Characteristics of the core protein of the aggregating proteoglycan from the Swarm rat chondrosarcoma.
A ternary complex of hyaluronic acid-binding region and link protein bound to hyaluronic acid was isolated from limit clostripain digests of proteoglycan aggregates isolated from the Swarm rat chondrosarcoma. Under these conditions, the hyaluronic acid-binding region has a molecular weight of approximately equal to 65,000 (HA-BR65). N-terminal amino acids in the complex were selectively 14C-carbamylated. The resulting derivatized HA-BR65 was isolated, and tryptic peptide maps were prepared and developed on two-dimensional TLC sheets. A single, labeled peptide was obtained which gave a Mr by approximately equal to 8,000 by SDS-PAGE. Chymotrypsin digestion of the ternary complex reduced the molecular weight of HA-BR65 to a polypeptide of approximately equal to 55,000 (HA-BR55) which still retains the same N-terminal tryptic peptide. Partial digestion of proteoglycan aggregates with clostripain generated a series of larger intermediates with the hyaluronic acid-binding region. Direct SDS-PAGE analysis revealed one major intermediate with approximately equal to 109,000 (HA-BR109) as well as HA-BR65. After chondroitinase digestion, two additional prominent intermediates were observed on a SDS-PAGE gel at Mr approximately equal to 120,000 (HA-BR120) and approximately equal to 140,000 (HA-BR140). All the intermediates were recognized by a monoclonal antibody specific for the hyaluronic acid-binding region, and all of them contained the same N-terminal tryptic peptide. The results indicate that the N terminus of the core protein is at the hyaluronic acid-binding end of the proteoglycan and that the chondroitin sulfate chains are first present on the core protein in a region between 109,000 and 120,000 molecular weight away from the N terminus.
D000595:Amino Acid Sequence; D000818:Animals; D000911:Antibodies, Monoclonal / Q000276:immunology; D001665:Binding Sites; D002813:Chondrosarcoma / Q000032:analysis*; D006820:Hyaluronic Acid / Q000378:metabolism; D008970:Molecular Weight; D010446:Peptide Fragments / Q000032:analysis; D011509:Proteoglycans / Q000032:analysis* / Q000276:immunology; D051381:Rats
D000911:Antibodies, Monoclonal; D010446:Peptide Fragments; D011509:Proteoglycans; D006820:Hyaluronic Acid