猪脑谷氨酸脱羧酶的分离纯化以及生物学活性鉴定

L-谷氨酸脱羧酶是γ-氨基丁酸合成的关键限速酶,广泛的存在于脊椎动物神经细胞以及β-胰腺细胞,是胰岛素依赖型糖尿病(IDDM)病人以及僵硬综合症(SMS)病人血清的关键抗原。运用sephamryl S-200以及DEAEsepharose可以从猪脑中分离纯化出谷氨酸脱羧酶。纯化的GAD在变性条件下电泳,经考马斯亮蓝R250染色以及Western-Blot鉴定主要有两条带,分子量分别为67kD和44kD。根据L-谷氨酸脱羧酶能够分解谷氨酸产生γ-氨基丁酸和CO2的特性,通过测定产物γ-氨基丁酸推断酶活。以上实验结果表明从猪脑中分离纯化到的是具有生物学活性以及免疫原性的谷氨酸脱羧酶,可进一步改良为IDDM检测试剂盒,用于IDDM的预防和预测。

Purification and Biological Analysis of L-glutamic Acid Decarboxylase from Porcine Brain

L-Glutamic acid decarboxylase (GAD)is the enzyme that synthesizes the neurotransmitter γ-aminobutyric acid (GABA)in neurons and in pancreatic β cell in vertebrate. Also GAD has been identified as the autoantigen to sera of insulin-dependent diabetes mellitus (Type Ⅰ diabetes,abbreviate to IDDM)and stiff-Man syndrome (SMS).In our experiment sephancryl S200 and DEAE-sepharose were exploited to purify GAD from porcine brain. The purified GAD showed two main bands,67 kD and 44 kD,on SDS-PAGE by Coomassie Brilliant Blue R-250 and Western-Blot under reducing condition. Activity of GAD was assayed by measuring the formation of GABA.Above all,it could be concluded that the purified GAD had biological activity and immunocompetence which could be developed to Kits in the detection of people of or recent-onset of Type Ⅰ diabetes and used in predicting IDDM.