Biosynthesis of chondroitin sulfate side chains and hyaluronate. Time-course studies with cartilage slices of calf ribs under different conditions.

The time course of double labeling with 35SO24- and [3H]glucosamine was followed in a semi-in vitro system of cartilage slices from calf ribs whose chondroitin sulfate peptide pool consists of (A) less than 1% of very short under sulfated side chains of less than 10 disaccharide units length, (B) 3--5% of short under sulfated longer side chains (16 to 25 disaccharide units), (C) 3--5% of short, slightly oversulfated side chains (16--23 dissacharide units, very probably containing some dermatan sulfate), (D) the bulk material (74--82% of total uronate) of longest, slightly undersulfated or equally sulfated side chains (22--42 disaccharide units). After 10 min incubation rapid chain elongation with [3H]glucosamine and prelabeling with 35SO24- of endogenous acceptors are apparent. Chains of type A exhibit highest specific radioactivities. During 30--60 min incubation it is mainly chains of type B that show highest specific radioactivities, after 90 min chains of type C. On the after hand, chains of type D always incorporated the highest total amount of both precursors. Preincubation of slices for 40 min at 37 degres C strongly enhances labeling rates of all types A and B. After 10 min preincubation followed by 35SO24- labeling for 60 min, a decrease of radioactivity of type A and a distinct increase with type B are observed during the post incubation period. After pulse chase experiments type B exhibits highest specific radioactivities. The data make it evident that undersulfated short chondroitin sulfate side chains form very rapidly in a well organised manner and grow, by elongation and proceeding sulfation processes, to longer higher sulfated chains. The labeling of the hyaluronate pool is about half of that of the chondroitin sulfate pool after a lag phase of 10 min. The latter increases linearly after 35--45 min incubation time. However, after preincubation and chase experiments the hyaluronate pool is more highly labeled. The data indicate different precursor pools of both biosynthesis mechanisms, probably located in different cell compartments and/or different cartilage cells.