Mucopolysaccharide Sulfation in Chick Embryo Cartilage

The sulfate acceptor present in boiled, dialyzed preparations of the 105,000 x g supernatant obtained from chick embryo cartilage was purified and characterized. Pretreatment of this supernatant with Pronase or alkali resulted in increased sulfate incorporation, while hyaluronidase treatment abolished or markedly reduced sulfate incorporation. The acceptor was purified by protamine chloride precipitation, Pronase digestion, and fractionation on Ecteola cellulose and Sephadex G-50. Ecteola fractionation and electrophoresis showed the most active acceptor fractions to be slightly deficient in sulfate, whereas Sephadex fractionation showed acceptor ability to be relatively independent of molecular size. This acceptor thus appears to be a molecule of the chondroitin sulfate type, the ability of which to accept sulfate is strongly influenced by its charge properties, but not by molecular size. A mechanism of sulfation was proposed, which is in agreement with the data and which involves sulfation occurring concurrently with polysaccharide elongation. Acid hydrolysis of the 35SO4=-labeled acceptor indicated that the primary site of sulfation was an axial hydroxyl group, which in this case would be at position 4 of the N-acetylgalactosamine moiety. However, pretreatment of acceptor with Pronase or alkali resulted in sulfation predominantly of an equatorial hydroxyl. Thus, it is possible that in the native proteinpolysaccharide complex the conformation might be such that the 4-positions of the acetylgalactosamine residues are most readily available to the sulfotransferase.