N-terminal carbamylation of the hyaluronic acid-binding region and the link protein from the chondrosarcoma proteoglycan aggregate.
The ternary complex consisting of a 65-kDa peptide originating from the proteoglycan core protein and a 43-kDa link protein bound to hyaluronic acid was purified from a clostripain digest of the rat chondrosarcoma aggregating proteoglycan and 14C-carbamylated with potassium [14C]cyanate. At a pH of 8.0, 14C-carbamylation of the alpha-NH2 groups in the N-terminal amino acids was favored over carbamylation of epsilon-NH2 groups in the lysinyl residues for both the 65- and 43-kDa species. Two-dimensional tryptic peptide maps revealed a single major, distinctly different, fluorographic spot for each. These tryptic peptides had approximate masses of 4.5 kDa (from the 65-kDa species) and 3.0 kDa (from the 43-kDa species) on sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels and each contained greater than 60% of the total radioactivity associated with its original polypeptide. Primary amino acid sequencing of the 65-kDa species gave a defined sequence for the first 4 N-terminal residues, whereas sequencing through the first 4 residues of a fully carbamylated species gave no dabsylated derivative for the first residue but identical residues in position 2-4 as for the noncarbamylated species and loss of radioactive derivative. Digests of 14C-carbamylated ternary complex with alpha-chymotrypsin resulted in a limit 14C-carbamylated 55-kDa species which contained greater than 85% of the radiolabel originally in the 65-kDa peptide. Similarly, trypsin generated two radiolabeled species, 60 and 58 kDa. These limit digest peptides (55, 60, 58 kDa) all contained the 4.5-kDa N-terminal tryptic peptide. Thus peptides removed from the 65-kDa peptide digestion with either alpha-chymotrypsin or trypsin were on the carboxyl end of the molecule.
D000818:Animals; D002813:Chondrosarcoma / Q000032:analysis*; D002918:Chymotrypsin / Q000378:metabolism; D003485:Cyanates / Q000378:metabolism; D003546:Cysteine Endopeptidases; D004591:Electrophoresis, Polyacrylamide Gel; D010450:Endopeptidases / Q000378:metabolism; D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid / Q000378:metabolism*; D008970:Molecular Weight; D010446:Peptide Fragments / Q000378:metabolism*; D011506:Proteins / Q000378:metabolism*; D011509:Proteoglycans / Q000378:metabolism*; D051381:Rats; D014357:Trypsin / Q000378:metabolism; D000099249:Proteoglycan Link Protein
D002918:Chymotrypsin; D003485:Cyanates; D003546:Cysteine Endopeptidases; D010450:Endopeptidases; D016326:Extracellular Matrix Proteins; D006820:Hyaluronic Acid; D010446:Peptide Fragments; D011506:Proteins; D011509:Proteoglycans; D014357:Trypsin; D000099249:Proteoglycan Link Protein; C019913:clostripain; C024835:potassium cyanate