Structural changes in the large proteoglycan in differentiating chondrocytes from the chick embryo tibiotarsus

35SO4(2-)- or [3H]GlcN-labeled heavy proteochondroitin sulfate was isolated from monolayer cultures of chondrocytes from the zones of dividing, elongated, and hypertrophying cells of chick embryo tibias, and the keratan sulfate (KS) component was characterized. The KS glycopeptides remaining after digestion of the proteoglycans with thermolysin and chondroitinases were isolated and depolymerized by hydrazinolysis and nitrous acid cleavage. The resulting KS disaccharides had nonreducing terminal D-galactose (Gal) residues and reducing terminal anhydro-D-mannose (AMan) residues. The KS fractions from all cultures had identical disaccharide compositions, with 18-20% Gal—-AMan, 72-79% Gal—-AMan(6-SO4), and 7-9% Gal(6-SO4)—-AMan(6-SO4). The ratios of chondroitin sulfate (CS) to KS synthesized by cultures of dividing, elongated, and hypertrophied chondrocytes were 15, 27, and 30, respectively. Approximately 30% of the CS chains of the proteochondroitin sulfate in the cell matrix pools had nonreducing terminal GalNAc(4,6-diSO4) residues, but none of the CS chains in the proteochondroitin sulfate recovered from the culture medium pools were terminated with these residues.