Absence of covalently linked core protein from newly synthesized hyaluronate.
1. Primary cultures of chondrocytes from the Swarm rat chondrosarcoma were labelled with either [3H]glucosamine or [14C]glucosamine, and hyaluronate synthesized by the cells was isolated from the cell layer. Parallel cultures were labelled with either [3H]serine or [3H]lysine, and identical fractions were isolated from the cell layer. Some cultures were dual-labelled. 2. In cultures labelled with [3H]serine for between 30 min and 24 h and extracted with 4.0 M-guanidine, a procedure that solubilizes predominantly extracellular macromolecules, small amounts of [3H]serine-labelled molecules were found associated with the hyaluronate fraction purified from the extract by dissociative CsCl-density-gradient centrifugation and dissociative Sepharose CL-2B chromatography. About 75% of the [3H]serine-labelled molecules in the fraction were specifically associated with hyaluronate, since they could be removed by prior treatment with proteinase-free Streptomyces hyaluronidase. The association of the [3H]serine-labelled molecules with hyaluronate was non-covalent, since they could be separated from it by further centrifugation in CsCl density gradients containing 4 M-guanidinium chloride and a zwitterionic detergent. 3. In other experiments the cultures were extracted with a sequential zwitterionic-detergent/guanidinium chloride procedure that completely solubilized the cell layer and enabled fractions containing newly synthesized cell-associated hyaluronate to be isolated. Zwitterionic detergent was present throughout. No [3H]lysine was incorporated into these fractions, irrespective of whether the cultures were pulsed concurrently with [3H]lysine and [14C]glucosamine or sequentially with [3H]lysine to prelabel the protein pool (24 h) followed by [14C]-glucosamine to label hyaluronate (1 h). 4. The results show that newly synthesized hyaluronate is not associated with covalently bound protein, and suggest that chain synthesis is initiated by a mechanism other than on to a core protein. Small amounts of [3H]serine-labelled molecules are, however, non-covalently associated with extracellular hyaluronate. Their identity is at present unknown, but they are probably of low molecular weight.
D000818:Animals; D001622:Betaine / Q000031:analogs & derivatives; D002478:Cells, Cultured; D002499:Centrifugation, Density Gradient; D002813:Chondrosarcoma / Q000378:metabolism*; D002850:Chromatography, Gel; D019791:Guanidine; D006146:Guanidines; D006820:Hyaluronic Acid / Q000096:biosynthesis*; D006821:Hyaluronoglucosaminidase / Q000494:pharmacology; D008239:Lysine / Q000378:metabolism; D000644:Quaternary Ammonium Compounds; D051381:Rats; D012694:Serine / Q000378:metabolism
D006146:Guanidines; D000644:Quaternary Ammonium Compounds; C034730:zwittergent 3-12; D001622:Betaine; D012694:Serine; D006820:Hyaluronic Acid; D006821:Hyaluronoglucosaminidase; D019791:Guanidine; D008239:Lysine