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.     

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

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

Release of O-sulfate groups under mild acid hydrolysis conditions used for estimation of N-sulfate content

The treatment of chondroitin sulfate isolated from cultured B16 mouse melanoma cells with 0.04 M HCl at 100°C for 90 min released up to 45% of O-sulfate residues as free inorganic sulfate. In addition to the release of inorganic sulfate, extensive degradation of this polysaccharide as well as of cartilage chondroitin sulfate, pig rib cartilage proteoglycan, heparin and hyaluronic acid was also evident under these conditions. The above hydrolysis conditions are used for characterizing ³⁵S-labeled heparan sulfates synthesized by cultured cells and to calculate ratio of N- and O-sulfates in these molecules. Our results suggest that caution in necessary in interpreting the results of mild acid hydrolysis of glycosaminoglycans.     

Hydraulic conductivity of chondroitin sulfate proteoglycan solutions

The hydraulic conductivity of solutions of Swarm rat chondrosarcoma proteoglycan subunit and of chondroitin 4- and 6-sulfate up to concentrations of 80 mg ml-1 have been measured under physiological conditions using sedimentation velocity and membrane ultrafiltration techniques. This study establishes the very high flow resistance of the proteoglycan and that this resistance is due to its constituent chondroitin sulfate chains. We have also demonstrated little difference in the hydraulic conductivity of chondroitin 4-sulfate as compared to chondroitin 6-sulfate. Studies of hydraulic conductivity of chondroitin sulfate and proteoglycan subunit over a range of salt concentrations demonstrate that the chondroitin sulfates exhibit only a small degree of electrolyte dissipation indicating that their constituent charge groups do not significantly contribute to flow resistance at high mechanical pressures. It appears that the shape and conformation of the polysaccharide backbone and its glycosidic linkages are the factors that primarily govern flow resistance. This is also consistent with the fact that hydraulic conductivity of the proteoglycans and chondroitin sulfates is considerably lower than that of its more charged counterpart heparin but has similar values to hyaluronate. Qualitative agreement between sedimentation analysis and ultrafiltration measurements is also established although the latter technique suffers from not knowing over what distance, adjacent to the membrane, ultrafiltration takes place. It is predicted that the proteoglycans will significantly contribute to flow resistance of cartilagenous tissues which confirms the Maroudas correlation that high proteoglycan concentration in cartilage yields high flow resistance. Further, we establish through a comparison of hydraulic conductivity measurements on hyaluronate, desulfated chondroitin sulfate, chondroitin sulfate, and proteoglycan subunit and osmotic pressure measurements of hyaluronate and proteoglycan that the sulfate groups of the chondroitin sulfate chain play only a small role in the net movement of water relative to the proteoglycan.     

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)     

Mucopolysaccharide-polypeptide interactions: Effect of the position of the sulfate group

The differences in the interaction in solution of poly(l-lysine) with chondroitin 6-sulfate (chondroitin sulfate C) and with chondroitin 4-sulfate (chondroitin sulfate A) have been studied by circular dichroism spectroscopy. Both mucopolysaccharides force the poly(l-lysine) to adopt the α-helix in solution rather than the charged coil form expected at neutral pH. The observed spectra indicates that the polypeptide is at least 80% helical when the 6-sulfate form is present, but only about 20% α-helical in the presence of chondroitin 4-sulfate. Thus chondroitin66-sulfate has a stronger conformation directing effect on poly(l-lysine) than does the 4-sulfate, which is probably due to the different positions of the sulfate group on the polysaccharide c chain.     

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.     

Stimulation of human arterial smooth muscle cell chondroitin sulfate proteoglycan synthesis by transforming growth factor-beta

Summary Human platelet-derived transforming growth factor-beta (TGF-beta) is a cell-type specific promotor of proteoglycan synthesis in human adult arterial cells. Cultured human adult arterial smooth muscle cells synthesized chondroitin sulfate, dermatan sulfate, and heparan sulfate proteoglycans, and the percent composition of these three proteoglycan subclasses varied to some extent from cell strain to cell strain. However, TGF-beta consistently stimulated the synthesis of chondroitin sulfate proteoglycan. Both chondroitin 4- and chondroitin 6-sulfate were stimulated by TGF-beta to the same extent. TGF-beta had no stimulatory effect on either class of [³⁵S]sulfate-labeled proteoglycans which appeared in an approximately 1:1 and 2:1 ratio of heparan sulfate to dermatan sulfate of the medium and cell layers, respectively, of arterial endothelial cells. Human adult arterial endothelial cells synthesized little or no chondroitin sulfate proteoglycan. Pulse-chase labeling revealed that the appearance of smooth muscle cell proteoglycans into the medium over a 36-h period equaled the disappearance of labeled proteoglycans from the cell layer, independent of TGF-beta. Inhibitors of RNA synthesis blocked TGF-beta-stimulated proteoglycan synthesis in the smooth muscle cells. The incorporation of [³⁵S]methionine into chondroitin sulfate proteoglycan core proteins was stimulated by TGF-beta. Taken together, the results presented indicate that TGF-beta stimulates chondroitin sulfate proteoglycan synthesis in human adult arterial smooth muscle cells by promoting the core protein synthesis. Supported in part by grants from the Public Health Service, U.S. Department of Health and Human Services, Washington, DC (CA 37589 and HL 33842), RJR Nabisco, Inc., and Chang Gung Biomedical Research Foundation (CMRP 291).     

