The Sialic Acids: XVI. ISOLATION OF A MUCIN SIALYLTRANSFERASE FROM SHEEP SUBMAXILLARY GLAND

Abstract

Abstract A sialyltransferase that catalyzes the synthesis of mucin from cytidine 5'-monophospho-N-acetylneuraminic acid and sialidase-treated sheep submaxillary mucin has been isolated from sheep submaxillary glands. The partially purified transferase incorporated approximately 70% of the sialic acid cleaved from the mucin by sialidase. Kinetic studies showed that the approximate Km values were 1.9 mm for the sialidase-treated mucin (calculated in terms of the concentration of N-acetylgalactosamine acceptor sites), and 0.57 mm for the CMP-N-acetylneuraminic acid. No metal ion requirement could be demonstrated for the reaction. The purified enzyme could utilize CMP-N-glycolylneuraminic acid in place of CMP-N-acetylneuraminic acid. Active acceptors included polymers containing terminal N-acetylgalactosamine residues such as sialidase-treated mucins from the submaxillary glands of sheep, pig, and cow, as well as fetuin, a glycopeptide from milk, and erythrocyte hemagglutination inhibitor. Crude extracts contained an endogenous acceptor for sialic acid, which was separated from the sialyltransferase and partially characterized as an incomplete mucin. A tissue survey showed the presence of the sialyltransferase in extracts of submaxillary glands of sheep, pig, and cow, and each of the extracts was active with the mucins obtained from any of the species. The requirements for acceptor activity are discussed. The ovine submaxillary mucin synthesized by the purified enzyme was purified and characterized, and periodate oxidation studies showed that the sialic acid was linked to the N-acetylgalactosamine residue at C-6.