Distribution of keratan sulfate in cartilage proteoglycans.

After chondroitinase digestion of bovine nasal and tracheal cartilage proteoglycans, subsequent treatment with trypsin or trypsin followed by chymotrypsin yielded two major types of polypeptide-glycosaminoglycan fragments which could be separated by Sepharose 6B chromatography. One fragment, located close to the hyaluronic acid-binding region of the protein core, had a high relative keratan sulfate content. This fragment contained about 60% of the total keratan sulfate, but less than 10% of the total chondroitin sulfate present in the original proteoglycan preparation. The weight average molecular weight of the keratan sulfate-enriched fragment was 122,000, as determined by sedimentation equilibrium centrifugation. The chemical and physical data indicate that this fragment contains an average of 10 to 15 keratan sulfate chains, if the average molecular weight of individual chains is assumed to be about 8,000, and about 5 chondroitin sulfate chains attached to a peptide of about 20,000 daltons. The other population of fragments was derived from the other end of the proteoglycan molecule, the chondroitin sulfate-enriched region, and contained mainly chondroitin sulfate chains. About 90% of the total chondroitin sulfate, but only 20 to 30% of the total keratan sulfate was recovered in these fragments. On the average, approximately 5 chondroitin sulfate chains and 1 keratan sulfate chain could be linked to the same peptide. Another 10 to 20% of the total keratan sulfate, originally found in or near the hyaluronic acid-binding region, was not separated from the chondroitin sulfate-enriched fragments. Hydroxylamine could be used to liberate a large molecular size, chondroitin sulfate-enriched fragment (Kav 0.54 on Sepharose 2B) from the proteoglycan aggregates. The remainder of the protein core, containing the keratan sulfate-enriched region, was bound to hyaluronic acid with the link proteins and recovered in the void volume on the Sepharose 2B column.     

Isolation and biochemical characterization of the tryptic fragments of bovine nasal-cartilage proteoglycan monomer of high buoyant density.

Relatively homogeneous fractions of proteoglycan fragments were prepared from tryptic digests of the 4M-guanidinium chloride extract of bovine nasal cartilage. Glycosaminoglycan-containing fragments were separated from non-proteoglycan contaminants by ion-exchange chromatography and fractionated by equilibrium density-gradient centrifugation under dissociative conditions. The fractions of highest buoyant density were chromatographed on a column of Sepharose 4B, digested with chondroitinase ABC and chromatographed on a column of Sepharose 6B, yielding two distinct fractions: fraction B/6B-4 contained fragments from the chondroitin sulphate-bearing region of the proteoglycan monomer, and fraction B/6B-2 fragments from the keratan sulphate-rich region, most probably including a chondroitin sulphate-bearing monomer segment. By dansyl chloride analysis, fraction B/6B-2 had alanine and leucine as sole and fraction B/6B-4 had isoleucine and leucine as greatly predominant N-terminal amino acids, indicative of the relative homogeneity of these preparations of cartilage proteoglycan monomer fragments.     

Structural studies on proteoglycan from human chondrosarcoma.

Proteoglycan was isolated from a human chondrosarcoma which contained all glycosaminoglycans found in articular cartilage. Proteoglycans extracted by associative (67% of total uronate) and subsequent dissociative (27% of total uronate) solvents were identical as assessed by chromatography on Sepharose 2B (K_av 0.43), electrophoresis on acrylamideagarose gels, and in their ability to bind to hyaluronate. In addition there were no differences in chondroitin sulfate size, ratio of chondroitin 4- to 6-sulfate, or in size or form of keratan sulfate present. Two forms of keratan sulfate were identified following treatment with alkaline borohydride: A larger species (～-23 monosaccharides) was isolated from the keratan sulfate-enriched region only; a smaller oligosaccharide (～-13 monosaccharides) was recovered from all peptidoglycans released by trypsin, chymotrypsin treatment.     