Isolation and Purification of Chondroitin 6-Sulfate Protein From Umbilical Cord

Intact chondroitin 6-sulfate protein can be extracted from umbilical cord with dilute saline. Hyaluronic acid which is also extracted, is removed by precipitation with cetylpyridinium chloride followed by washing of the precipitate with aqueous sodium chloride. Subsequent purification is effected by passage through cation and anion exchange resins. Elution from the latter with salt solutions of increasing concentration yields chondroitin 6-sulfate proteoglycan in two fractions. The product is isolated from each of the fractions as the calcium salt by fractional precipitation with ethanol. The protein moiety can be cleaved from the mucopolysaccharide either by proteolytic digestion or treatment with alkali. The results obtaired on reaction with alkali and with sodium borohydride indicate that the polysaccharide is covalently linked to the protein through a serine unit.     

Effect of beta-xylosides on synthesis of cartilage-specific proteoglycan in chondrocyte cultures.

Monolayer cultures of embryonic chick chondrocytes were incubated with ³⁵SO₄ in the presence and absence of 1.0 mM p-nitrophenyl-β-D-xylose for 2 days. The relative amounts of chondroitin sulfate proteoglycan and free chondroitin sulfate chains were measured following gel filtration on Sephadex G-200. Synthesis of β-xyloside-initiated polysaccharide chains was accompanied by an apparent decrease in chondroitin sulfate proteoglycan production by the treated cultures. The amount of core protein was determined from equivalent numbers of β-xyloside-treated and untreated cells by a radioimmune assay. Similar amounts of core protein were found in both types of cultures, indicating that decreased synthesis of cartilage-specific core protein is not responsible for the observed decrease in overall chondroitin sulfate proteoglycan production.     

Effect ofin vitro differentiation on proteoglycan structure in cultured human monocytes

Parellel toin vitro differentiation of human monocytes into macrophage-like cells, the cells change their synthesis of glycosaminoglycans from chondroitin 4-sulfate to highly sulfated chondroitin sulfate, containing 4,6-disulfatedN-acetylgalactosamine units [Kolsetet al. (1983) Biochem J 210:661–67]. After exposure of monocyte cultures to [³⁵S]sulfate for 24h either from the onset of cultivation, prior to differentiation, or from day 4, after differentiation,³⁵S-macromolecules from medium and cell-layer were isolated and characterized. The cell-layer of day 5 cultures contained both proteoglycans and free polysaccharide chains, while the³⁵S-macromolecules present in the cell-layer of day 1 cultures and in medium of both monocytes and macrophage-like cells were almost exclusively of proteoglycan nature. Proteoglycans produced by macrophage-like cells were of larger size than the monocyte proteoglycans, most likely due to an increased polysaccharide chain length. These proteoglycans, in contrast to the monocyte-derived species, also showed affinity for fibronectin at physiological ionic strength.     

Purification and structural properties of the precursors of the globin messenger RNAs

X-ray diffraction data have been obtained from sodium and calcium salts of a proteoglycan rich in chondroitin 4-sulfate isolated from the Swarm rat chondrosarcoma. When sodium is the only countercation associated with the proteoglycan, the oriented polysaccharide chains adopt a 3-fold helical conformation in the solid state and pack in a trigonal unit cell with dimensions a = b = 1.45 nm and c = 2.88 nm. Addition of small amounts of calcium or full conversion of the polyanion from a sodium to a calcium salt form results in a conformational transition to a somewhat more extended 2-fold structure.For the calcium salt X-ray intensity data were used to refine the polysaccharide conformation and packing arrangement in the unit cell. Two antiparallel chains were found to crystallize in an orthorhombic unit cell with space group P22₁2₁ and dimensions a = 0.745 nm, b = 1.781 nm and c = 1.964 nm. The individual helix axes intersect the base plane of the unit cell at (x_f = 0, y_f = 0) and ($\text{x}{}_{\mathrm{f}}\text{= 0, y}{}_{\mathrm{f}}\text{=}\text{1}\text{2}$), and the polyanions are crystallographically equivalent, being related by the symmetry of the space group.The conformation of chondroitin 4-sulfate is stabilized intramolecularly by O.3 … O.5 hydrogen bonds across the β(1 → 4) linkage as well as by OSO^?₃ … Ca²⁺ … ^?OOC co-ordination across the β(1 → 3) linkage. Within the lattice adjacent parallel chains interact through COO^? … Ca²⁺ … ^?OOC bridges, and each calcium co-ordination shell is completed with an additional five water molecules to form a distorted, square antiprism. These water molecules are hydrogen-bonded to neighboring polyanions, and all intermolecular interactions involve water bridges or calcium ion co-ordination.On the basis of the refined packing model and the known structural features of the proteoglycan, models are considered for proteoglycan organization in connective tissue. Consideration of the conformational directing influence and relative abundance of calcium in the intercellular matrix suggest that the secondary structure of chondroitin 4-sulfate in vivo is likely to be similar to the conformation described in this study.     

