scholarly journals The electrophoretic heterogeneity of bovine nasal cartilage proteoglycans

1976 ◽  
Vol 157 (2) ◽  
pp. 357-367 ◽  
Author(s):  
P J Roughley ◽  
R M Mason
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Ionic Strength ◽  
Gel Electrophoresis ◽  
Proteolytic Enzymes ◽  
Gradient Centrifugation ◽  
Hydrodynamic Size ◽  
Nasal Cartilage ◽  
Large Pore ◽  
Low Ionic Strength

1. Proteoglycans were extracted from bovine nasal cartilage with 2.0M-CaC2 or with 0.15M-KCl followed by 2.0M-CaC2. Proteoglycan fractions were prepared from the extracts by density-gradient centrifugation in CsCl under ‘associative’ and ‘dissociative’ conditions. 2. The heterogeneity of the proteoglycan fractions was investigated by large-pore-gel electrophoresis. It was concluded that extracts made with 2.0M-CaCl2 or sequential 2.0M-CaCl2 contain two major species of proteoglycan ‘subunit’ of different hydrodynamic size, together with proteoglycan aggregates. Both ‘subunits’ have mobilities that are greater than those of proteoglycans obtained from pig articular cartilage McDevitt & Muir (1971) Anal. Biochem. 44, 612-622] and are therefore probably smaller in size than the latter. 3. Proteoglycan fractions isolated from cartilage extracted lith 0.15M-KCl separated into two main components on large-pore-gel electrophoresis with mobilities greater than those of proteoglycans extracted with 2.0M-CaCl2. Proteoglycans extracted at low ionic strength from bovine nasal cartilage are of similar hydrodynamic size to those extracted from pig articular cartilage under the same conditions [McDevitt & Muir (1971) Anal. Biochem. 44, 612-622]. 4. The role of endogenous proteolytic enzymes in producing proteoglycan heterogeneity, particularly in low-ionic-strength cartilage extracts is discussed. 5. Hyaluronic acid and ‘link proteins’ were present in the proteoglycan fraction separated from KCl extracts as well as in the fraction separated from CaCl2 extracts. Hyaluronic acid can only be identified in proteoglycan fractions by large-pore-gel electrophoresis after proteolysis and further purification of the fraction. 6. Collagen was extracted by both salt solutions and was tentatively identified as type II. Small amounts of collagen appear to be associated with the proteoglycan-aggregate fraction from the high-ionic-strength extract but not with the corresponding fraction from the KCl extract.

scholarly journals The properties of proteoglycan prepared from human articular cartilage by using associative caesium chloride gradients of high and low starting densities

1985 ◽  
Vol 232 (1) ◽  
pp. 111-117 ◽  
Author(s):  
M T Bayliss ◽  
P J Roughley
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Proteinase Inhibitors ◽  
Density Gradient Centrifugation ◽  
Gradient Centrifugation ◽  
Human Articular Cartilage ◽  
Adult Human ◽  
Cscl Density Gradient ◽  
Proteoglycan Aggregates ◽  
Hyaluronic Acid Binding

Proteoglycan was extracted from adult human articular cartilage from both the knee and the hip, and A1 preparations were prepared by CsCl-density-gradient centrifugation at starting densities of 1.69 and 1.5 g/ml. Irrespective of whether the cartilage was diced to 1 mm cubes or sectioned to 20 micron slices there was always a lower proportion of both protein and proteoglycan aggregate in the A1 preparation prepared at 1.69 g/ml. Furthermore, the addition of exogenous hyaluronic acid to the extracts before centrifugation did not improve the yield of aggregate at 1.69 g/ml. These results were not affected by the presence of proteinase inhibitors in the extraction medium. It appears that adult human articular cartilage contains a high proportion of low-density proteoglycan subunits and hyaluronic acid-binding proteins that make most of the re-formed proteoglycan aggregates of a lower density than is usually encountered with younger human and mammalian hyaline cartilages.

scholarly journals Immunochemical analysis of cartilage proteoglycans. Cross-reactivity of molecules isolated from different species

1981 ◽  
Vol 199 (1) ◽  
pp. 81-87 ◽  
Author(s):  
J Wieslander ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Chondroitin Sulphate ◽  
Limb Bud ◽  
Binding Region ◽  
Human Cartilage ◽  
Nasal Cartilage ◽  
Cartilage Proteoglycans ◽  
Rabbit Articular Cartilage ◽  
Hyaluronic Acid Binding

