A novel low-molecular weight chondroitin sulphate proteoglycan isolated from cartilage.

Proteoglycans were isolated from cartilage by extraction with 4M-guanidinium chloride followed by direct centrifugation in 4M-guanidinium chloride/CsCl at a low starting density, 1.34 g/ml. N-Ethylmaleimide was included in the extraction solvent as a precaution against contamination of proteoglycans with unrelated proteins mediated by disulphide exchange. A novel, discrete, low-buoyant-density proteoglycan (1.40--1.35 g/ml) was demonstrated by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. Its proteoglycan nature was revealed by the shift in the molecular size observed on gel electrophoresis after treatment with chondroitinase ABC. The core protein was monodisperse. The proteoglycan was further purified by gel chromatography with and without addition of hyaluronate. The proteoglycan constitutes less than 2% (by weight) of the total extracted proteoglycans and is not capable of interacting with hyaluronate. The same proteoglycan was purified in larger quantities by sequential associative and dissociative CsCl-density-gradient centrifugation, zonal rate sedimentation in a sucrose gradient and gel chromatography on Sepharose CL-4B. The pure proteoglycan had a molecular weight of 76 300 determined by sedimentation-equilibrium centrifugation and an apparent partial specific volume of 0.59 ml/g. It contained about 25% protein (of dry weight) and had remarkably high contents of leucine and cysteine as compared with other proteoglycans. The proteoglycan contained two to three large chondroitin sulphate chains and some oligosaccharides.     

Isolation and characterization of dermatan sulphate proteoglycan from human uterine cervix.

Proteoglycans were extracted from human uterine cervix with 4 M-guanidinium chloride in the presence of proteinase inhibitors. They were purified by density-gradient centrifugation in 4 M-guanidinium chloride/CsCl (starting density 1.32 g/ml) followed by DEAE-cellulose and Sepharose chromatography. Only one polydisperse proteoglycan was found. s020,w was 2.1S and the weight-average molecular weight was 73 000 (sedimentation-equilibrium centrifugation) to 110 500 (light-scattering). The core protein was monodisperse, with an apparent molecular weight of 47 000. The proteoglycan contained about 30% protein and probably two or three glycosaminoglycan side chains per molecule. High contents of aspartate, glutamate and leucine were found. The glycan moiety of the proteoglycan was exclusively dermatan sulphate, with a co-polymeric structure with approximately equal quantities of iduronic acid- and glucuronic acid-containing disaccharides.     

Isolation and partial characterization of proteoglycans from rat incisors.

Newly synthesized proteoglycans of rat incisors were labelled in vivo for 6h with [35S]-sulphate in order to facilitate their detection during purification and characterization. Proteoglycans were extracted from non-mineralized portions (predentine) of rat incisors with 4M-guanidinium chloride and subsequently from dentine by demineralization with a 0.4M-EDTA solution containing 4M-guanidinium chloride. Both extractions were performed at 4 degrees C in the presence of proteinase inhibitors. Purification of proteoglycans was achieved with a procedure involving gel-filtration chromatography, selective precipitation of phosphoproteins, affinity chromatography and ion-exchange chromatography. Two proteoglycan populations were found in the initial extract (Pd-PG I and Pd-PG II), whereas only one fraction (D-PG) was obtained after demineralization. The minor proteoglycan fraction from the first extract, Pd-PG I, although not totally characterized, differed sharply from the other proteoglycans in that it had a larger molecular size with larger glycosaminoglycan chains composed of chondroitin 4- and 6-sulphate isomers. In contrast, the major proteoglycans Pd-PG II and D-PG had smaller hydrodynamic sizes with smaller glycosaminoglycan chains (but larger than those from bovine nasal cartilage proteoglycans) composed exclusively of chondroitin 4-sulphate. The major proteoglycans were incapable of interacting with hyaluronic acid. In general, the amino acid compositions of the major proteoglycans of rat incisors resembled that of bovine nasal cartilage proteoglycans, but the former had lower proline, valine, isoleucine, leucine, and higher aspartic acid, contents.     