Proteochondroitin sulfate synthesized in cartilages induced in vivo and in vitro by bone matrix gelatin

Implanted allogeneic demineralized bone matrix gelatin induced sequential development of cartilage and bone in the recipient rat muscle tissue. Proteoglycans of the implants labeled in vivo with [³⁵S]sulfate at different stages of development were analyzed by sucrose density gradient centrifugation. The major proteoglycan synthesized in day-5 implant, just prior to onset of chondrogenesis, was a dermatan sulfate-containing proteoglycan with relatively slow sedimentation rate. Additionally, a small amount of a faster sedimenting component could be detected. The faster sedimenting proteoglycan, in which chondroitin 4-sulfate accounted for 85% of total radioactivity, became predominant in day-10 sample when cartilage formation was maximal. By day 30, when cartilage had been replaced by newly formed bone, the synthesis of this faster sedimenting component had ceased. A similar, if not identical, proteoglycan was found to be a major one synthesized by the in vitro-induced cartilage. This proteoglycan was smaller in overall size and shorter in length of its chondroitin sulfate chains than a major proteoglycan component obtained from neonatal rat epiphyseal cartilage. Concurrent with these changes in proteoglycan type, there appeared to be a change in collagen type, since type II collagen, in addition to type I collagen, was synthesized in day-10 implant. These results indicate that the proteoglycan can be used as a molecular marker for chondrogenesis by bone matrix gelatin.     

Low-density lipoprotein binding affinity of arterial wall proteoglycans: characteristics of a chondroitin sulfate proteoglycan subfraction

The characteristics of an arterial wall chondroitin sulfate proteoglycan (CS-PG) subfraction that binds avidly to low-density lipoproteins (LDL) was studied. A large CS-PG was extracted from bovine aorta intima-media under dissociative conditions, purified by density-gradient centrifugation and gel filtration chromatography, and further subfractionated by affinity chromatography on LDL-agarose. A proteoglycan subfraction, representing 25% of the CS-PG, showed an elution profile (with dissociation from LDL-agarose occurring between 0.5 and 1.0 M NaCl) corresponding to that of heparin, heretofore considered to be the most strongly binding glycosaminoglycan with LDL. The proteoglycan subfraction which migrated as a single band on composite agarose-polyacrylamide gel electrophoresis contained chondroitin 6-sulfate, chondroitin 4-sulfate and dermatan sulfate in a proportion of 70:22:8. The core protein of the proteoglycan had an apparent molecular weight of 245,000, and contained approx. 33 glycosaminoglycan chains with an average molecular weight of 32,000. The CS-PG subfraction, like heparin, formed insoluble complexes in the presence of 30 mM Ca2+. Complexing of LDL with proteoglycan resulted in two classes of interactions with 0.1 and 0.3 proteoglycan monomer bound per LDL particle characterized by an apparent Kd of 4 and 21 nM, respectively. This indicates that multiple LDL particles bind to single proteoglycan monomers even at saturation. In contrast, LDL-heparin interactions showed a major component characterized by an apparent Kd of 151 nM and a Bmax of 9 heparin molecules per LDL particle. The occurrence of a potent LDL-binding proteoglycan subfraction within the family of arterial CS-PG may be of importance in terms of lipid accumulation in atherogenesis.     

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.     

Chromatography of glycosaminoglycans on hydrophobic gel. Correlation between chromatographic behavior of glycosaminoglycans on phenyl-sepharose CL-4B and their solubility in the presence of high concentrations of ammonium sulfate

