronate binding region. Within limits of detection, all core proteins belong to the high-molecular-weight category, with weights in excess of 212 kD. Antibodies directed against chondroitin 4-/dermatan sulfate and against keratan sulfate primarily bind to extracellular matrix material located in the extracellular spaces and to matrix elements in the pericellular regions of fibroblastic stromal cells. The antibody that recognizes chondroitin 6-sulfate binds to sites on surfaces of fibroblastic stromal cells and also to extracellular matrix material. Little or no antibody binding is detected on surfaces of granulocytic cells. These studies indicate that chondroitin sulfate and keratan sulfate chains are both present in the proteoglycan extract.     

An immunological study of glycosaminoglycans in the connective tissue of bovine and cod skeletal muscle

The presence of sulfated glycosaminoglycans (GAGs) was demonstrated in the connective tissue of bovine and cod skeletal muscle by histochemical staining using Alcian blue added MgCl₂ (0.06 M and 0.4 M, respectively). For further identification of the sulfated GAGs, a panel of monoclonal antibodies, 1B5, 2B6, 3B3 and 5D4 was used that recognizes epitopes in chondroitin-0-sulfate (C0S), chondroitin-4-sulfate/dermatan sulfate (C4S/DS), chondroitin-6-sulfate (C6S) and keratan sulfate (KS), respectively. Light microscopy and Western blotting techniques showed that in bovine and cod muscle C0S and C6S were primarily localized pericellularly, whereas cod exhibited a more intermittent staining. C4S was expressed around the separate cells and also in the perimysium and myocommata. In contrast to bovine muscle, which hardly expressed highly sulfated KS, cod exhibited a very strong and consistent staining. Western blotting showed that C0S and C6S were mainly associated with proteoglycans (PGs) of high molecular sizes in both species. Contrary to bovine muscle, C4S in cod was associated with molecules of various sizes. Both cod and bovine muscle contained KSPGs of similar sizes as C4S. KSPGs of different sizes and buoyant densities, sensitive to keratanase I and II were found expressed in cod.     

Comparison of carbohydrate-containing substances from non-calcified and calcified portions of bovine costal cartilage.

Carbohydrate-containing substances were extracted from non-calcified (NCC) and calcified (CC) portions of bovine costal cartilage with 0.5 M LaCl3 by the method of Mason and his co-workers, followed by dilution of the extract with 9 volumes of water. The precipitate formed on dilution yielded Fr. P, while Fr. S was obtained from the supernatant. Fr. P was separated into two subfractions by gel filtration on Sepharose 2B. The experimental results showed that Fr. P contained proteoglycans with different molecular sizes and compositions, while Fr. S contained proteoglycan, hyaluronic acid, glycoproteins, and glycogen. The present data suggest that in the proteoglycan of Fr. P, the relative content of chondroitin sulfate decreases with a concomitant increment in that of keratan sulfate on calcification. In addition, elevation of the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate, together with a small increment of non-sulfated disaccharide units in the chondroitin sulfate chains appear to occur on calcification. The glycogen content in Fr. S diminished on calcification. The present observations suggest therefore that the remodeling of proteoglycan consumption of glycogen in bovine costal cartilage occur on calcification.     

Isolation and characterization of the sulfated glycosaminoglycans of the vitreous body

Ester sulfate containing glycosaminoglycans comprising approx. 3% of the total glycosaminoglycan content, have been isolated from protease-digested bovine vitreous body by stepwise fractionation on AG-1X2(Cl^?) and gel filtration on Bio-Gel P-300. Two heparan sulfate and two chondroitin-4-sulfate fractions were isolated in nearly pure form. The heparan sulfate fractions were undersulfated and contained the same relative proportions of N- and O-sulfate (1 : 2), although the total sulfate content differed by approx. 100%. No chondroitin-6-sulfate was present in the isolates, based on evidence obtained from chondroitin ABC lyase experiments.     

Analysis of the proteoglycans synthesized by corneal explants from embryonic chicken. II. Structural characterization of the keratan sulfate and dermatan sulfate proteoglycans from corneal stroma

Radioisotopically labeled proteoglycans were isolated from a 4 M guanidine HCl, 2% Triton X-100 extract of corneal stroma from day 18 chicken embryos by anion-exchange chromatography. Two predominant proteoglycans in the sample were separated by octyl-Sepharose chromatography using a gradient elution of detergent in 4 M guanidine HCl. One proteoglycan had an overall mass of approximately 125 kDa, a single dermatan sulfate chain (approximately 85-90% chondroitin 4-sulfate, low iduronate content) of approximately 65 kDa, and a core protein after chondroitinase ABC digestion of approximately 45 kDa which also contained one to three N-linked oligosaccharides and one O-linked oligosaccharide. The other proteoglycan had an overall size of approximately 100 kDa, two to three keratan sulfate chains of approximately 15 kDa each, and a core protein following keratanase digestion of approximately 51 kDa which included two to three N-linked but no O-linked oligosaccharides. A larger size, a greater overall hydrophobicity (as measured by its interaction with octyl-Sepharose) and an absence of O-linked oligosaccharides argue that this core protein is a distinct gene product from the core protein of the dermatan sulfate proteoglycan.