The influence of environmental agents on prostaglandin biosynthesis and metabolism in the lung. Inhibition of lung 15-hydroxyprostaglandin dehydrogenase by exposure of guinea pigs to 100 per cent oxygen at atmospheric pressure.

The methods of Hascall & Sajdera (1969) were used to compare the proteoglycans of human intervertebral disc with those of bovine nasal cartilage. In contrast with cartilage, most of the hexuronate of disc could be extracted at low shear with water or dilute salt solutions. Extracts of disc with 4M-guanidinium chloride were centrifugated in 0.4M-guanidinium chloride in a CsCl gradient. Analytical ultracentrifugation of the hexuronate-containing heavy component revealed two fractions. both more polydisperse than those of cartilage. Also the more rapidly sediminting component was a much smaller fraction of the total. After prior extraction with 0.4M-guanidinium chloride, 4M-guanidinium chloride extracts of disc were found, by ultracentrifugal analysis, to be enriched in components resembling the proteoglycan monomer and aggregating factors of cartilage.     

Structure of proteoglycans from different layers of human articular cartilage

Full-depth plugs of adult human articular cartilage were cut into serial slices from the articular surface and analysed for their glycosaminoglycan content. The amount of chondroitin sulphate was highest in the mid-zone, whereas keratan sulphate increased progressively through the depth. Proteoglycans were isolated from each layer by extraction with 4M-guanidinium chloride followed by centrifugation in 0.4M-guanidinium chloride/CsCl at a starting density of 1.5 g/ml. The efficiency with which proteoglycans were extracted depended on slice thickness, and extraction was complete only when cartilage from each zone was sectioned at 20 microns or less. When thick sections (250 microns) were extracted, hyaluronic acid was retained in the tissue. Most of the proteoglycans, extracted from each layer under optimum conditions, could interact with hyaluronic acid to form aggregates, although the extent of aggregation was less in the deeper layers. Two pools of proteoglycan were identified in all layers by gel chromatography (Kav. 0.33 and 0.58). The smaller of these was rich in keratan sulphate and protein, and gradually increased in proportion through the cartilage depth. Chondroitin sulphate chain size was constant in all regions. The changes in composition and structure observed were consistent with the current model for hyaline-cartilage proteoglycans and were similar to those observed with increasing age in human articular cartilage.     

Purification and characterization of human platelet proteoglycan.

Freshly prepared platelets were shown to contain glycosaminoglycans equivalent to 530 micrograms of hexuronate/10(11) platelets. When the platelets were extracted with 4 M-guanidinium chloride containing proteinase inhibitors, and the extract was dialysed extensively against 7 M-urea solution, almost all of proteoglycan was recovered in the urea-soluble fraction. The proteoglycan was purified from the urea-soluble fraction with a yield of 47% by DEAE-Sephacel chromatography, CsCl-density-gradient centrifugation, Bio-Gel A-15m gel filtration and then rechromatography on DEAE-Sephacel. The purified proteoglycan contained 30% glucuronic acid, 32% N-acetylgalactosamine, 14% sulphate and 15% protein. Serine, glutamic acid, glycine, aspartic acid and leucine accounted for 64% of the total amino acids. The Mr of the proteoglycan was assessed to be approx. 136000 by sedimentation-equilibrium methods. The galactosaminoglycan released by alkaline-borohydride treatment of the proteoglycan was converted stoichiometrically into 4-sulphated unsaturated disaccharide by digestion with chondroitinase AC-II, indicating that the galactosaminoglycan was fully sulphated chondroitin 4-sulphate. The apparent Mr of the chondroitin sulphate was assessed to be 28000 by gel filtration on Bio-Gel A-0.5m (KD 0.18). On two-dimensional electrophoresis on a cellulose acetate membrane, the chondroitin sulphate gave a single compact spot co-migrating with a reference chondroitin sulphate, indicating that the chondroitin sulphate chains were homogeneous in both length and charge density. On the basis of these results, the proteoglycan in human platelets was concluded to be a macromolecule of Mr 136000 containing four chondroitin 4-sulphate chains each with the apparent Mr of 28000.     