Antibodies directed against whole bovine nasal-cartilage proteoglycan and against the hyaluronic acid-binding region and chondroitin sulphate peptides from the same molecule were used in immunodiffusion and immunoelectromigration experiments. Proteoglycans from bovine nasal and tracheal cartilage showed immunological identity, with all three antisera. Proteoglycans from pig hip articular cartilage, dog hip articular cartilage, human tarsal articular cartilage and rat chondrosarcoma reacted with all the antisera and showed immunological identity with the corresponding structures isolated from bovine nasal-cartilage proteoglycans. In contrast, proteoglycans from rabbit articular cartilage, rabbit nasal cartilage and cultured chick limb buds did not react with the antibodies directed against the hyaluronic acid-binding region, though reacting with antibodies raised against whole proteoglycan monomer and against chondroitin sulphate peptides. All the proteoglycans gave two precipitation lines with the anti-(chondroitin sulphate peptide) antibodies. Similarly, the proteoglycans reacting with the anti-(hyaluronic acid-binding region) antibodies gave two precipitation lines. The results indicate the presence of at least two populations of aggregating proteoglycan monomers in cartilage. The relative affinity of the antibodies for cartilage proteoglycans and proteoglycan substructures from various species was determined by radioimmunoassay. The affinity of the anti-(hyaluronic acid-binding region) antibodies for the proteoglycans decreased in the order bovine, dog, human and pig cartilage. Rat sternal-cartilage and rabbit articular-cartilage proteoglycans reacted weakly, whereas chick limb-bud and chick sternal-cartilage proteoglycans did not react. In contrast, the affinity of antibodies to chondroitin sulphate peptides for proteoglycans increased in the order bovine cartilage, chick limb bud and chick sternal cartilage, dog cartilage, rat chondrosarcoma, human cartilage, pig cartilage, rat sternal cartilage and rabbit cartilage.

scholarly journals The degradation of cartilage proteoglycans by tissue proteinases. Proteoglycan structure and its susceptibility to proteolysis

1977 ◽  
Vol 167 (3) ◽  
pp. 629-637 ◽  
Author(s):  
P J Roughley ◽  
A J Barrett
Keyword(s):  
Ionic Strength ◽  
Cathepsin D ◽  
Core Protein ◽  
Degradation Products ◽  
Gradient Centrifugation ◽  
Limited Proteolysis ◽  
Extraction Procedure ◽  
Cathepsin G ◽  
Nasal Cartilage ◽  
Cscl Density Gradient

1. Proteoglycan was obtained from bovine nasal cartilage by a procedure involving sequential extraction with a low-ionic-strength KCl solution, then a high-ionic-strength CaCl2 solution. Purification was by CsCl-density-gradient centrifugation. 2. The CaCl2- extracted proteoglycan was subjected to proteolytic degradation by papain, trypsin, cathepsin D, cathepsin B, lysosomal elastase or cathepsin G. Degradation was allowed to proceed until no further decrease in viscosity was detectable. 3. The size and chemical composition of the final degradation products varied with the different proteinases. Cathepsin D and cathepsin G produced glycosaminoglycan-peptides of largest average size, and papain produced the smallest product. 4. The KCl-extracted proteoglycan was intermediate in molecular size and composition between the CaCl2-extracted proteoglycan and the largest final degradation products, and may have been formed by limited proteolysis during the extraction procedure. 5. It is postulated that the glycosaminoglycan chains are arranged in groups along the proteoglycan core protein. Proteolytic cleavage between the groups may be common to the majority of proteinases, whereas clevage within the groups is dependent on the specificity of each individual proteinase.

scholarly journals N-terminal sequence of proteoglycan fragments isolated from medium of interleukin-1-treated articular-cartilage cultures. Putative site(s) of enzymic cleavage

1992 ◽  
Vol 284 (2) ◽  
pp. 589-593 ◽  
Author(s):  
P Loulakis ◽  
A Shrikhande ◽  
G Davis ◽  
C A Maniglia
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Amino Acid ◽  
Interleukin 1 ◽  
Human Articular Cartilage ◽  
Terminal Amino Acid ◽  
Protein Core ◽  
Nasal Cartilage ◽  
The Media ◽  
Terminal Amino

