Mulder H, Schachter H, De Jong-Brink M, Van der Ven J G, Kamerling J P, Vliegenthart J F
Department of Bio-Organic Chemistry, Utrecht University, The Netherlands.
Eur J Biochem. 1991 Oct 15;201(2):459-65. doi: 10.1111/j.1432-1033.1991.tb16303.x.
Connective tissue of the freshwater pulmonate Lymnaea stagnalis was shown to contain galactosyltransferase activity capable of transferring Gal from UDP-Gal in beta 1-3 linkage to terminal GalNAc of GalNAc beta 1-4GlcNAc-R [R = beta 1-2Man alpha 1-O(CH2)8COOMe, beta 1-OMe, or alpha,beta 1-OH]. Using GalNAc beta 1-4GlcNAc beta 1-2Man alpha-1-O(CH2)8COOMe as substrate, the enzyme showed an absolute requirement for Mn2+ with an optimum Mn2+ concentration between 12.5 mM and 25 mM. The divalent cations Mg2+, Ca2+, Ba2+ and Cd2+ at 12.5 mM could not substitute for Mn2+. The galactosyltransferase activity was independent of the concentration of Triton X-100, and no activation effect was found. The enzyme was active with GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe (Vmax 140 nmol.h-1.mg protein-1; Km 1.02 mM), GalNAc beta 1-4GlcNAc (Vmax 105 nmol.h-1.mg protein-1; Km 0.99 mM), and GalNAc beta 1-4GlcNAc beta 1-OMe (Vmax 108 nmol.h-1.mg protein-1; Km 1.33 mM). The products formed from GalNAc beta 1-4GlcNAc beta 1-2Man alpha 1-O(CH2)8COOMe and GalNAc beta 1-4GlcNAc beta 1-OMe were purified by high performance liquid chromatography, and identified by 500-MHz 1H-NMR spectroscopy to be Gal beta 1-3GalNAc beta 1-4GlcNAc 1-OMe, respectively. The enzyme was inactive towards GlcNAc, GalNac beta 1-3 GalNAc alpha 1-OC6H5, GalNAc alpha 1--ovine-submaxillary-mucin, lactose and N-acetyllactosamine. This novel UDP-Gal:GalNAc beta 1-4GlcNAc-R beta 1-3-galactosyltransferase is believed to be involved in the biosynthesis of the hemocyanin glycans of L. stagnalis.
已证明淡水肺螺类椎实螺的结缔组织含有半乳糖基转移酶活性，该酶能够将来自UDP - Gal的Gal以β1 - 3连接方式转移至GalNAcβ1 - 4GlcNAc - R的末端GalNAc上[R = β1 - 2Manα1 - O(CH2)8COOMe、β1 - OMe或α,β1 - OH]。以GalNAcβ1 - 4GlcNAcβ1 - 2Manα - 1 - O(CH2)8COOMe作为底物时，该酶对Mn2 +有绝对需求，最佳Mn2 +浓度在12.5 mM至25 mM之间。12.5 mM的二价阳离子Mg2 +、Ca2 +、Ba2 +和Cd2 +不能替代Mn2 +。半乳糖基转移酶活性与Triton X - 100的浓度无关，未发现激活作用。该酶对GalNAcβ1 - 4GlcNAcβ1 - 2Manα1 - O(CH2)8COOMe（Vmax 140 nmol·h - 1·mg蛋白 - 1；Km 1.02 mM）、GalNAcβ1 - 4GlcNAc（Vmax 105 nmol·h - 1·mg蛋白 - 1；Km 0.99 mM）和GalNAcβ1 - 4GlcNAcβ1 - OMe（Vmax 108 nmol·h - 1·mg蛋白 - 1；Km 1.33 mM）有活性。由GalNAcβ1 - 4GlcNAcβ1 - 2Manα1 - O(CH2)8COOMe和GalNAcβ1 - 4GlcNAcβ1 - OMe形成的产物通过高效液相色谱法纯化，并通过500 - MHz 1H - NMR光谱鉴定，分别为Galβ1 - 3GalNAcβ1 - 4GlcNAc 1 - OMe。该酶对GlcNAc、GalNacβ1 - 3GalNAcα1 - OC6H5、GalNAcα1 - 绵羊 - 颌下 - 粘蛋白、乳糖和N - 乙酰乳糖胺无活性。这种新型的UDP - Gal:GalNAcβ1 - 4GlcNAc - Rβ1 - 3 - 半乳糖基转移酶被认为参与了椎实螺血蓝蛋白聚糖的生物合成。