Isolation of proteoglycans from human articular cartilage.

Proteoglycans were extracted from normal human articular cartilage of various ages with 4M-guanidinium chloride and were purified and characterized by using preformed linear CsCl density gradients. With advancing age, there was a decrease in high-density proteoglycans of low protein/uronic acid weight ratio and an increase in the proportion of lower-density proteoglycans, richer in keratan sulphate and protein. Proteoglycans of each age were also shown to disaggregate in 4M-guanidinium chloride and at low pH and to reaggregate in the presence of hyaluronic acid and/or low-density fractions. Osteoarthrotic-cartilage extracts had an increased content of higher-density proteoglycans compared with normal cartilage of the same age, and results also suggested that these were not mechanical or enzymic degradation products, but were possibly proteoglycans of an immature nature.     

Chondroitin sulphate proteoglycan in the substratum adhesion sites of Balb/c 3T3 cells. Fractionation on various ion-exchange and affinity columns.

Proteoglycans on the cell surface play critical roles in the adhesion of fibroblasts to a fibronectin-containing extracellular matrix, including the model mouse cell line Balb/c 3T3. In order to evaluate the biochemistry of these processes, long-term [35S]sulphate-labelled proteoglycans were extracted quantitatively from the adhesion sites of 3T3 cells, after their EGTA-mediated detachment from the substratum, by using an extractant containing 1% octyl glucoside, 1 M-NaCl and 0.5 M-guanidinium chloride (GdnHCl) in buffer with many proteinase inhibitors. Greater than 90% of the material was identified as a large chondroitin sulphate proteoglycan (Kav. = 0.4 on a Sepharose CL2B column), and the remainder was identified as a smaller heparan sulphate proteoglycan; only small amounts of free chains of glycosaminoglycan were observed in these sites. These extracts were fractionated on DEAE-Sepharose columns under two different sets of elution conditions: with acetate buffer (termed DEAE-I) or with acetate buffer supplemented with 8 M-urea (termed DEAE-II). Under DEAE-I conditions about one-half of the material was eluted as a single peak and the remainder required 4 M-GdnHCl in order to recover it from the column; in contrast, greater than 90% of the material was eluted as a single peak from DEAE-II columns. Comparison of the elution of [35S]sulphate-labelled proteoglycan with that of 3H-labelled proteins from these two columns, as well as mixing experiments, indicated that the GdnHCl-sensitive proteoglycans were trapped at the top of columns, partially as a consequence of their association with proteins in these adhesion-site extracts. Affinity chromatography of these proteoglycans on columns of either immobilized platelet factor 4 or immobilized plasma fibronectin revealed that most of the chondroitin sulphate proteoglycan and the heparan sulphate proteoglycan bound to platelet factor 4 but that only the heparan sulphate proteoglycan bound to fibronectin, providing a ready means of separating the two proteoglycan classes. Affinity chromatography on octyl-Sepharose columns to test for hydrophobic domains in their core proteins demonstrated that a high proportion of the heparan sulphate proteoglycan but none of the chondroitin sulphate proteoglycan bound to the hydrophobic matrix. These results are discussed in light of the possible functional importance of the chondroitin sulphate proteoglycan in the detachment of cells from extracellular matrix and in light of previous affinity fractionations of proteoglycans from the substratum-adhesion sites of simian-virus-40-transformed 3T3 cells.     

Proteoglycans of the intervertebral disc. Absence of degradation during the isolation of proteoglycans from the intervertebral disc.

