猪脑谷氨酸脱氢酶的分离纯化及性质和功能基团研究
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摘要
谷氨酸脱氢酶(glutamate dehydrogenase, GDH)是参与谷氨酸新陈代谢的关键酶之一,它以NAD/NADP作为辅酶,可逆的催化谷氨酸脱氨,使之形成α-酮戊二酸和氮,从而参与谷氨酸的合成与分解。作为一种脱氢酶,它广泛的存在于各种动植物以及微生物体内,常以四聚体或六聚体的形式存在,在生物体的碳、氮代谢中起着非常重要的作用。该酶在医药卫生以及化学分析等领域被广泛应用,到目前为止,已有多种来源的GDH得到了详细研究,但未见猪脑GDH的研究报道。因此,本文以来源广泛、廉价的猪脑为材料,对GDH进行了分离纯化以及部分酶学性质研究,为GDH的深入研究和应用提供了数据支持,为该酶的获得提供了一条新的途径。其结果如下：
1.猪脑谷氨酸脱氢酶的分离纯化
新鲜猪脑通过冷冻、匀浆、硫酸铵沉淀DEAE-Sepharose离子交换层析以及Superdex-200凝胶过滤层析等方法,获得了电泳纯的猪脑谷氨酸脱氢酶。其回收率为24.61%,纯化倍数为70.07倍,酶比活力为13.63U/mg。
2.猪脑谷氨酸脱氢酶的性质研究
理化性质研究表明,猪脑谷氨酸脱氢酶的全酶相对分子质量和亚基相对分子质量分别为330.08kD和56.04kD,推测该酶可能由6个相同亚基构成。它的最适反应温度为55-C,最适pH值为8.2。该酶在40℃以下以及pH6-8有非常好的稳定性,其对NADH的表观Km值为0.084mmol／L。3.有机溶剂、化学试剂及金属离子对猪脑谷氨酸脱氢酶的影响
甲醇、乙醇、异丙醇、SDS、草酸、抗坏血酸等对该酶有较强的抑制作用,而EDTA对该酶则表现出一定的激活作用。Ca2+、Mg2+、Mn2+、Ba2+对猪脑谷氨酸脱氢酶表现出一定的抑制作用,而Cu2+、Co2+、Zn2+表现出的抑制作用却非常明显,最终都使酶几乎完全失活,Na+、Li+、K+则对该酶没有表现出明显的作用。
4.猪脑谷氨酸脱氢酶的功能基团研究
功能基团的修饰结果表明：半胱氨酸的巯基、甲硫氨酸的硫醚基可能是猪脑谷氨酸脱氢酶活性中心的必需基团,参与了该酶活性中心的构成。而丝氨酸残基、组氨酸咪唑基、精氨酸胍基、二硫键、酪氨酸酚羟基、赖氨酸ε-氨基等上述几种基团可能不是猪脑谷氨酸脱氢酶活性中心的必需基团或者只有极少数的上述几种基团是该酶活性中心的必需基团。
Glutamate dehydrogenase (GDH) is one of key enzymes of the glutamate metabolism and catalyzes the reversible oxidative deamination of L-glutamate toα-ketoglutaric acid and ammonia when couples with the coenzyme NAD/NADP, which would take part in the synthesis and decomposition of ammonia. As a dehydrogenase, GDH exists wildly in the animals, plants and microorganism. The enzyme exists in the form of tetramer or hexamer and plays a very important role in the metabolism of carbon and ammonia. Because of the extensive application of the enzyme in the area of medicine and chemical analysis, there are lots of researches about GDH from different organism except porcine brain so far. We have isolated and purified the GDH from porcine brain and researched on the partial properties of the enzyme. Our works establish a basis for researching on the metabolism of carbon and ammonia in living organisms, provide the data for the deeply investigation and application of GDH, and found a new pathway to obtain GDH. The results were as follows:
1. Separation and purification of GDH from porcine brain
The glutamate dehydrogenase which showed a single band on SDS-PAGE had been purified from porcine brain by frozen, homogenization, ammonium sulfate precipitation, DEAE-Sepharose chromatography and Superdex-200 chromatography. Its multiple of purification was 70.07and its specific activity was13.63U/mg.24.61% of the glutamate dehydrogenase activity was recovered.
2. Properties of GDH from porcine brain
The enzyme had a relative molecular mass of 330.08KD. The relative molecular mass of the subunit was about 56.04KD, so the porcine brain GDH may have the same of six subunits. The optimum temperature and pH of this enzyme were 55℃and 8.2 respectively. The glutamate dehydrogenase displayed excellent stability at temperature below 40℃and pH 6-8. Its apparent Km towards NADH was 0.084mmol/L.