Structural studies of two populations of keratan sulphate chains from mature bovine articular cartilage

Two discrete peptido-keratan sulphate fragments were isolatedvia chondroitinase ABC and trypsin digestion of a proteoglycan aggregate fraction prepared from bovine femoral head cartilage (six year old animals). The larger fragments (K_av=0.07, CL-6B) contained peptides substituted with several keratan sulphate (KS) chains from the KS-rich region of the proteoglycan and the smaller fragments (K_av=0.5, CL-6B) contained peptides with, perhaps, only one KS chain and the stubs of post-chondroitinase-treated chondroitin sulphate chains.The two peptido-KS samples and the KS chains derived from these by alkaline borohydride reduction were characterised by¹³C-NMR spectroscopy. The two populations of KS chains were also examined by chromatography (Sephadex G-75), and keratanase digestion followed by chromatography on Bio-Gel P-10. From the results it was concluded that the KS chains from the two major trypsin-derived peptido-KS fragments had similar sulphation levels, distributions of hydrodynamic sizes and susceptibilities to keratanase.Abbreviations KS keratan sulphate - A1 proteoglycan aggregate - T diphenyl carbamyl chloride (DPCC)-trypsintreated - CB chondroitinase ABC-treated - C chymotrypsin-treated - P papain-treated - R alkaline borohydride-reduced - TSP sodium 3-trimethylsilylpropionate     

Maturation-related changes in proteoglycans of fetal articular cartilage

Proteoglycans of the articulating and growing zones of maximum and minimum contact of bovine fetal articular cartilage were studied and compared to proteoglycans of immature calf and adult steer. During fetal maturation, localized changes were observed as early as the second trimester of fetal life but were restricted to the most superficial zones. Proteoglycans extracted from the growing zones were purified by density-gradient ultracentrifugation. The majority of proteoglycan monomers were able to interact with endogenous hyaluronate to form aggregates. Monomers had, at all fetal stages, similar elution profiles on Sepharose 2B and similar ratios of chondroitin sulfate chains/keratan sulfate chains/O-glycosidically linked oligosaccharides. Keratan sulfate chains were of similar size at all stages, but chondroitin sulfate chain size decreased markedly with fetal maturation. In the first and second trimesters of fetal life, the proteoglycans were poorly substituted with glycosaminoglycans. A major increase in the absolute number of glycosaminoglycans and oligosaccharides attached to core protein was detected during the third trimester of fetal life. No further changes in substitution occurred in early postnatal life. Enzymatic digestion of proteoglycan monomer demonstrated that the increase in substitution with keratan sulfate occurred to the same extent in the main polysaccharide attachment region and in the keratan sulfate-rich region.     

Age-related changes in the chemical composition of bovine articular cartilage. The structure of high-density proteoglycans

Proteoglycans were extracted from the articular cartilage of foetal, calf and adult bovine metacarpal–phalangeal joints with 4m-guanidinium chloride. After extraction, the high-density proteoglycans (PG-I fractions) were prepared by sedimentation in two sequential CsCl-density-gradient procedures [Swann, Powell & Sotman (1979) J. Biol. Chem. 254, 945–954]. The PG-I fractions from foetal, calf and adult tissues accounted for 75%, 52% and 46% respectively of the extracted components. The glucosamine, galactose, N-acetylneuraminic acid and protein contents increased with age. The overall amino acid compositions of PG-I fractions were similar. Fractionation of PG-I-fraction samples on a Bio-Gel A-50m column indicated that the molecular weight decreased with age. The PG-I fractions were specifically ³H-labelled by treatment with galactose oxidase followed by reduction with NaB³H₄. The ³H radioactivity was incorporated into both galactose and galactosamine residues of different carbohydrate side chains. The elution profiles of alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples on a Sepharose 6B column showed that the molecular weights of chondroitin sulphate chains were 13500, 12000 and 10500 in foetal, calf and adult tissues respectively. Fractionation of the alkaline borohydride-treated foetal, calf and adult PG-I-fraction samples and ³H-labelled calf and adult PG-I-fraction samples on a Bio-Gel P-10 column showed that there was an inverse relationship between the low-molecular-weight O-linked oligosaccharides and the higher-molecular-weight sialic acid-containing constituents at different ages. The oligosaccharide components of foetal, calf and adult PG-I-fraction samples represented 79%, 69% and 36% respectively of the total sialic acid content of the proteoglycans. Similarly in the ³H-labelled calf and adult samples 75% and 30% of the total radioactivity were present in the oligosaccharide components respectively. Digestion with chondroitinase AC-II and infrared analyses showed that the PG-I-fraction F and C samples contained primarily chondroitin 4-sulphate chains whereas PG-I-fraction sample A was 6-sulphated. These studies show that the major proteoglycans (PG-I fractions) in the articular cartilage of foetal, calf and adult animals differ in the content, types and structure of the chondroitin sulphate, keratan sulphate and oligosaccharide constituents. These changes in proteoglycan structure reflect the gross age-related changes in the chemical composition of the tissue.     

