Sadao. Sato, F. Rahemtulla, C. Prince
Connective tissue research
Proteoglycans of bovine compact bone were purified by chromatography of the formic acid precipitate of an EDTA extract. The sequential chromatographic steps consisted of gel filtration on Sepharose CL-6B in 4-M guanidine HCl, ion-exchange chromatography on DEAE-Sephacel in 4-M urea and rechromatography on Sepharose CL-6B in 4-M guanidine HCl. The preparation consisted of a relatively small proteoglycan (Kav = 0.4 on Sepharose CL-6B) containing about 40% protein, 21% hexuronic acid, 23% galactosamine and lesser amounts of other monosaccharides. The core protein was shown by gradient NaDodSO4 gel electrophoresis, electrotransfer and immunodetection to be monodispersed with an Mr = 45,000. Analysis of glycopeptides obtained after papain digestion of the proteoglycan and separation from glycosaminoglycan chains by gel chromatography, indicated that both N-linked and O-linked oligosaccharides were present. The glycosaminoglycan chains liberated by papain digestion eluted from Sepharose CL-6B as a broad peak with Kav = 0.50, slightly ahead of the position of elution of bovine nasal cartilage glycosaminoglycans (Kav = 0.52); the bone glycosaminoglycans are thus slightly larger than those from cartilage and smaller than the ones attached to fetal bone proteoglycans. These chains were totally susceptible to chondroitinase AC II, a procedure that yielded unsaturated disaccharides corresponding predominantly to chondroitin-4-sulfate, and to a lesser extent chondroitin-6-sulfate. Antisera raised against adult bone proteoglycans cross-reacted with core protein of bone proteoglycan (obtained after chondroitinase digestion) but not with papain digested proteoglycan. In addition, they cross-reacted with core protein and trypsin-liberated, chondroitin sulfate rich region (AlTAl) derived from cartilage proteoglycans and, to a lesser extent, rat bone proteoglycans. No cross-reactivity could be detected to Smith-degraded cartilage proteoglycans, bone acidic glycoproteins or serum proteins.