title: Polypeptide N-acetylgalactosaminyltransferase activity in tracheal epithelial microsomes.
abstract: Pig tracheal epithelium, a site of extensive mucin biosynthesis, contained polypeptide N-acetylgalactosaminyltransferase activity directed towards L-threonine residues. The enzyme preparation was broadly similar in properties to preparations from other tissues, e.g. pig and bovine submaxillary glands, bovine colostrum, BW5147 mouse lymphoma and baby-hamster kidney cells. Enzyme was membrane-bound and was released from microsomal preparations by extraction with Triton X-100. Extracted enzyme had a pH optimum of 7.5, had a requirement for Mn2+ (10 mM) and was inhibited by Na2EDTA. The Km for UDP-N-acetylgalactosamine was 110 microM and that for an octapeptide acceptor (VTPRTPPP) was 3.0 mM at 37 degrees C. Using a range of synthetic peptides of known structure related to TPPP it was established that L-threonine residues were specifically O-glycosylated probably in the alpha-configuration. Synthetic peptides containing the TPPP sequence required a peptide length of five or more for significant acceptor activity. In VTPRTPPP the two threonine residues were similarly glycosylated, as revealed by tryptic cleavage of the glycosylated product and separation of the 3H-labelled fragments. The enzyme preparation also specifically catalysed the transfer of N-acetylgalactosaminyl residues from UDP-N-acetyl[1-3H]galactosamine to bovine submaxillary mucin core protein and to myelin basic protein.
authors.affiliation: Bayer U.K. Ltd., Pharma Research, Stoke Poges, Bucks, U.K.
authors.forename: J M
authors.initials: JM
authors.lastname: Cottrell
authors.forename: R L
authors.initials: RL
authors.lastname: Hall
authors.forename: R G
authors.initials: RG
authors.lastname: Sturton
authors.forename: P W
authors.initials: PW
authors.lastname: Kent
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D004848:Epithelium / Q000201:enzymology; D005700:Galactosyltransferases / Q000378:metabolism*; D006031:Glycosylation; D007700:Kinetics; D008861:Microsomes / Q000201:enzymology*; D008969:Molecular Sequence Data; D009077:Mucins / Q000378:metabolism; D004676:Myelin Basic Protein / Q000378:metabolism; D017350:N-Acetylgalactosaminyltransferases; D013363:Submandibular Gland / Q000378:metabolism; D013552:Swine; D014132:Trachea / Q000201:enzymology*; D000097763:Polypeptide N-acetylgalactosaminyltransferase
chemical_list: D005700:Galactosyltransferases; D009077:Mucins; D004676:Myelin Basic Protein; D017350:N-Acetylgalactosaminyltransferases; D000097763:Polypeptide N-acetylgalactosaminyltransferase