Proteoglycans extracted with 4M-guanidinium chloride from pig intervetebral discs, and purified by equilibrium density-gradient centrifugation in CsCl, were of smaller hydrodynamic size than those extracted and purified in the same way from the laryngeal cartilage of the same animal. Whether this difference in size arose from degradation during the extraction and purification of the proteoglycans of the disc was investigated. Purified proteoglycans labelled either in the chondroitin sulphate chains or in the core protein were obtained from laryngeal cartilage by short-term organ culture. These labelled proteoglycans were added at the beginning of the extraction of the disc proteoglycans, and labelled cartilage and unlabelled disc proteoglycans were isolated and purified together. There was no appreciable loss of radioactivity after density-gradient centrifugation nor decrease in hydrodynamic size of the labelled cartilage proteoglycans on chromatography on Sepharose 2B, when these were present during the extraction of disc proteoglycans. It is concluded that disc proteoglycans are intrinsically of smaller size than cartilage proteoglycans and this difference in size does not arise from degradation during the extraction.     

Electron-microscopic and electrophoretic studies of bovine femoral-head cartilage proteoglycan fractions.

Proteoglycans (A1D1) extracted from bovine femoral-head cartilage were examined by electron microscopy using benzyldimethylammonium chloride as a spreading agent. The preparation contained a mixture of particles, some with a 'beaded' structure and a contiguous filamentous 'tail' at one end and others which appeared as round 'blobs', some of which also had filamentous tails. Previous electron-microscopic studies of proteoglycan monomers have indicated that their length distributions were apparently unimodal, a finding that contrasted with agarose/polyacrylamide-gel-electrophoresis results, which generally indicated two bands. In the present study proteoglycans isolated from the slowly migrating electrophoretic band were shown to be predominantly the larger molecules of beaded appearance, whereas the rapidly migrating proteoglycans were predominantly molecules with the 'blob-like' appearance. Gel-filtration, isopycnic-density-gradient-centrifugation and rate-zonal-centrifugation techniques were evaluated as means of proteoglycan fractionation by electron microscopy and agarose-gel electrophoresis. Rate-zonal centrifugation in mixed-salt gradients of caesium chloride/4 M-guanidinium chloride yielded the most effective fractionation.     

Isolation and characterization of proteoglycans synthesized by mouse osteoblastic cells in culture during the mineralization process.

Proteoglycans in mineralized (0.5 M-EDTA/4 M-guanidinium chloride-extractable) and non-mineralized (4 M-guanidinium chloride-extractable) matrices synthesized by a mouse osteoblastic-cell line MC3T3-E1 were characterized at different phases of mineralization in vitro. Cell cultures were labelled with [35S]sulphate and either [3H]glucosamine or 3H-labelled amino acids. At the mineralization phase a large majority of proteoglycans were extracted with 4 M-guanidinium chloride (G extract), and at least five species of labelled proteoglycans were identified; dermatan sulphate proteoglycans (DSPG), apparent Mr approx. 120,000 and 70,000), heparan sulphate proteoglycans (HSPG, apparent Mr approx. 200,000 and 120,000) and DS chains with very little core protein. DSPGs weakly bound to an octyl-Sepharose CL-4B column and HSPGs bound more tightly, whereas DS chains did not bind to the column. Amounts of labelled proteoglycans extracted with 0.5 M-EDTA/4 M-guanidinium chloride (EDTA extract) were much less than those in G extract. Although the predominant species in the EDTA extract were comparable with the DS or DSPGs in the G extract, none of them bound to octyl-Sepharose CL-4B, indicating their lack of hydrophobicity. At the nonmineralizing phase a large chondroitin sulphate proteoglycan (Mr greater than 600,000) was found in the matrix in addition to the five proteoglycan species similar to those at the mineralization phase. Although DS chains at the early phase were similar in size to those at the mineralization phase, the ratio of 2-acetamido-2-deoxy-3-O-(beta-D-gluco-4-enepyranosyluronic acid)-4-O-sulpho-D-galactose to 2-acetamido-2-deoxy-3-O-(beta-D-gluculo-4-enepyranosyluronic acid)-6-O-sulpho-D-galactose was less than that at the mineralization phase. These results agree with those of previous studies performed in vivo and suggest that alteration in the synthesis of proteoglycans is involved in the mineralization process. They also suggest that at the osteoblastic mineralization front proteoglycans undergo partial degradation and lose their hydrophobicity.     

Isolation and characterization of two sialoproteins present only in bone calcified matrix.