Bovine articular cartilage was cultured both in the presence and in the absence of human recombinant interleukin-1 alpha (IL-1) (100 units/ml). Addition of this cytokine stimulated matrix degradation approx. 3-fold. This increased degradation permitted characterization of the large chondroitin sulphate proteoglycan (aggrecan) fragments accumulating in the media. When compared with controls, the proteoglycans isolated from the medium of cultures treated with IL-1 exhibited a decrease in the Kav. (control 0.25; IL-1-treated 0.37), determined by Sepharose CL-2B chromatography. This decrease in proteoglycan size was accompanied by a decreased ability of these monomers to associate with hyaluronic acid. Thus only 20% of the proteoglycans isolated from the medium of IL-1-treated cultures, compared with 39% for control cultures, had the capacity to form high-M(r) aggregates with hyaluronic acid. SDS/PAGE analysis of the proteoglycans from the media of IL-1-treated cultures demonstrated several large proteoglycan protein-core bands (M(r) 144,000-380,000). The protein-core bands with M(r) 144,000-266,000 exhibited a significantly decreased reactivity with monoclonal antibody 1-C-6 (specific for domains G1 and G2). The N-terminal amino acid sequence of four of these protein-core bands (M(r) 144,000, 173,000, 214,000 and 266,000) yielded sequences LGQRPPV-Y-PQLF(E), AGEGP(S)GILEL-GAP(S)-AP(D)M, GLG-VEL-LPGE and (A)RGSVIL-AKPDFEV-P-A. A comparison of these N-terminal amino acid sequences with the published proteoglycan sequence for bovine nasal cartilage [Oldberg, Antonsson & Heinegård (1987) Biochem. J. 243, 255-259], rat chondrosarcoma [Doege, Sasaki, Horigan, Hassell & Yamada (1987) J. Biol. Chem. 262, 17757-17769] and human articular cartilage [Doege, Sasaki, Kimura & Yamada (1991) J. Biol. Chem. 266, 894-902] permitted assignment of their relative positions on the core protein. Furthermore, on the basis of this similarity to published sequence, putative sites of enzymic cleavage were constructed. These theoretical cleavage sites revealed a glutamic acid residue in the P1 position and an uncharged polar or non-polar residue in the P1′ position.

The antigenicity of myosin and C-protein

1976 ◽  
Vol 192 (1109) ◽  
pp. 439-449 ◽  
Keyword(s):  
Ionic Strength ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecylsulphate ◽  
C Protein ◽  
The Past ◽  
Homologous Antiserum ◽  
Conventional Manner ◽  
Low Ionic Strength ◽  
Repeated Precipitation

Rabbit myosin prepared in the conventional manner by repeated precipitation at low ionic strength was recently shown to contain substantial amounts of impurities; the principal impurity is a component of the myofibril called C-protein. Because antiserum to such conventionally prepared myosin has been used in the past for labelling studies of muscle, it was necessary to study the immunological characteristics of myosin and C-protein and in particular to test the specificity of this antiserum. Antisera to both rabbit myosin and C-protein have been successfully elicited in goats. These antisera have been analysed by immunodiffusion and by precipitin reactions in solution. The analysis has been helped by the examination of immunoprecipitates by polyacrylamide gel electrophoresis in the presence of sodium dodecylsulphate. It is concluded that: ( a ) C-protein and myosin are antigenically distinct and therefore that C-protein is not derived from myosin. ( b ) Purified myosin can behave as a classically simple antigen giving a single precipitin line when diffused against its homologous antiserum. ( c ) C-protein is a powerful immunogen; the amount present as an impurity in myosin prepared in the conventional way by repeated precipitation at low ionic strength is capable of eliciting a large amount of antibody. Consequently the pattern obtained by labelling myofibrils with antiserum to conventionally prepared myosin would contain information about the location of C-protein superimposed on information about the location of myosin.

Detection of multiple forms of proteolytic enzymes by starch gel electrophoresis

1968 ◽  
Vol 46 (10) ◽  
pp. 1317-1320 ◽  
Author(s):  
E. kaminski ◽  
W. Bushuk
Keyword(s):  
Ionic Strength ◽  
Gel Electrophoresis ◽  
Direct Detection ◽  
Proteolytic Enzymes ◽  
Urea Concentration ◽  
Starch Gel Electrophoresis ◽  
Electrophoretic Separation ◽  
Multiple Forms ◽  
Starch Gel ◽  
Sensitive Method

A rapid and sensitive method for the direct detection of multiple forms of proteolytic enzymes by starch gel electrophoresis is described. The location of the enzyme components is identified by the degradation of hemoglobin which is included in the starch gel. This method was used to identify the enzyme components of the 10 commercial proteolytic enzymes bromelain, chymotrypsin, ficin, papain, pepsin, pronase B, protease, proteinase, and two preparations of trypsin. The effects of urea concentration and the ionic strength of aluminium lactate buffer were also examined. The best results were obtained with 3 M urea and with an ionic strength of 0.1 for the lactate buffer. It was observed that the number of enzyme components decreased with increasing concentrations of urea or increasing ionic strength of lactate buffer. The number of enzyme components did not always correspond to the number of protein bands. Self-digestion occurred in some of the protein bands in the starch gel after electrophoretic separation of the proteolytic enzymes.

