Effect of d-Glucosamine Concentration on the Kinetics of Mucopolysaccharide Biosynthesis in Cultured Chick Embryo Vertebral Cartilage

The relative rates of synthesis of chondroitin 4-sulfate, chondroitin 6-sulfate, and hyaluronic acid by intact 8-day chick embryo vertebral cartilage are found to be influenced in a complex fashion by varying concentrations of d-glucosamine in the culture medium. At 0.17 mm d-glucosamine the intracellular pool of UDP-N-acetylhexosamine is less than 5 nmoles per mg of tissue protein and the rate of chondroitin 6-sulfate synthesis is approximately twice the rate of chondroitin 4-sulfate synthesis. When the concentration of d-glucosamine in the culture medium is increased to 2 mm, the UDP-N-acetylhexosamine level rapidly comes to a steady state level of 30 to 35 nmoles per mg of protein and the overall rate of mucopolysaccharide synthesis is approximately doubled over the rate observed at the lower d-glucosamine level. At 4 and 25 mm d-glucosamine, the pool size of UDP-N-acetylhexosamine remains at 30 to 35 nmoles per mg of protein, but the rate of synthesis of chondroitin 4-sulfate and chondroitin 6-sulfate is lowered, as is the rate of incorporation of l-[3H]leucine into protein. The rate of hyaluronic acid synthesis, on the other hand, increases progressively as the d-glucosamine concentration is raised to 25 mm. At 0.17 mm d-glucosamine, hyaluronic acid synthesis represents approximately 5 % of the total mucopolysaccharide synthesis; at 25 mm d-glucosamine, hyaluronic acid synthesis is 40 % of the total mucopolysaccharide synthesis. The incorporation of d-[14C]glucosamine into chondroitin 4-sulfate and chondroitin 6-sulfate proceeds with a lag. The lag is more pronounced for chondroitin 4-sulfate, and at all d-glucosamine concentrations chondroitin 6-sulfate is formed in excess of chondroitin 4-sulfate in the early stages of the incubation. However, at a time in the incubation which varies with the d-glucosamine concentrations, the rate of chondroitin 4-sulfate synthesis begins to exceed the rate of chondroitin 6-sulfate synthesis and after a 20-hour incubation, the total amount of chondroitin 4-sulfate that accumulates is in excess of the amount of chondroitin 6-sulfate. At all d-glucosamine concentrations, the sum of the rates of chondroitin 4-sulfate, chondroitin 6-sulfate, and chondroitin synthesis is linear; thus, the change in rates of synthesis of individual mucopolysaccharides is not accompanied by a diminution in the over-all rate of mucopolysaccharide synthesis. d-Galactosamine is incorporated into chondroitin sulfate at approximately one-tenth the rate of d-glucosamine incorporation. Increasing concentrations of d-glucosamine in the culture medium have the same qualitative effects on mucopolysaccharide synthesis as corresponding levels of d-glucosamine.