| Abstract: | Bovine aortic endothelial cells were cultured in medium containing 3H]glucosamine and concentrations of 35S]sulfate ranging from 0.01 to 0.31 mM. While the amount of 3H]hexosamine incorporated into chondroitin sulfate and heparan sulfate was constant, decreasing concentrations of sulfate resulted in lower 35S]sulfate incorporation. Sulfate concentrations greater than 0.11 mM were required for maximal 35S]sulfate incorporation. Chondroitin sulfate was particularly affected so that the sulfate to hexosamine ratio in 3H]chondroitin 35S]sulfate dropped considerably more than the sulfate to hexosamine ratio in 3H] heparan 35S]sulfate. Sulfate concentration had no effect on the ratio of chondroitin 4-sulfate to chondroitin 6-sulfate. The ratios of sulfate to hexosamine in cell-associated glycosaminoglycans were essentially identical with the ratios in media glycosaminoglycans at all sulfate concentrations. DEAE-cellulose chromatography confirmed that sulfation of chondroitin sulfate was particularly sensitive to low sulfate concentrations. While cells incubated in medium containing 0.31 mM sulfate produced chondroitin sulfate which eluted later than heparan sulfate, cells incubated in medium containing less than 0.04 mM sulfate produced chondroitin sulfate which eluted before heparan sulfate and near hyaluronic acid, indicating that many chains were essentially unsulfated. At intermediate concentrations of sulfate, chondroitin sulfate was found in very broad elution patterns suggesting that most did not fit an "all or nothing" mechanism. Heparan sulfate produced at low concentrations of sulfate eluted with narrower elution patterns than chondroitin sulfate, and there was no indication of any "all or nothing" sulfation. |
