Purification to homogeneity and enzymatic characterization of an alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase from porcine submaxillary glands.
By means of affinity chromatography on CDP-hexanolamine-agarose, a CMP-N-acetylneuraminate: alpha-N-acetylgalactosaminide alpha 2 leads to 6 sialyltransferase (EC 2.4.99.1) has been purified 117,000-fold to homogeneity from Triton X-100 extracts of porcine submaxillary glands. The enzyme consists of several electrophoretic forms that can be partially resolved by chromatography on Sephadex G-200, the largest of which has a molecular weight of approximately 160,000 as estimated by sodium dodecyl sulfate-gel electrophoresis. Periodate oxidation studies show that the linkage formed by this enzyme with ovine submaxillary asialo-mucin as the acceptor substrate is NeuAc alpha 2 leads to 6GalNAc alpha 1 leads to O-Thr/Ser. On the basis of initial rate studies and the patterns of inhibition observed with alternate acceptor substrates, the transferase is proposed to have either a random equilibrium kinetic mechanism or an ordered steady state mechanism with the acceptor substrate binding first. Among a wide variety of oligosaccharides, glycoproteins, and simple glycosides (including p-nitrophenyl-alpha-N-acetylgalactosaminide), the only acceptor substrates for this enzyme are those glycoproteins containing the structure, R leads to 3GalNAc alpha 1 leads to O-Thr/Ser, where R may be H or a beta-galactoside.
D000116:Acetylgalactosamine; D000818:Animals; D005615:Freezing; D006023:Glycoproteins; D007700:Kinetics; D012799:Sialyltransferases / Q000302:isolation & purification / Q000378:metabolism*; D013363:Submandibular Gland / Q000201:enzymology*; D013379:Substrate Specificity; D013552:Swine; D014166:Transferases / Q000378:metabolism*
D006023:Glycoproteins; D014166:Transferases; D012799:Sialyltransferases; D000116:Acetylgalactosamine