Two different sialoproteins were isolated from the mineralized matrix of bovine bone by using extraction with guanidinium chloride first without and then with EDTA. The sialoproteins were purified by chromatography on DEAE-cellulose eluted with a sodium acetate gradient in 7 M-urea, pH 6. Two sialoproteins (I and II) were then separated by chromatography on DEAE-cellulose eluted with a sodium chloride gradient in 7 M-urea, pH 4. The ratio between recovered sialoprotein I and II was 1:5. The chemical analysis of the two sialoproteins showed that they differed. Both, however, had very high contents of aspartic acid/asparagine and glutamic acid/glutamine though they differed markedly in contents of leucine and glycine. Both sialoproteins contained phosphate, sialoprotein I more than sialoprotein II. Content of sialic acid was substantially higher in the more prominent sialoprotein II (13.4% of dry weight) than in sialoprotein I (4.8% of dry weight). The peptide patterns produced by trypsin digests of [125I]iodinated sialoproteins I and II showed both structural similarities and structural differences. Sialoprotein II, being the major component, was characterized further. Its molecular mass was 57300 Da determined by sedimentation-equilibrium centrifugation in 6 M-guanidinium chloride, and its sedimentation coefficient (S0(20),w) was 2.53 S. Upon rotary shadowing, sialoprotein II appeared as an extended rod, having a core with an average length of 40 nm. Two types of oligosaccharides, N-glycosidically and O-glycosidically linked to the core protein, were isolated from sialoprotein II. Contents of mannose and sialic acid in the O-linked oligosaccharide were surprisingly high. Antibodies against sialoprotein II were raised in rabbits and an enzyme-linked immunosorbent assay was developed. Antigenicity of sialoprotein II was not affected by reduction and alkylation, was only partially lost upon trypsin digestion and was completely lost upon fragmentation of the core protein by alkaline-borohydride treatment, indicating that all antigenic sites were located in the protein portion. Sialoprotein I expectedly showed only partial immunological cross-reactivity with sialoprotein II. The quantity of sialoprotein II in bone extracts was found to be about 1.5 mg/g wet wt. of bone, but the protein was not detected in extracts of a number of other bovine tissues i.e. aorta, cartilage, dentine, kidney, liver, muscle, sclera, skin and tendon.     

Matrix proteins bound to associatively prepared proteoglycans from bovine cartilage.

Proteoglycans were extracted from bovine tracheal cartilage by high-speed homogenization, the use of dissociative solvents being avoided. The homogenate was fractionated by gel chromatography, sucrose-density-gradient centrifugation and ion-exchange chromatography. A previously unrecognized protein, cartilage matrix protein, was identified by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis. It cofractionated with the proteoglycans in all systems, indicating an interaction. The cartilage matrix protein-proteoglycan complex was dissociated by treatment with 4M-guanidinium chloride. The complex again formed when the guanidine was removed. The cartilage matrix protein has a mol.wt. of more than 200000. On reduction it yields subunits with a mol.wt. of approx. 60000.     

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.     

Non-collagenous proteins of predentine from dentinogenically active bovine teeth.

Predentin(e) was dissected out from unerupted permanent bovine teeth. The non-collagenous proteins were extracted at -13 degrees C by 4 M-guanidinium chloride containing proteinase inhibitors and separated by DEAE-Sepharose and Sephadex G-100 chromatography. In addition to a few minor constituents, the only major non-collagenous components that could be demonstrated were albumin and proteoglycan. The localization of the former, demonstrated by optical-microscopical immunochemistry, was such that it was concluded that albumin is not a constituent of predentin matrix. Very low amounts of phosphoprotein were found in predentin matrix. This was of two types, high- and low-phosphorylated. Larger amounts of phosphoprotein were not present until the dissection was carried deeper into newly formed dentin(e). On the basis of the present results and previously obtained morphological data the conclusion was drawn that predentin matrix, containing virtually only collagen type I and proteoglycan, is similar in composition to that of loose connective tissue and primarily aimed at the production and maturation of collagen fibres. Only immediately before the mineralization front are the non-collagenous protein components secreted that initiate and govern calcium-phosphate mineral formation.     

