| Budget Amount *help |
¥3,000,000 (Direct Cost: ¥3,000,000)
Fiscal Year 2004: ¥700,000 (Direct Cost: ¥700,000)
Fiscal Year 2003: ¥2,300,000 (Direct Cost: ¥2,300,000)
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| Research Abstract |
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.
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