3. Effect of different organic solvent, chemical compound and metal ions on the activity of GDH
The enzyme activity could be strongly inhibited when interacting with methanol, ethanol, isopropanol, SDS, oxalic acid and ascorbic acid. Ca2+,Mg2+, Mn2+, Ba2+ partially inhibited the enzymatic activity, Cu2+, Co2+, Zn2+ showed strong inhibitory action on the enzymatic activity, which deactivated the enzyme at last. Na+, Li+, K+ had no obvious effect on the activity of GDH. 4. Chemical modification of GDH from porcine brain
The modification results showed that:Sulfydryl and Met should be considered as indispensable function groups of GDH, but disulfide bonds, Ser, His, Arg, Tyr and Lys residues should not be.
1.猪脑谷氨酸脱氢酶的分离纯化
新鲜猪脑通过冷冻、匀浆、硫酸铵沉淀DEAE-Sepharose离子交换层析以及Superdex-200凝胶过滤层析等方法,获得了电泳纯的猪脑谷氨酸脱氢酶。其回收率为24.61%,纯化倍数为70.07倍,酶比活力为13.63U/mg。
2.猪脑谷氨酸脱氢酶的性质研究
理化性质研究表明,猪脑谷氨酸脱氢酶的全酶相对分子质量和亚基相对分子质量分别为330.08kD和56.04kD,推测该酶可能由6个相同亚基构成。它的最适反应温度为55-C,最适pH值为8.2。该酶在40℃以下以及pH6-8有非常好的稳定性,其对NADH的表观Km值为0.084mmol／L。3.有机溶剂、化学试剂及金属离子对猪脑谷氨酸脱氢酶的影响
甲醇、乙醇、异丙醇、SDS、草酸、抗坏血酸等对该酶有较强的抑制作用,而EDTA对该酶则表现出一定的激活作用。Ca2+、Mg2+、Mn2+、Ba2+对猪脑谷氨酸脱氢酶表现出一定的抑制作用,而Cu2+、Co2+、Zn2+表现出的抑制作用却非常明显,最终都使酶几乎完全失活,Na+、Li+、K+则对该酶没有表现出明显的作用。
4.猪脑谷氨酸脱氢酶的功能基团研究
功能基团的修饰结果表明：半胱氨酸的巯基、甲硫氨酸的硫醚基可能是猪脑谷氨酸脱氢酶活性中心的必需基团,参与了该酶活性中心的构成。而丝氨酸残基、组氨酸咪唑基、精氨酸胍基、二硫键、酪氨酸酚羟基、赖氨酸ε-氨基等上述几种基团可能不是猪脑谷氨酸脱氢酶活性中心的必需基团或者只有极少数的上述几种基团是该酶活性中心的必需基团。
Glutamate dehydrogenase (GDH) is one of key enzymes of the glutamate metabolism and catalyzes the reversible oxidative deamination of L-glutamate toα-ketoglutaric acid and ammonia when couples with the coenzyme NAD/NADP, which would take part in the synthesis and decomposition of ammonia. As a dehydrogenase, GDH exists wildly in the animals, plants and microorganism. The enzyme exists in the form of tetramer or hexamer and plays a very important role in the metabolism of carbon and ammonia. Because of the extensive application of the enzyme in the area of medicine and chemical analysis, there are lots of researches about GDH from different organism except porcine brain so far. We have isolated and purified the GDH from porcine brain and researched on the partial properties of the enzyme. Our works establish a basis for researching on the metabolism of carbon and ammonia in living organisms, provide the data for the deeply investigation and application of GDH, and found a new pathway to obtain GDH. The results were as follows:
1. Separation and purification of GDH from porcine brain
The glutamate dehydrogenase which showed a single band on SDS-PAGE had been purified from porcine brain by frozen, homogenization, ammonium sulfate precipitation, DEAE-Sepharose chromatography and Superdex-200 chromatography. Its multiple of purification was 70.07and its specific activity was13.63U/mg.24.61% of the glutamate dehydrogenase activity was recovered.
2. Properties of GDH from porcine brain
The enzyme had a relative molecular mass of 330.08KD. The relative molecular mass of the subunit was about 56.04KD, so the porcine brain GDH may have the same of six subunits. The optimum temperature and pH of this enzyme were 55℃and 8.2 respectively. The glutamate dehydrogenase displayed excellent stability at temperature below 40℃and pH 6-8. Its apparent Km towards NADH was 0.084mmol/L.
3. Effect of different organic solvent, chemical compound and metal ions on the activity of GDH
The enzyme activity could be strongly inhibited when interacting with methanol, ethanol, isopropanol, SDS, oxalic acid and ascorbic acid. Ca2+,Mg2+, Mn2+, Ba2+ partially inhibited the enzymatic activity, Cu2+, Co2+, Zn2+ showed strong inhibitory action on the enzymatic activity, which deactivated the enzyme at last. Na+, Li+, K+ had no obvious effect on the activity of GDH. 4. Chemical modification of GDH from porcine brain
The modification results showed that:Sulfydryl and Met should be considered as indispensable function groups of GDH, but disulfide bonds, Ser, His, Arg, Tyr and Lys residues should not be.
引文
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