Synthesis of cartilage-specific proteoglycan by suspension cultures of adult chondrocytes.

Chondrocytes isolated from larynges of adult pigs were cultured as cell suspensions for at least 4 days before use. During continuous-labelling experiments in nutrient medium for 18h with 35SO42- and [3H]glucosamine as precursors, some macromolecular polyanionic material was synthesized which behaved on gel chromatography as proteoglycan. Gel chromatography on Sepharose 2B showed that a proportion of the proteoglycans in the medium appeared to be aggregated, and was dissociated in 4M-guanidinium chloride. Moreover the dissociated proteoglycan interacted with hyaluronic acid. Newly synthesized proteoglycan was larger than the average total cetylpyridinium chloride-precipitable material assayed as uronic acid alone.     

A biochemical and immuno-electron microscopical analysis of chondroitin sulphate-rich proteoglycans in human alveolar bone

This study used biochemical and immunohistochemical methods to characterize the chondroitin sulphate-rich proteoglycans from human alveolar bone obtained from an oral source. Proteoglycans were extracted from bone by a sequential 4 m guanidine HCl extraction process, and purified by DEAE-ion exchange chromatography. SDS-PAGE and Western blot analysis, using CS-56 monoclonal antibody, demonstrated one major proteoglycan species with a core protein of 58 kDa, glycosaminoglycan chains of 45--66 kDa and a mean molecular weight of 205 kDa. This work confirmed the biochemistry of chondroitin sulphate-rich proteoglycans from a novel source of adult human alveolar bone, and pointed towards a proteoglycan with a high glutamate, glycine, aspartate, alanine, serine and leucine content. Sections of alveolar bone were embedded in LR White resin, labelled with CS-56 antibody and examined with the light and electron microscopes. At the light microscope level, labelling was restricted to the osteocyte lacunae and canaliculi. Ultrastructural observations showed that the labelling was localized to fine filamentous material in the walls of the osteocytes and canaliculi. Sparse labelling was associated with the collagen fibres immediately subjacent to the lamina limitans, but no labelling of the mineralized matrix was observed. These findings also indicated subtle differences in the distribution of chondroitin sulphate compared with previously reported work, which may indicate species or age differences in the samples used in this study. Ultrastructural analysis confirmed and extended observations of glycosaminoglycan localization at the osteocyte cell membrane of mature human alveolar bone     

Hyaluronic acid in cartilage and proteoglycan aggregation

1. Dissociation of purified proteoglycan aggregates was shown to release an interacting component of buoyant density higher than that of the glycoprotein-link fraction of Hascall & Sajdera (1969). 2. This component, which produced an increase in hydrodynamic size of proteoglycans on gel chromatography, was isolated by ECTEOLA-cellulose ion-exchange chromatography and identified as hyaluronic acid. 3. The effect of pH of extraction showed that the proportion of proteoglycan aggregates isolated from cartilage was greatest at pH4.5. 4. The proportion of proteoglycans able to interact with hyaluronic acid decreased when extracted above or below pH4.5, whereas the amount of hyaluronic acid extracted appeared constant from pH3.0 to 8.5. 5. Sequential extraction of cartilage with 0.15m-NaCl at neutral pH followed by 4m-guanidinium chloride at pH4.5 was shown to yield predominantly non-aggregated and aggregated proteoglycans respectively. 6. Most of the hyaluronic acid in cartilage, representing about 0.7% of the total uronic acid, was associated with proteoglycan aggregates. 7. The non-aggregated proteoglycans were unable to interact with hyaluronic acid and were of smaller size, lower protein content and lower keratan sulphate content than the disaggregated proteoglycans. Together with differences in amino acid composition this suggested that each type of proteoglycan contained different protein cores.     

