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Kinetics of mucopolysaccharide and glycoprotein synthesis by chick embryo chondrocytes. Effect of D-glucose concentration in the culture medium.
Late log cultures of chick embryo vertebral chondrocytes in Dulbecco's Modified Eagle's Medium with 10% fetal calf serum consume D-glucose from the culture medium at a rate of approximately 0.40 mumol per h per 10(6) cells. When the D-glucose concentration in the medium drops below 1 mumol per ml the glycogen stores are rapidly exhausted, and cell growth ceases. 35SO4(2)- is incorporated into chondroitin-6-SO4 and chondroitin-4-SO4 at linear rates of 1.2 and 0.4 nmol per h per 10(6) cells, respectively, until the D-glucose level in the medium drops below 1 mumol per ml, but there is always a slight lag in the initial appearance of chondroitin-4-SO4. Throughout the period of 35SO4 2- labeling, the amount of chondroitin-6-SO4 that is recovered in the cells exceeds the amount that is recovered in the medium, but the opposite is true for chondroitin-4-SO4. However, when cells prelabeled with 35SO4(2-) are then transferred to a label-free medium, the secretion of chondroitin sulfates proceeds at much slower rates, and the kinetics of chondroitin-6-SO4 and chondroitin-4-SO4 secretion are very similar. In this chase experiment the chondroitin sulfates are recovered quantitatively after a 24-h incubation period, indicating that these embryonic chondrocytes do not degrade the chondroitin sulfates under these culture conditions. The rate of incorporation of counts from D-[14C]glucosamine into mucopolysaccharides and glycoproteins increase with time. This nonlinear rate results from a progressive increase in the specific activity of the UDP-N-acetyl-D-[14C]hexosamine pool as the D-glucose in the culture medium is depleted. A linear relationship is demonstrated between the logarithm of the 14C counts per min per nmol of UDP-N-acetylhexosamine and the logarithm of the concentration of D-glucose in the culture medium over a range of 1 to 20 mumol of D-glucose per ml. The relative rates of appearance of counts from 35SO4(2-) and D-[14]glucosamine in chondroitin 4-SO4 and chondroitin-6-SO4 are used to calculate the specific activity of the UDP-N-acetyl-D-[14C]hexosamine pool at each stage in the labeling period. The resulting values are then used to calculate the rates of synthesis of the nonsulfated polymers, namely, chondroitin, hyaluronic acid, and glycoprotein.
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