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β1—4半乳糖基转移酶受体底物的动力学

ACCEPTOR SUBSTRATES' KINETICS OF β1-4 GALACTOSYLTRANSFERASE
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摘要 研究β1-4半乳糖基转移酶(β1-4GT)与受体底物的亲和性、其底物的天线偏爱性以及糖基对亲和性的影响,以及β1-4GT对不同底物的天线特异性(branchspecificity)。用两种荧光标记的底物1(Gnβ1-2Mal-6(Gnβ1—2Mal-3)Mβ1-4Gnβ1—4Gn-PA)和底物2(Gnβ1-2Mal—6(Gnβ1-2Mal-3)Mβ1—4Gnβ1-4(Fucal-6)Gn-PA),用正常雄性大鼠的肝组织的高尔基体作为酶,测定β1-4GT的不同底物的Km值[1]。结果:底物1和2的Km值分别为2.31×10-3mol/L和3.12×10-3mol/L;对底物1,α1,3天线的N-乙酰氨基葡萄糖基比α1,6天线的反应性强,形成Gnβ1-2Mal-6(Gβ1-4Gnβ1-2Mal—3)Mβ1—4Gnβ1-4Gn—PA产物是Gβ1-4Goβ1—2MQl一6(Goβ1-2Mal-3)Mβ1-4Gnβ1-4Gn-PA的2.10倍左右;对底物2,α1,3天线的N-乙酰氨基葡萄糖基比α1,6天线的反应性更强,形成Gnβ1-2Mal-6(Gβ1-4Gnβ1-2Mal-3)Mβ1-4Gnβ1-4(Fucα1-6)Gn-PA的产物是Gβ1-4Gnβ1-2Mal-6(Gnβ1-2Mal-3)Mβ1-4Gnβ1-4(Fucα1—6)Gn-PA的4.59倍左右。结论:核心岩藻糖对酶与受体底物的亲和性有一定影响。 PURPOSE TO study the affinity to acceptor substrates by β1-4 galactosyltransferase, the enzymaticalbranch specificity and the effects on the affinity by glycosyil groups.METHODS The kinetic experiments were carried out using crude enzyme extracts of Golgi complex fromthe rat liver with a fluorescene assay method for β1 - 4 galactosyltransferase (β1- 4GT), involving a pyridylaminated sugar as an acceptor substrate. Two fluorescent sugar chains, in which the reducing end of the Gn β1- 2Ma1-6(Gn β1- 2Ma1- 3)Mβ1-4 Gn β1-4 Gn(substrate 1) and Gn β1-2 Ma1-6(Gn β1-2 Mal-3)Mβ1-4 Gn β1-4 (Fuc a1-6)Gn (substrate 2) has been aminated with 2-aminopyridine. Then the fluorescent reaction product is separated by reversephase high-performance liquid chromatography.RESULTS The Km for substrate 1 and substrate 2 was found to be 2. 31 ×10-3 mol/L and 3. 12 ×10-3mol/L respectively Furthermore, our research has revealed that the product of Gnβ1-2 Ma1-6(Gβ1-4Gnβ1- 2Ma1-3)Mβ1-4Gnβ1 - 4Gn - PAwas2.10timesasmuchasthatofGβ1- 4Gnβ1 - 2Ma1 - 6 (Gnβ1- 2Ma1-3)Mβ1-4 Gn β1-4 Gn-PA; the product of Gn β1-2 Ma1-6(Gβ1-4 Gnβ1 - 2 Ma1- 3)Mβ1- 4Gnβ1 - 4(Fuca1- 6)Gn - PA4. 59± 0. 29timesasmuchasthatofGβ1-4Gnβ1 - 2Ma1 - 6(Gnβ1 - 2Ma1- 3)Mβ1-4 Gnβ1 -4(Fuc a1- 6) Gn- PA.CONCLUSIONS The affinity of β1-4 GT to acceptor Substrate I is more than that to Substrate 2. The core fucosecould decrease the affinity. β1-4 GT has branch sgecificity on two Substrates. The Gn of a1,3 mannose branch of substrates is more susceptible to be galactosylated than that of a1,6 branch.
出处 《上海医科大学学报》 CSCD 1997年第5期339-342,共4页 Journal of Fudan University(Medical Science)
基金 国家自然科学基金!39570176 39470164
关键词 天线特异性 高效液相色谱法 糖蛋白 β1-4GT β1-4 galactosyltransferase branch specificity high-performance liquid chromatography
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  • 1顾建新,生物化学与生物物理学报,1997年,29卷,1期,1页
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