Heterotypic tetramer (A2D2) complexes of non-epidermal keratins isolated from cytoskeletons of rat hepatocytes and hepatoma cells

Cytoskeletal residues obtained after extraction of rat liver and cultured rat hepatoma cells (line MH1C1) were used to isolate cytokeratin subunit complexes by solubilization in low salt buffer containing 4 M-urea. Alternatively, the complexes were prepared by solubilization of total cytoskeletal proteins in 9.5 M-urea or 6 M-guanidinium hydrochloride (Gu . HCl), followed by separation using reversed phase high pressure liquid chromatography and dialysis first against either 9.5 M-urea or 6 M-Gu . HCl and then against buffers containing either 4 M-urea or 2 M-Gu . HCl, respectively. The complexes contained only two cytokeratin polypeptides in a 1 : 1 ratio as demonstrated by electrophoresis and isoelectric focusing, i.e. components A (Mr 55,000; isoelectric point in 9.5 M-urea, pH 6.4) and D (Mr 49,000; isoelectric point, pH 5.38) which were separated from each other at urea concentrations higher than 7 M. The complex had a sedimentation coefficient S25,w of 4.96 S in 2 M-Gu . HCl. Sedimentation equilibrium analysis gave an average Mr value of 207,000 which was interpreted as a tetramer containing two chains each of A and D. This complex was also directly demonstrated by gel electrophoresis under non-dissociating conditions. Using dimethyl suberimidate to cross-link the complex in solution of 4 M-urea or 2 M-Gu . HCl, we identified covalently linked heterodimers of A and D, and a tetrameric unit containing equal amounts of A and D which was the largest cross-link product obtained. This complex was similar to the tetrameric complex of rat and human vimentin formed under the same conditions. The constituents of the cross-linked products were identified by two-dimensional ("diagonal") gel electrophoresis, involving the cleavage of the bis(amidine) cross-links after the initial separation in the first dimension. Identical cross-link products were recognized when cytokeratin filaments were used. By electron microscopy the complexes appeared as threads of 2 to 3 nm diameter with a mean length of approximately 48 nm. On dialysis to low salt buffer, the complexes formed 2 to 3 nm protofilaments, intertwisted 3 to 4 nm protofilaments and typical 7 to 11 nm intermediate-sized filaments. Complexes formed from equivalent cytokeratins of other species such as man and cow, as well as heterologous recombinations such as human component A mixed with bovine component D and vice versa, showed the same characteristics.(ABSTRACT TRUNCATED AT 400 WORDS)     

Isolation and characterization of a low molecular weight chondroitin sulfate proteoglycan from rabbit skeletal muscle

Proteoglycans may be implicated in the process of aggregation of acetylcholine receptors in the basal lamina of skeletal muscle and possibly in the mechanism of reinnervation at the neuromuscular junction. In order to further deduce the role of such proteoglycans, we have sought to isolate them and define their molecular structures. In this study, proteoglycans were extracted from rabbit skeletal muscle by using 4 M guanidine hydrochloride and were purified by sequential cesium chloride density gradient ultracentrifugation, DEAE-cellulose ion-exchange chromatography, and Sepharose CL-6B and CL-2B gel filtration under dissociative conditions. A chondroitin sulfate proteoglycan which constituted about 44% of the total hexuronic acid content of the muscle tissue was isolated. This proteoglycan was found to have an apparent molecular weight [by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE)] of 95,000, consistent with its small hydrodynamic size (Kav = 0.8 on Sepharose CL-2B), and to consist of peptide and glycosaminoglycan in a weight ratio of 1.0/0.8. The average molecular weight of its core protein-oligosaccharide remnants is 50,000, as estimated by SDS-PAGE of the chondroitinase ABC digested proteoglycan. Alkaline NaB3H4 treatment of the intact proteoglycan released chondroitin sulfate chains with an average molecular weight of 21,000. Pronase digestion of the intact proteoglycan generated glycosaminoglycan-peptides with an average of two chondroitin sulfate chains per peptide. These two saccharide units account for the total glycosaminoglycans per molecule and appear to be closely spaced on the core protein.(ABSTRACT TRUNCATED AT 250 WORDS)