Anatomically determined polydispersity of proteoglycans of immature articular cartilage

Proteoglycans of the articulating and growing zones of minimum- and maximum-contact areas of calf articular cartilage were studied. Material was extracted sequentially (0.15 m sodium acetate, 2 m CaCl₂, and 4 m guanidinium chloride) in the presence of protease inhibitors. The very small proportion of material extracted by 0.15 m sodium acetate was poor in carbohydrates, but rich in serine, glycine, and glutamic acid, and had a K_av of 0.42 on Sepharose 2B. Proteoglycan extracted from the articulating zone was of smaller average hydrodynamic size (K_av of monomer, 0.42) than that from the growing zone (K_av of monomer, 0.32), but the attached chondroitin sulfate chains were of similar size. Proteoglycan prepared from the articulating area of minimum contact was chondroitin sulfate enriched (molar ratio of GalN:GlcN, 27) in comparison to that prepared from other regions of the articular cartilage (GalN:GlcN, 9–12). It is suggested that age-related maturation may be modified by physiological load or stress.     

The structural characterization of proteoglycans of culture aortic smooth muscle cells and arterial wall of the pig

Aortic proteoglycans, from the growth medium of cultured smooth muscle cells and from sequential associative and dissociative extracts of the tissue of origin, the pig aorta, were isolated and purified by precipitation with cetylpiridinium chloride. After isopycnic CsCl gradient centrifugation under associative conditions 94% of the cell-secreted proteoglycans were recuperated in the bottom one fifth (?_av = 1.62 g/ml) fraction. In contrast 80% of the tissue proteoglycans of both extracts, fractionated into two fractions: the bottom one fifth (?_av = 1.60 g/ml) fraction and three fifths (?_av = 1.42 g/ml) fraction. Fractionated tissue proteoglycans were composed predominantly of chondroitin sulfate-dermatan sulfate (83–90%) with lower proportions of heparan sulfate (5–11%) and hyaluronic acid (3–6%) whilst cell-secreted proteoglycans showed a similar glycosaminoglycan composition but with a higher proportion of hyaluronic acid (11–13%). Sepharose 2B and C1-2B chromatography of tissue proteoglycans of high buoyant density showed the presence of only subunit proteoglycans whilst those of intermediate density contained a complex species, partially dissociable in 4 M guanidinium chloride, along with K_av 0.50 subunit species. The latter was also observed for cell-secreted proteoglycans although obtained at high buoyant density. The cell-secreted subunit proteoglycans became separated into two distinct populations when chromatographed on Sepharose 4B and C1-4B, half of which eluted in the column V_o and the rest at a K_av of 0.34.. The majority of subunit macromolecules eluted at the V_o fractions of Sepharose 6B and C1-6B columns. Unlike the major species of cartilage proteoglycans, only approx. 20% of purified arterial proteoglycans formed complexes. This proportion could be increased by only 4–7% by interaction, of a mixture of subunit cell-secreted and tissue-extracted proteoglycans, with hyaluronic acid. These results suggest that proteoglycans secreted by cultured aortic smooth muscle cells and present in the aortic tissue possess certain similar physicochemical properties and are present in the form of complex and several subunit species.     

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.     

