Purification, composition, molecular weight, and subunit structure of ovine submaxillary mucin.

Abstract

Ovine submaxillarymucin was isolated by modifications of published methods to give preparations with properties similar to those reported earlier.The last step in purification is the removal of small amounts of contaminating protein by gel filtration of mucin previously reacted with dansyl (5-dimethylaminonaphthalene-1-sulfonyl) chloride.The contaminating protein contained all of the protein-bound fluorescent dansyl groups.Asialomucin and apomucin were prepared from mucin by similar means after treatment with protease-free glycosidases.Each form of mucin contained a blocked NH,-terminal residue and was devoid of amino acids reactive with dansyl chloride.Threonine, serine, proline, glycine, and alanine accounted for about 75% of the amino acid content of each type of mucin.The composition of mucin suggests that the hydroxyl group of each threonine and serine residue in the molecule is in 0-glycosidic linkage with N-acetylgalactosamine and that about 86% of the Nacetylgalactosaminyl groups are in glycosidic linkage with sialic acid.The molecular weights of mucin, asialomucin, and apomucin were examined by sedimentation-equilibrium in the ultracentrifuge.Apomucin in 0.5 M sodium chloride behaved as a pure protein with a molecular weight of 58,300.Mucin and asialomucin appeared to aggregate in the same solvent, suggesting that aggregation is dependent upon the carbohydrate content of mucin.A more detailed ultracentrifugal analysis of mucin in 2 M sodium chloride indicates that mucin forms a self-associating system in which the extent of association is a function of ionic strength and protein concentration.It is concluded that association occurs among molecules of M,. = 154,000, which is close to the expected size of apomucin containing sialyl-cu2 + &N-acetylpalactosamine in 0-glycosidic linkage at each of the threonyl and seryl residues in the molecule. Mucinis the predominant sialic acid-containing glycopro-t&n in the mucous secretions of submaxillary glands.Its function, along with similar mucoproteins found in the respi-