No abstract

A fraction containing chondroitin sulphate, isolated from bovine cortical bone under mild conditions, was separated by ion-exchange chromatography into three fractions with apparent homogeneity on electrophoresis and ultracentrifugation. Two of these appeared to consist of chondroitin sulphate bound to a glycoprotein ;core' that had similarities to the bone sialoprotein described previously. The differences in composition of the two fractions were considered to be due to variation in the number or lengths of the polysaccharide chains. The presence of xylose and the alkali-lability of the bond between protein and polysaccharide suggested the presence of a xylosylserine linkage. The third fraction had the properties of a relatively pure chondroitin sulphate which contained a small amount of peptide. These fractions differed considerably from the protein-polysaccharide complexes of epiphysial and other cartilages, and their relevance to the possible role of glycosaminoglycans is discussed.

1.A fraction (G2) which contained 300/, of the total non-collagenous proteins was prepared from neutral EDTA extracts of bovine cortical bone. Three hitherto undescribed proteins (G2B-glycoprotein, GZC-glycoprotein, G~C F ) were identified in this fraction together with collagen, serum albumin, immunoglobulin G and transferrin.2 . The G2B-glycoprotein was prepared from the G2 fraction and shown to be homogenous by electrophoretic and immunochemical criteria.3. The G2B-glycoprotein was demonstrated to be present in bovine plasma at a concentration of approximately 20 mg/lOO ml. It was found that in the collagenase digest of decalcified bone matrix the ratio of the GZB-glycoprotein concentration to the serum albumin or immunoglobin G concentration was upto 250 times higher than the ratio in plasma.hexosamines and 14O/, neutral sugars, and had an apparent molecular weight of around 50000. 4.The G2B-glycoprotein contained approximately 7S0/, protein, 4O/, sialic acids, 3Analyses of mature cortical bone demonstrate that collagen constitutes OOo/, by weight of the organic matrix [l]. This has led to the frequent assumption that the early stages of calcification must occur in a highly collagenous matrix. It has been shown, however, that in the osteoid border where the first mineralized deposits are formed, non-collagenous protein constitute between 30°/, and 50°/, (w/w) of the total protein, and that during the course of mineralization the non-collagenous protein content decreases while the amount of collagen remains constant [2,3]. A similar decrease in the amount of protein polysaccharides during mineralization has been demonstrated [4].Plasma proteins [5,6], peptides [7], proteoglycans and glycoproteins [S -111 have been identified as constituents of the non-collagenous protein pool present in compact bone. The proteoglycans and acidic glycoproteins of bovine and rabbit bone have been isolated and characterized in some detail but the presence of considerable amounts of other glycoproteins has also been demonstrated [ll, 121. The present studies were directed toward the isolation and characterization of components from the major glycoprotein fraction prepared from the EDTA extract of bovine cortical bone. Abbreviation. IgG, immunoglobulin G.Enzyme. Collagenase (EC 3.4.4.19). MATERIALS AND METHODS Preparation of Bone FractionsAdult bovine cortical bone was powdered and extracted with sodium-EDTA solutions a t pH 7.5 [13] and the EDTA extract fractionated according to the method of Herring [S], represented schematically in Scheme 1. Recoveries of protein, hydroxyproline and sialic acid were determined a t each step.A sample (240 mg) of the G2 fraction was separated into several components by chromatography on a column (1 x I0 cm) of DEAE-cellulose (DE32, W & R. Balston, Kent, England) which had been equilibrated with 0.05M Tris-HC1 buffer pH 7.2. The column was eluted with this equilibration buffer (100 ml), followed by a linear salt gradient of 0 to 0.4 M NaCl in 0.05 M Tris-HC1 buffer pH 7.2 (total volume 200 ml). ...

1. An analysis of bovine bone sialoprotein, a homogeneous glycoprotein isolated from cortical bone, is presented. 2. Analytical results agree with earlier physical measurements indicating a molecular weight of about 23000. 3. Mild acid hydrolysis and treatment with neuraminidase showed that fucose and sialic acid occupy terminal positions on oligosaccharide chains. 4. Treatment of the sialic acid-free glycoprotein with beta-galactosidase showed that much of the galactose occupies a sub-terminal location in the intact glycoprotein. 5. The polypeptide chain is rich in aspartic acid, glutamic acid, serine, threonine and glycine, and has no detectable free terminal amino group. 6. Glycopeptides were studied after proteolytic digestion. 7. It is considered that the carbohydrate moiety is highly branched and is probably linked by an acid- and alkali-stable glycosylamine bond involving aspartic acid.