Enzymatic sulfation of galactose residue of keratan sulfate by chondroitin 6-sulfotransferase

We have previously found that the purified chondroitin 6-sulfotransferase(C6ST), which transfers sulfate from 3'-phosphoadenosine 5'-phosphosulfate(PAPS) to position 6 of N-acetylgalactosamine in chondroitin,catalyzed the sulfation of keratan sulfate, and that both theC6ST activity and the keratan sulfate sulfotransferase (KSST)activity were expressed in COS-7 cells when C6ST cDNA was transfected.In this report we describe some properties of the KSST activitycontained in the purified C6ST, and characterize the sulfatedproducts formed from keratan sulfate and partially desulfatedkeratan sulfate. Optimal pH, requirement for cationic activators,and K_m value for PAPS of the KSST activity were very similarto those of the C6ST activity. ³⁵S-Labeled glycosaminoglycansformed from keratan sulfate and partially desulfated keratansulfate were N-deacetylated by treatment with hydrazine/hydrazinesulfate and then cleaved with HNO₂ at pH 4, and the resultingproducts were reduced with NaB³H₄. Analysis of the degradationproducts with paper chromatography and high performance liquidchromatography provided evidence that C6ST transferred sulfateto position 6 of galactose residue which was glycosidicallylinked to N-acetylglucosamine 6-sulfate residue or to N-acetylglucosamineresidue. Northern blot analysis using poly (A)⁺ RNA from 12-d-oldchick embryos indicated that the message of C6ST was expressednot only in the cartilage but also in the cornea in which keratansulfate is actively synthesized. chondroitin sulfate keratan sulfate glycosaminoglycan sulfotransferase hydrazinolysis deaminative cleavage     

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.     

Affinity binding of the cartilage proteoglycan protein-keratan sulfate core to immobilized hyaluronic acid.

The protein-keratan sulfate core of bovine nasal cartilage proteoglycan was purified by affinity chromatography on a column of immobilized hyaluronic acid. The hyaluronic acid was immobilized by reaction with a hydrazido-alkyl derivative of Sepharose in the presence of borohydride. Proteoglycan was digested with chondroitinase ABC and the entire mixture was passed over a column of the Sepharose-hyaluronic acid maintained at 4°C. After the digested chondroitin sulfate chains were washed from the column, the bound protein-keratan sulfate core was eluted with 4m guanidinium chloride. The protein-keratan sulfate core interacts with the affinity matrix through its hyaluronic acid binding site as shown by the inhibition of binding by free hyaluronic acid and hyaluronic acid decasaccharide.     

Liver alcohol dehydrogenase subunit equivalence studied by rapid sampling of alcohol product formed from sequentially bound [4alpha-3H]NADH.

Proteoglycans of calf and steer articular cartilage were studied with a view of assessing structure and changes occurring as a result of the aging process. The average reduction in hydrodynamic size noted in steer was associated with a diminution in size of the chondroitin sulfate-rich region of the core protein as well as the chondroitin sulfate chains themselves. By contrast the keratan sulfate-rich region was hydrodynamically larger in steer although the keratan sulfate chains were only slightly longer than in calf. The proteoglycans showed a maturation-related decrease in chondroitin sulfate content (shorter chains, fewer chains, smaller chondroitin sulfate-rich region) and an enrichment in keratan sulfate chains in both the chondroitin sulfate-rich and keratan sulfate-rich regions. Proteoglycans from both age groups contained an oligosaccharide which was recovered mainly from outside of the keratan sulfate-rich region. There were no significant differences in size between keratan sulfate chains recovered from the keratan sulfate-rich region and the chondroitin sulfate-rich region.     

Isolation and characterization of proteoglycans from growing antlers of wapiti (Cervus elaphus)

Proteoglycans were extracted with 4 M guanidine–HCl from the zone of maturing chondrocytes, the site of endochondral ossification of growing antlers of wapiti (Cervus elaphus). Proteoglycans were isolated by DEAE-Sephacel chromatography and separated by Sepharose CL-4B chromatography into three fractions. Fraction I contained a high molecular mass (>1000 kDa) chondroitin sulfate proteoglycan capable of interacting with hyaluronic acid. Its amino acid composition resembled that of the cartilage proteoglycan, aggrecan. Fraction II contained proteoglycans with intermediate molecular weight which were recognized by monoclonal antibodies specific to chondroitin sulfate and keratan sulfate. Fraction III contained a low molecular mass (<160 kDa) proteoglycan, decorin, with a glucuronate-rich glycosaminoglycan chain.     

Two linkage-region fragments isolated from skeletal keratan sulphate contain a sulphated N-acetylglucosamine residue.

