Abstract
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.
Highlights
The K, is 8 PM for UDP-GalNAc and 2.5 mg/ml for ascites hepatoma enzyme in several aspects
H and F indicate that the colostrum enzymeis a gly- and describe an antibody raised against the purified colostrum coprotein containing two N-linked oligosaccharides. transferase that cross-reacts with the intracellular murine
In contrast to Asn-linked oligosaccharide synthesis which is initiated by the en bloc transfer of a preassembled oligosaccharide from a lipid carrier to the nascent protein [2], 0linked oligosaccharide synthesis begins with the transfer of N-acetylgalactosamine from its nucleotide sugar donor to serine or threonine residues on the protein [3]
Summary
In the partially purified preparation and in a crude microsomal extract from BW5147cells. Characterization of Antiserum-Rabbit antibodies raised against purified bovine colostrum N-acetylgalactosaminyltransferase precipitate transferase activity from a partially purified (Apomucin Sepharose I1 eluate) preparation of the colostrum enzyme (Fig. 1OA).In thisexperiment, 72 pg ofIgG (corresponding to 50 pl of antiserum) added to anincubation mixture containing 69 units of N-acetylgalactosaminyltransferase precipitated 81%of the activity or approximately 30 ng of transferase (based on a specific activity of 1860units/ mg). 100 pl of a 1:l shown in lanes 1 and 2 of Fig. 11,an immunoreactive species suspension of protein A-Sepharose in PBS was added and the was observed which had a M , of 71,000, slightly greater than incubation continued for an additional 50 min on ice during which that of the soluble bovine enzyme. Radioactivity precipitable with preimmune serum has been subtracted from each value
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