同型半胱氨酸对谷胱甘肽合成的抑制作用

Inhibitive effects of homocysteine on glutathione synthesis

目的:探讨同型半胱氨酸对谷胱甘肽合成的影响及其发生的可能原因。方法:实验于2004-08/2005-02在新乡医学院生物化学与分子生物学实验室进行。选用健康家兔20只,取抗凝血,离心收集红细胞,制备含有γ-谷氨酰半胱氨酸合成酶和谷胱甘肽合成酶体系的红细胞裂解液,设置对照组,高、低剂量同型半胱氨酸处理组,每组均设8个平行管。各组均加入100mmol/L的Tris-Hcl缓冲液(pH8.0)60μL,1mol/LMgCl28μL,10mmol/LATP40μL,50mmol/L谷氨酸(调pH为中性)40μL,红细胞裂解液40μL。低、高浓度同型半胱氨酸处理组再分别加入100mmol/L同型半胱氨酸20μL和60μL,其余实验条件相同。以反应中生成无机磷的mmol数表示γ-谷氨酰半胱氨酸合成酶活性大小。分别用紫外分光光度法和荧光法检测各组无机磷和谷胱甘肽含量。结果:①无机磷的生成量:低浓度同型半胱氨酸组和高浓度同型半胱氨酸组明显低于对照组犤(1.476±0.240),(0.751±0.085),(3.279±0.470)mmol/L,P<0.01犦,高浓度同型半胱氨酸组明显低于低浓度同型半胱氨酸组(P<0.05)。②还原型谷胱甘肽含量:低同型半胱氨酸组和高同型半胱氨酸组明显低于对照组犤(3.058±0.264),(1.908±0.301),(4.476±0.462)μmol/L,P<0.01犦,高浓度同型半胱氨酸组明显低于低浓度同型半胱氨酸组(P<0.01)。结论:同型半胱氨酸有可能通过竞争性抑制γ-谷氨酰半胱氨酸合成酶的活性,减少细胞内还原型谷胱甘肽的生成。

AIM： To explore the effects of homocysteine on the glutathione synthesis and the possible reason of occurrence. METHODS： The experiment was conducted in the Laboratory of Biochemistry and Molecular Biology, Xinxiang Medical College from August 2004 to February 2005. Twenty healthy rabbits were selected. The antiblood coagulation was gained, and the erythrocyie was collected after centrifugation to prepare the erythrocyte splitting liquor with γ-glutamylcysteine synthetase and gluthetase. It was divided into control group, homocysteine group of high and low dosage with 8 equal tubes in each group. 100 mmol/L Tris-Hcl buffer was added in every group （pH 8.0） 60 μL,1 mol/L MgC12 8 μL,10 mmol/L ATP 40 μ L,50 mmol/L glutaminol （pH as neutrality）40 μL, and 40 μL erythrocyte splitting liquor. 100 mmol/L homocysteine 20 μI, and 60 μL were added into the low and high homocysteine group, respectively, and the other experiment was the same. The activity of γ-glutamylcysteine synthetase was showed by the content of inorganic phosphate formed in reaction. The contents of phosphate and glutathione were respectively detected by ultraviolet spectrnphntnmetry and fluorumetry. RESULTS： ① Content of phosphate： It was lower significantly in the low and high homocysteine group than that in the control group [（1.476±0.240）, （0.751±0.085）, （3.279±0.470） mmol/L, P（0.01]. It was lower significantly in the high homocysteine group than that in the low homocysteine group （P 〈 0.05 ）. ② The content of reduced glutathione： It was lower significantly in the low and high homocysteine group than that in the control group [（3.058±0.264）, （1.908±0.301）,（4.476±0.462）μmol/L,P 〈 0.01]. It was lower in the high homocysteine group than that in the low homocysteine group （P 〈 0.01 ）. CONCLUSION： Homocysteine can decrease the generation of reduced glutathione in cell by means of inhibiting the activity of γ-glutamylcysteine synthetase competitively.