Peptido-keratan sulphate fragments were isolated from the nucleus pulposus of bovine intervertebral discs (6-year-old animals) after chondroitin ABC lyase digestion followed by digestion of A1D1 proteoglycans by diphenylcarbamoyl chloride-treated trypsin and gel-permeation chromatography on Sepharose CL-6B. Treatment of these peptido-keratan sulphate fragments with alkaline NaB3H4 yielded keratan sulphate chains with [3H]galactosaminitol end-labels, and these chains were further purified by gel-permeation chromatography on Sephadex G-50 and ion-exchange chromatography on a Pharmacia Mono-Q column in order to exclude any contamination with O-linked oligosaccharides. The chains were then treated with keratanase, and the digest was chromatographed on a Bio-Gel P-4 column followed by anion-exchange chromatography on a Nucleosil 5 SB column. Two oligosaccharides, each representing 18% of the recovered radiolabel, were examined by 500 MHz 1H-n.m.r. spectroscopy, and shown to have the following structures: [formula: see text] The structure of oligosaccharide (I) confirms the N-acetylneuraminylgalactose substitution at position 3 of N-acetylgalactosamine in the keratan sulphate-protein linkage region found by Hopwood & Robinson [(1974) Biochem. J. 141, 57-69] but additionally shows the presence of a 6-sulphated N-acetylglucosamine. Electron micro-probe analysis specifically confirmed the presence of sulphur in this sample. This sulphate ester group differentiates the keratan sulphate linkage region from similar structures derived from O-linked oligosaccharides [Lohmander, De Luca, Nilsson, Hascall, Caputo, Kimura & Heinegård (1980) J. Biol. Chem. 255, 6084-6091].     

Isolation and characterization of an asparagine-linked keratan sulfate from the skin of a marine teleost, Scomber japobicus

Keratan sulfate was isolated from the skin of Pacific mackerel (Scomber japonicus) after exhaustive digestion with pronase followed by ethanol precipitation and fractionation on a cellulose column with 0.3% recovery of dried material. The keratan sulfate preparation was separated into four major fractions by Dowex-1 column chromatrography. The chemical and infrared spectrum analyses of the four fractions showed a high degree of heterogeneity in sulfation. Since the carbohydrate-peptide linkage in the teleost skin keratan sulfate was found to be stable in alkali, and asparagine was the predominant amino acid, the asparagine residue in the peptide backbone was most likely to be involved in the N-glycosyl linkage with the carbohydrate moiety. Besides the type of carbohydrate-peptide linkage, the teleost skin keratan sulfate is very similar to corneal keratan sulfate, (keretan sulfate I) in two respects: (1) The teleost skin and bovine corneal keratan sulfates were hydrolyzed much faster by endo-β-galactosidase that the whale nasal cartilage keratan sulfate (keratan sulfate II). (2) Although the teleost skin keratan sulfate showed considerable polydispersity, the molecular weight was in the same range as the corneal keratan sulfate, and it was relatively higher than that of the cartilage keratan sulfate.     

In vitro synthesis of the glycosaminoglycans in estrogen-induced medullary bone in Japanese quail

A short-term incubation system has been developed for the study of glycosaminoglycan synthesis during the early stages of medullary bone formation in estrogenized male Japanese quail. Quail were injected with estradiol-17β and killed at different times thereafter. Femoral shafts were incubated in BGJ_b medium (Fitton-Jackson modification) for 2 h prior to being labeled with H₂³⁵SO₄ for 2 h in the same medium. Glycosaminoglycans were extracted by a 24-h papain digest and chromatographed on a DEAE Bio-Gel A column. Material from birds that had been treated with estrogen for 34 h prior to incubation produced an elution profile showing two distinct peaks (I and II). Elution profiles of material from control animals had a single peak corresponding to peak II in the estrogenized samples. The estrogen-induced glycosaminoglycan peak I was present after 20 h of estrogen treatment and increased dramatically between 25 and 30 h after treatment. Identification of the peak material was achieved by digestion with chondroitinase ABC, keratan sulfate β-endogalactosidase, or nitrous acid followed by chromatography on a Sephadex CL-6B column. Peak I was keratan sulfate and peak II was predominantly chondroitin sulfate. The in vitro production of a unique marker for medullary bone matrix provides an excellent opportunity for studying the dynamics of matrix synthesis.     

