Isolation and characterization of proteoglycans from chick limb bud chondrocytes grown in vitro

Chick limb bud mesenchyme cells from stage 23-24 embryos were isolated and grown in culture under conditions facilitating chondrogenic development. Dissociative extraction methods were used to isolate proteoglycans from Day 8 cultures, at which time the incorporation of [35S]sulfate into these macromolecules occurred at maximal rates. The monomer (D1) fraction contained 85 to 90% of the proteoglycans originally present in the matrix of the cultures. The composition of this fraction was approximately 7 to 8% protein, 7% keratan sulfate, and 85% chondroitin sulfate. The proportions of nonsulfated, 4-sulfated, and 6-sulfated disaccharides in chondroitinase digests were about 11%, 31%, and 58%, respectively. The D1 fraction exhibited a single, polydisperse component on Sepharose 2B chromatography and in the ultracentrifuge (so = 19 S). In associative density gradients about 35% of the proteoglycans were recovered in a gel at the top of the gradient. The remainder were recovered at the bottom of the gradient in the aggregate (A1) fraction. The A1 fraction exhibited two components, aggregate (about 70% of the total) and monomer, upon Sepharose 2B chromatography and in the ultracentrifuge (so = 120 S for aggregate; 18 S for monomer). The aggregate preparation contained only one of the two link proteins (molecular weight of about 45,000) which occur in proteoglycan preparations from many hyaline cartilages.