The biosynthesis in vitro of chondroitin sulphate in neonatal rat epiphysial cartilage

Abstract

1. A system is described, which was used to incubate neonatal rat epiphysial cartilage in vitro with [U-14C]glucose and [35S]sulphate. 2. The acid glycosaminoglycans of neonatal rat epiphyses were extracted and fractionated on cetylpyridinium chloride–cellulose columns. The major components were chondroitin 4-sulphate (65%), chondroitin 6-sulphate (15%), hyaluronic acid (4%) and keratan sulphate (2%). 3. The acid-soluble nucleotides and intermediates of glycosaminoglycan synthesis were separated on a Dowex 1 (formate) system. The tissue contents and cellular concentrations of these metabolites were determined. 4. The rates of synthesis of UDP-glucuronic acid and UDP-N-acetyl-hexosamine from [U-14C]glucose were found to be 0.79±0.16 and 3.2±0.08nmol/min per g wet wt. respectively. 5. The incorporation of [U-14C]glucose into the uronic acid and hexosamine moieties of the polymers was also measured and the turnover rates of the glycosaminoglycans were calculated. It was found that chondroitin sulphate was turning over in about 70h and hyaluronic acid in about 120h. 6. The relative rates of synthesis of the sulphated glycosaminoglycans were calculated from [35S]sulphate incorporation and were found to be in good agreement with those obtained from [U-14C]glucose labelling.