Solubilization and partial purification of steroid sulfatase of human placenta

Steroid sulfatase of human placenta has been solubilized by treatment of the microsomal fraction with an amphoteric surface active agent, Miranol H2M and ultrasound. Criteria of solubility include non-sedimentation of the activity following centrifugation at 160,000 × g, its retention on Sepharose 6B and a single peak of activity after polyacrylamide gel electrophoresis. Enzyme activity was located in the same gel fractions for the two substrates tested; cholesterol sulfate and dehydroisoandrosterone sulfate. The addition of dithiothreitol was found necessary to maintain the stability of the enzyme indicating the presence of sulfhydryl groups in the molecule. A molecular weight of approximately 330,000 has been estimated from the elution volume of the enzyme system on a column of Sepharose 6B. It is believed that this protein represents a sulfatase enzyme complex composed of subunits with different specificities. From kinetic studies, a K_m of 6.2 × 10^?5M for the cleavage of dehydroisoandrosterone sulfate and a K_m of 2 × 10^?6M for the cleavage of cholesterol sulfate have been calculated.     

Glycosaminoglycans and glycoproteins isolated from rabbit femur.

1. Complex carbohydrate fractions were extracted successively with 40% aqueous EDTA (pH 7.4) and 6M urea (PH 7.8) FROM ACETONE-DRIED bone powder of rabbit femur. 2. The carbohydrate fraction extracted with EDTA (E=Fr) was separated into five fractions,D1approximatelyD5 by DEAE-Dephadex A-50 column chromatography. Chemical and infrared spectral analyses, and enzymatic digestion indicate that D2 contained lessacidic glycoprotein, D3 contained sialoglycoprotein, D4 contained a low sulfated proteokeratan sulfate-like substance, and d5 contained glycoprotein-bound chondroitin sulfate A plus protein-free chondroitin sulfate A. 3. Two fractions, HU-D1 and HU-D2, were isolated from the carbohydrate fraction extracted with urea (HU-Fr) by successive digestion with collagenase [EC 3.4.99.5] and pronase, followed by gel-filtration on Sephadex G-100 and then DEAE-Sephadex A-50 column chromatography. HU-D1 and HU-D2 contained a low sulfated keratan sulfate-like substance linked to peptide and glycopeptide-bound chondroitin sulfated keratan sulfate-like substance linked to peptide and glycopeptide-bound chondroitin sulfate A, respectively. 4. The present findings indicate that rabbit femur contains low sulfated proteokeratan sulfate-like substances with varying sulfate contents and glycoprotein-bound chondroitin sulfate A as the principal glycosaminoglycans. The macromolecules bound more tightly to the tissue contain much more sulfate than the corresponding loosely bound ones.     

Heterogeneity of proteoglycans in monkey corneal stroma

The proteoglycans of the cynomolgus monkey corneal stroma were isolated and characterized by using a combination of physiochemical and biochemical methods. Proteoglycans were biosynthetically radiolabeled by incubating whole corneas in medium containing [³⁵S]sulfate and either [³H]serine or [³H]mannose as precursors. Macromolecules were extracted from the corneal stromas with 4 m guanidine-HCl. After dialysis into 8 m urea, proteoglycans in the extracts were initially purified by DEAE-cellulose chromatography. A portion of the proteoglycan fraction was digested with chondroitinase ABC, and the keratan sulfate proteoglycans were then isolated by rechromatography of the digest on DEAE-cellulose. Another portion of the proteoglycan fraction was digested with endo-β-galactosidase and the dermatan sulfate-proteoglycans were then isolated by chromatography of the digest on Sepharose CL-4B. Each proteoglycan population was further fractionated by chromatography on concanavalin A-Sepharose and by CsCl density gradient centrifugation. Four subpopulations for both the keratan sulfate proteoglycans and the dermatan sulfate proteoglycans were isolated. Based on differences in binding to concanavalin A-Sepharose, buoyant densities, and glycosaminoglycan content, subpopulations of each proteoglycan differ by the number and properties of both the glycosaminoglycan chains and the mannose-containing oligosaccharides attached to their protein core.