The effect of bound sulfate groups and uronic acid residues of glycosaminoglycans on their behavior in chromatography on hydrophobic gel was examined by the use of several pairs of depolymerized chondroitin, chondroitin 4- or 6-sulfate, and dermatan sulfate having comparable degree of polymerization. Chromatography on Phenyl-Sepharose CL-4B in 4.0-2.0 ammonium sulfate containing 10m hydrochloric acid showed that: (a) The retention of depolymerized chondroitin 4- or 6-sulfate on the gel varies with the temperature, whereas the depolymerized samples of chondroitin and dermatan sulfate does not show a temperature dependence (this is not the case for hyaluronic acid or dextrans). (b) Among depolymerized samples of chondroitin and chondroitin 4- and 6-sulfate that have a similar degree of polymerization, chondroitin 4- and 6-sulfate showed the highest retention. (c) The retention on the gel of chondroitin 6-sulfate, chondroitin 4-sulfate, and dermatan sulfate decreased in this order. The solubility in ammonium sulfate solution of the polysaccharides agreed well with the chromatographic behavior, suggesting that the fractionation by the hydrophobic gel largely depends on the ability to precipitate on the gel rather than on the hydrophobic interaction between gel and polysaccharide.     

A monoclonal antibody that specifically recognizes a glucuronic acid 2-sulfate-containing determinant in intact chondroitin sulfate chain

Monoclonal antibodies produced against chick embryo limb bud proteoglycan (PG-M) were selected for their ability to recognize determinants on intact chondroitin sulfate chains. One of these monoclonal antibodies (IgM; designated MO-225) reacts with PG-M, chick embryo cartilage proteoglycans (PG-H, PG-Lb, and PG-Lt), and bovine nasal cartilage proteoglycan, but not with Swarm rat chondrosarcoma proteoglycan. The reactivity of PG-H to MO-225 is not affected by keratanase digestion but is completely abolished after chondroitinase digestion. Competitive binding analyses with various glycosaminoglycan samples indicate that the determinant recognized by MO-225 resides in a D-glucuronic acid 2-sulfate(beta 1----3)N-acetylgalactosamine 6-sulfate disaccharide unit (D-unit) common to antigenic chondroitin sulfates. A tetrasaccharide trisulfate containing D-unit at the reducing end is the smallest chondroitin sulfate fragment that can inhibit the binding of the antibody to PG-H. Decreasing the size of a D-unit-rich chondroitin sulfate by hyaluronidase digestion results in progressive reduction in its inhibitory activity. The results suggest that the epitope has a requirement for a long stretch of a disaccharide-repeating structure for a better fit to the antibody.     

A competitive assay of lipoprotein: proteoglycan interaction using a 96-well microtitration plate

A method for the microassay in vitro of lipoprotein: proteoglycan interactions is described. The wells of a plastic 96-well microtitration plate are coated with low density lipoprotein. A limiting quantity of biotin-conjugated proteoglycan is allowed to bind to each coated well, and the amount of the latter retained in wells is estimated spectrophotometrically through subsequent binding of alkaline phosphatase-conjugated avidin. Many of the incubation parameters (e.g., time, pH, salt concentration, divalent cations), which influence the extent of binding of biotin-conjugated proteoglycan, have been studied and optimized. The effect upon binding of introducing different levels of proteoglycans or lipoproteins at the interaction step can be measured readily. Thus, the orders of increasing relative binding affinities were found to be high density lipoprotein less than Lipoprotein (a) less than low density lipoprotein; rat chondrosarcoma proteoglycan less than bovine nasal cartilage proteoglycan less than human aorta proteoglycan; chondroitin 4-sulfate less than chondroitin 6-sulfate less than dermatan sulfate for lipoproteins, proteoglycans, and glycosaminoglycans, respectively.     

Modulation of cAMP-dependent protein kinase by derivatives of chondroitin sulfate

Here, we report investigations about the direct effect of glycosaminoglycans, such as dermatan sulfate, chondroitin 4- and 6-sulfate upon cAMP-dependent protein kinase activity. The results indicate that glycosaminoglycans strongly influence the phosphorylation activity of this enzyme against histone type IIa and [Val⁶,Ala⁷]-kemptide. While chondroitin 4-sulfate and dermatan sulfate exhibit inhibitory effects, chondroitin 6-sulfate shows a stimulating effect. In addition, the chondroitin 6-sulfate is also able to reduce the chondroitin 4-sulfate and dermatan sulfate specific inhibition.     

Effects of glycosaminoglycans and proteoglycan on the in vitro assembly and thermal stability of collagen fibrils

The effects of three glycosaminoglycans (chondroitin 6-sulfate, dermatan sulfate, and hyaluronate) and a proteoglycan on the kinetics of fibril formation and on the thermal stability of the in vitro assembled collagen fibrils, under physiological conditions of ionic strength and pH, have been examined. The glycosaminoglycans were found to influence the kinetics of collagen precipitation but not the thermal stability of the in vitro assembled fibrils. The proteoglycan was found to influence the kinetics of collagen precipitation and to reduce the thermal stability of the in vitro assembled fibrils. Comparison of the interaction occurring between chondroitin 6-sulfate and collagen under acidic conditions (0.05M acetic acid) and that occurring under physiological conditions showed that markedly different interaction products were formed under the different conditions.