Purification and properties of UDP-gal:N-acetylgalactosaminide mucin: beta 1,3-galactosyltransferase from swine trachea mucosa.

Abstract

A /31,3-galactosyltransferase that transfers galactose from UDP-galactose to N-acetylga1actosaminide:mucin to yield galactosyl /31,3-N-acetylgalactosaminide mucin was purified from swine trachea membrane homogenates.The enzyme which was present in the microsome fraction was solubilized and purified to homogeneity by procedures which included affinity chromatography on Sepharose 4B containing covalently bound asialo Cowper's gland mucin.The enzyme showed a high specificity for N-acetylgalactosaminide residues linked to serine and threonine, and the most active glycosyl acceptors were macromolecular mucin glycoproteins con- taining free N-acetylgalactosaminyl residues linked to a polypeptide chain.The purified enzyme did not transfer galactose to the terminal N-acetylgalactosaminyl residues of type A blood group glycoprotein.Only a single band with molecular weight of 84,000 was observed upon gel electrophoresis in the presence of dodecyl sulfate, and a molecular weight of 90,000 was obtained by exclusion chromatography on Sephadex G-100 in the presence of 0.1% Triton X-100.The solubilized enzyme was unstable in the absence of Triton X-100 or Nonidet P-40.The K, for asialo Cowper's gland mucin was 0.35 PM, and the K,,, for UDP-galactose was 20 PM.The presence of sialic acid-linked a2,6 to the terminal N-acetylgalactosaminide residues of Cowper's gland mucin completely inhibited the transfer of galactose.The product formed in a large scale incubation of asialo Cowper's gland mucin with UDP ['4C]galactose and the purified transferase was isolated.A disaccharide, ["C] galactose /31,3-N-acetylgalactosaminitol, containing more than 90% of the incorporated radioactivity was released by treatment with alkaline borohydride and characterized by enzymatic and methylation analysis.