| Abstract: | A particulate fraction from calf thyroid catalyzes the transfer of mannose from GDP-mannose to exogenous glycopeptides and methyl or aryl glycosides to form alpha-D-mannopyranosyl-D-mannose sequences. The transfer to the simple glycosides required a single nonreducing mannose residue linked to a lipophilic aglycone. Thus p-nitrophenyl-, 4-methylumbelliferyl-, phenyl- and methyl-alpha-D-mannopyranosides were effective acceptors while free mannose and glycosides of several other sugars were totally inactive. The Km value for methyl-alpha-D-mannopyranoside was 2.6 mM. Specificity for the anomeric configuration of the acceptor was glycosylated to the extent of 50% of the alpha anomer and mutual inhibition between these two acceptors was observed. Acetolysis or mild acid hydrolysis of the 14C-labeled products from the glycoside acceptors yielded the disaccharide, 2-O-alpha-D-mannopyranosyl-D-mannose, which represents the predominant linkage between mannose residues in the carbohydrate unit A of thyroglobulin. Glycopeptides with mannose sequences served as acceptors for the transfer reaction but only after dinitrophenylation of their peptide portion. The unit A glycopeptides of thyroglobulin with 10 mannose residues (Km equals 0.89 mM) were much better acceptors than glycopeptides containing the core portion of unit B which contains only three mannose components. Reduction in size of unit A glycopeptide acceptors by timed alpha-mannosidase treatment resulted in a progressive decrease in activity. Peptide-free unit A was inactive even after it was modified to carry dinitrophenyl groups on its glucosamine residues. GDP-mannose was the most effective glycosyl donor, with a Km value of 1.4 muM for methyl-alpha-D-mannopyranoside and 0.30 muM for dinitrophenyl unit A glycopeptides, although ADP- and UDP-mannose could substitute to the extent of 40 to 45%. The mannose transfer to the glycopeptides had a optimum of 6.3 while that to the simple glycopeptides was best at pH 7.0. Both types of transfer reactions required a divalent cation with manganese serving most effectively in that capacity. Mannoslytransferase activity for both groups of acceptors was found predominantly in particulate subcellular fractions. A number of aromatic compounds and reagents which are disruptive of membrane integrity caused loss of enzyme activity presumably by interfering with the function of the lipophilic substituents on the various acceptors. |
