Adamany A M, Spiro R G
J Biol Chem. 1975 Apr 25;250(8):2830-41.
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.
来自小牛甲状腺的微粒部分催化甘露糖从GDP-甘露糖转移到外源性糖肽以及甲基或芳基糖苷上，以形成α-D-甘露吡喃糖基-D-甘露糖序列。转移到简单糖苷需要一个与亲脂性糖苷配基相连的单一非还原性甘露糖残基。因此，对硝基苯基-α-D-甘露糖苷、4-甲基伞形酮基-α-D-甘露糖苷、苯基-α-D-甘露糖苷和甲基-α-D-甘露糖苷是有效的受体，而游离甘露糖和其他几种糖的糖苷则完全无活性。甲基-α-D-甘露糖苷的Km值为2.6 mM。受体异头构型的特异性表现为α异头物的糖基化程度达50%，并且观察到这两种受体之间存在相互抑制作用。糖苷受体的14C标记产物经乙酰解或温和酸水解后产生二糖2-O-α-D-甘露吡喃糖基-D-甘露糖，它代表甲状腺球蛋白碳水化合物单元A中甘露糖残基之间的主要连接方式。具有甘露糖序列的糖肽作为转移反应的受体，但仅在其肽部分经二硝基苯基化后才行。含有10个甘露糖残基的甲状腺球蛋白单元A糖肽（Km等于0.89 mM）比仅含有三个甘露糖成分的单元B核心部分的糖肽是更好的受体。通过定时用α-甘露糖苷酶处理来减小单元A糖肽受体的大小会导致活性逐渐降低。即使在其葡糖胺残基上修饰带有二硝基苯基基团后，无肽的单元A仍无活性。GDP-甘露糖是最有效的糖基供体，对甲基-α-D-甘露糖苷的Km值为1.4 μM，对二硝基苯基单元A糖肽的Km值为0.30 μM，不过ADP-甘露糖和UDP-甘露糖可在40%至45%的程度上替代。甘露糖向糖肽的转移在pH 6.3时达到最佳，而向简单糖苷的转移在pH 7.0时最佳。这两种类型的转移反应都需要二价阳离子，其中锰在此方面最有效。两组受体的甘露糖基转移酶活性主要存在于微粒亚细胞部分。许多破坏膜完整性的芳香族化合物和试剂会导致酶活性丧失，大概是通过干扰各种受体上亲脂性取代基的功能。
