Purification and characterization of insoluble SOD in the animal models of amyotrophic lateral sclerosis

将肌萎缩性侧索硬化症（ALS）动物模型的突变型SOD 1（G93A，H46R）转基因动物的脑和脊髓分级分离成三个级分，如Tris缓冲盐水（TBS）可溶性级分、NP 40可溶性级分和NP 40不溶性级分。大多数突变体SOD 1可溶于TBS中，在NP 40级分中只有10%至30%的蛋白质。所有动物的TBS可溶性和NP 40可溶性SOD 1均均匀分布于前脑、脊髓、小脑和脑干。相比之下，去污剂不溶性突变体SOD1，G93A和H46R，仅从脊髓和脑干分离，这是中枢神经系统的两个区域，特别是受ALS影响。NP 40不溶性组分中的G93A活性显著低于TBS可溶性组分。当突变SOD 1在三个馏分进行免疫亲和层析使用的多克隆抗体对SOD 1，我们发现，突变SOD 1在NP 40不溶性馏分具有更高的亲和力的多克隆抗体比在TBS和NP 40可溶性馏分。从亲和柱上纯化的三个组分的突变SOD 1具有相同的分子量，通过质谱法判断。野生型和突变型SOD 1（WT，A4V，G37R，H46R，G93A）通过sf21昆虫细胞系统过量产生并纯化。突变SOD 1几乎没有检测到三个单克隆抗体在Western印迹分析。DTT或热处理的突变SOD 1对单克隆抗体的免疫反应性较低。由于这些抗体的所有表位都映射在希腊关键环内，因此这些数据表明，在解折叠过程中，野生型和突变型SOD 1之间的环中发生了不同的构象变化。

The brain and spinal cord of mutant SOD1(G93A, H46R) transgenic animals, which are animal models of amyotrophic lateral sclerosis(ALS), were fractionated into three fractions such as Tris buffered saline(TBS) soluble, NP40 soluble and NP40 insoluble fractions. Most of the mutant SOD1s were soluble in TBS, with only 10 to 30% as much protein in the NP40 fraction. TBS-soluble and NP40-soluble SOD1s from all animals were evenly distributed in the forebrain, spinal cord, cerebellum and brain stem. In contrast, the detergent insoluble mutant SOD1,both G93A and H46R, was isolated only from the spinal cord and brainstem, which are two regions of the central nervous system specifically affected by ALS. The activity of G93A in the NP40 insoluble fraction was significantly less than that in TBS soluble fraction. When mutant SOD1s in three fractions were subjected to immunoaffinity chromatography using a polyclonal antibody against SOD1,we found that mutant SOD1 in NP40 insoluble fraction had a higher affinity for a polyclonal antibody than that in TBS and NP40 soluble fractions. Mutant SOD1s from the three fractions purified on an affinity column had the same molecular mass as judged by mass spectrometry. Wild type and mutant SOD1(WT, A4V, G37R, H46R, G93A) were overproduced by sf21 insect cell system and were purified. Mutant SOD1s were barely detected by three monoclonal antibodies in Western blot analyses. Mutant SOD1s by DTT or heat treatment showed a lower immunoreactivity against the monoclonal antibodies. Because all the epitopes of these antibodies are mapped within the Greek key loop, these data suggest that different conformational changes occur in the loop between wild and mutant SOD1s during the unfolding process.