Assembly of 2.5 nm filaments from giardin, a protein associated with cytoskeletal microtubules in Giardia

1985 ◽  
Vol 78 (1) ◽  
pp. 205-231 ◽  
Author(s):  
R. Crossley ◽  
D. Holberton
Keyword(s):  
Ionic Strength ◽  
Sodium Dodecyl Sulphate ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Sodium Dodecyl ◽  
Structural Features ◽  
Polyacrylamide Gel ◽  
Structural Proteins ◽  
Low Ionic Strength ◽  
Uniform Population

The giardins are a family of approximately 30000 Mr structural proteins found in microribbons attached to microtubules in the disc cytoskeleton of Giardia. After examining the solubility of giardins in various agents, a method has been developed to extract these polypeptides and subsequently precipitate them selectively. The giardin chains are soluble in 10 mM-HEPES/EDTA buffer at high pH and low ionic strength, but become insoluble in 10 mM-MES/EDTA buffer at pH 6.7 when the ionic strength is raised above 50 mM salt. By dialysing giardin extracts in turn against dissociating and reassembly buffers, the purification is obtained of a subset of giardin chains identified by sodium dodecyl sulphate/polyacrylamide gel electrophoresis as the cytoskeleton bands 14a, 14b and 15. The structures forming under assembly conditions are all composed of fine filaments, 2–3 nm in diameter. Filaments after the first cycle of assembly are found in bundles, narrow ribbons of two or three filaments, and large ordered tactoids. Assembly after a second cycle of solubilization yields a more uniform population of long ribbons. Both the tactoids and the second cycle ribbons are transversely banded at the 15 nm interval characteristic of microribbons in the cytoskeleton. Filaments in the tactoids are precisely placed at a centre-to-centre separation of 2.5 nm. Other structural features of the tactoids are discussed in relation to the association behaviour and possible dimensions of the giardin molecular subunit.

scholarly journals Four classes of cell-associated proteoglycans in suspension cultures of articular-cartilage chondrocytes

1986 ◽  
Vol 233 (3) ◽  
pp. 809-818 ◽  
Author(s):  
Y Sommarin ◽  
D Heinegård
Keyword(s):  
Hyaluronic Acid ◽  
Articular Cartilage ◽  
Cell Surface ◽  
Gel Electrophoresis ◽  
Polyacrylamide Gel Electrophoresis ◽  
Suspension Cultures ◽  
Polyacrylamide Gel ◽  
Side Chains ◽  
Dermatan Sulphate ◽  
Core Proteins

The characteristics of cell-associated proteoglycans were studied and compared with those from the medium in suspension cultures of calf articular-cartilage chondrocytes. By including hyaluronic acid or proteoglycan in the medium during [35S]sulphate labelling the proportion of cell-surface-associated proteoglycans could be decreased from 34% to about 15% of all incorporated label. A pulse-chase experiment indicated that this decrease was probably due to blocking of the reassociation with the cells of proteoglycans exported to the medium. Three peaks of [35S]sulphate-labelled proteoglycans from cell extracts and two from the medium were isolated by gel chromatography on Sephacryl S-500. These were characterized by agarose/polyacrylamide-gel electrophoresis, by SDS/polyacrylamide-gel electrophoresis of core proteins, by glycosaminoglycan composition and chain size as well as by distribution of glycosaminoglycans in proteolytic fragments. The results showed that associated with the cells were (a) large proteoglycans, typical for cartilage, apparently bound to hyaluronic acid at the cell surface, (b) an intermediate-size proteoglycan with chondroitin sulphate side chains (this proteoglycan, which had a large core protein, was only found associated with the cells and is apparently not related to the large proteoglycans), (c) a small proteoglycan with dermatan sulphate side chains with a low degree of epimerization, and (d) a somewhat smaller proteoglycan containing heparan sulphate side chains. The medium contained a large aggregating proteoglycan of similar nature to the large cell-associated proteoglycan and small proteoglycans with dermatan sulphate side chains with a higher degree of epimerization than those of the cells, i.e. containing some 20% iduronic acid.

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