Purification and characterization of UDP-N-acetylgalactosamine: polypeptide N-acetylgalactosaminyltransferase from bovine colostrum and murine lymphoma BW5147 cells.
UDP-GalNAc:polypeptide N-acetylgalactosaminyl-transferase has been purified from two sources. A soluble form, purified 517,000-fold to homogeneity from bovine colostrum, has a molecular mass of 70,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 69,000 daltons by gel filtration. A membrane-bound form, partially purified 2,500-fold from BW5147 mouse lymphoma cells, has a molecular mass of 70,000 daltons by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 71,500 daltons by gel filtration. The purified colostrum enzyme exhibits specificity for UDP-GalNAc, has its pH optimum between pH 7.2 and 8.6, and requires Mn2+ for activity. The Km is 8 microM for UDP-GalNAc and 2.5 mg/ml for deglycosylated bovine submaxillary mucin. Treatments with endo-beta-N-acetylglucosaminidase H and F indicate that the colostrum enzyme is a glycoprotein containing two N-linked oligosaccharides. On most enzyme molecules, both oligosaccharides are of the complex type, but some molecules contain one complex type and one high mannose type. Antibodies raised against homogeneous bovine enzyme cross-react on immunoblots with a single protein of 71,000 daltons in the partially purified preparation and in a crude microsomal extract from BW5147 cells.
Elhammer
A
A
Kornfeld
S
S
D000596:Amino Acids / Q000032:analysis; D000818:Animals; D002417:Cattle; D002460:Cell Line; D003126:Colostrum / Q000201:enzymology*; D003429:Cross Reactions; D004591:Electrophoresis, Polyacrylamide Gel; D005700:Galactosyltransferases / Q000302:isolation & purification*; D006863:Hydrogen-Ion Concentration; D007700:Kinetics; D008223:Lymphoma / Q000201:enzymology*; D008345:Manganese / Q000378:metabolism; D051379:Mice; D008970:Molecular Weight; D017350:N-Acetylgalactosaminyltransferases
D000596:Amino Acids; D008345:Manganese; D005700:Galactosyltransferases; D017350:N-Acetylgalactosaminyltransferases; C003742:UDPgalactosamine-galactose acetylgalactosaminyltransferase