Comparative Fractionation Studies of Cartilage Proteinpolysaccharides

Abstract

Abstract Proteinpolysaccharides from bovine embryonic rudiments and calf nasal septa were compared by conventional ultracentrifugation in isotonic salt solutions, equilibrium density gradient centrifugation, and free flow electrophoresis. These methods are all suitable for use with large amounts of material and give reliable materials balances. Fractions obtained by any one method could be isolated and then refractionated by the other methods. The main distinction between fractions was in their composition. All fractions of the rudiment proteinpolysaccharide contained more protein than the corresponding nasal septum fractions. In gradient centrifugation, where the distribution of the proteinpolysaccharide was most influenced by protein content, much more of the rudiment proteinpolysaccharide than of the nasal septum proteinpolysaccharide was found in the less dense section of the gradient. Moreover, during conventional ultracentrifugation in isotonic salt solution, much more of the rudiment proteinpolysaccharide separated from the solution as a gel phase. All proteinpolysaccharide fractions obtained by the methods used in this study, regardless of composition, migrate to a characteristic peak area on electrophoresis. The protein-rich fractions of proteinpolysaccharide could be localized in the upper part of the gradient on centrifugation, in the separated, gel phase on conventional ultracentrifugation, and in the more slowly migrating section of the characteristic peak area on electrophoresis. Collagen is found in all high protein fractions and even, to a lesser extent, in the dense fraction of the rudiment proteinpolysaccharide. However, the collagen is not responsible for the distribution pattern of the proteinpolysaccharides on fractionation since that distribution is not altered by the removal of collagen with collagenase. The major component of protein-rich fractions from either tissue was shown to be proteinpolysaccharide by both analytical and electrophoretic criteria. The remixing experiments, which have attributed a specific linking function to a glycoprotein for the aggregation of proteinpolysaccharide, implicitly depend on the absence of proteinpolysaccharide in the low density fractions. Hence, the isolation of proteinpolysaccharide from these fractions questions the validity of such experiments as an unequivocal demonstration of a definitive role for the glycoprotein as a linking agent.