• Journal of Life Science
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• v.16 no.5
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• pp.716-722
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• 2006

Superoxide dismutase (SOD) is physiologically important in regulating cellular homeostasis and apoptotic cell death, and its mutations are the cause of familial amyotrophic lateral sclerosis (FALS). Mitochondrial serine protease HtrA2 has a pro-apoptotic function and has known to be associated with neurodegenerative disorders. To investigate the relationship between genes associated with apoptotic cell death, such as HtrA2 and SOD1, we utilized the pGEX expression system to develop a simple and rapid method for purifying wild-type and ALS-associated mutant SOD1 proteins in a suitable form for biochemical studies. We purified SOD1 and SOD1 (G93A) proteins to approximately 90% purity with relatively high yields (3 mg per liter of culture). Consistent with the result in mammalian cells, SOD1 (G93A) was more insoluble than wild-type SOD1 in E. coli, indicating that research on the aggregate formation of SOD1 may be possible using this pGEX expression system in E. coli. We investigated the HtrA2 serine protease activity on SOD1 to assess the relationship between two proteins. Not only wild-type SOD1 but also ALS-associated mutant SOD1 (G93A) were cleaved by HtrA2, resulting in the production of the 19 kDa and 21 kDa fragments that were specific for anti-SOD1 antibody. Using protein gel electrophoresis and immunoblot assay, we compared the relative molecular masses of thrombin-cleaved GST-SOD1 and HtrA2-cleaved SOD1 fragments and can predict that the HtrA2-cleavage sites within SOD1 are the peptide bonds between leucine 9-lysine 10 (L9-K10) and glutamine 23-lysine 24 (Q23-K24). Our study indicates that SOD1 is one of the substrate for HtrA2, suggesting that both HtrA2 and SOD1 may be important for modulating the HtrA2-SOD1-mediated apopotic cell death that is associated with the pathogenesis of neurodegenerative disorder.

• Korean Journal of Agricultural Science
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• v.9 no.1
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• pp.284-302
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• 1982

Development of protein resources as food has been a big issue especially in Southeast Asia region, and intake of protein is also insufficient in Korea. To cope with this shortage of protein resources and its improvement together with increased production of high nutritive animal protein, studies were carried out on feeding of Korean native goats. In the experiments were made absorption of carbohydrate and volatile fatty acid in miniature rumen, and absorption of amino acid in rumen as in vivo were conducted as part of studies on nutritional absorption in rumen. Those nutritional for improvement of feeding and management as described above are summarized as following. 1. According to the result of test on the nutritional absorption of native goat by means of miniature rumen method, absorption ratio of VFA measured at 0.5, 1 and 2 hours after injection of nutrition showed propionic acid 70-86%, acetic acid 74-87%, and lactic acid 76-89%. In the absorption of organic substances, ethyl alcohol of 0.5% showed 29-87% and lactic acid of 0.1M showed 12-27% of absorption ratio. 2. Result of absorption measurement in rumen from L-type free amino acid injection in the content of rumen vein showed lower rate at menthionine-free group compared to whole-egg amino acid injection in the content of rumen vein showed lower rate at methioine-free group compared to whole-egg amino acid group, and high absorption ratio was observed at methionine 3 times group and urea added group. Deficiency of methionine caused no change of the content in mucous membranes. 3. Absorption of amino acid in rumen muscular layer showed equal tendency as in the mucous membrane without exerting any influence of methionine deficiency. At the methionine3-times group, content of methionine and glutamine were increased by 14.7 and 4.4 times as compared to whole-egg amino acid group, an absorption ratio of glutamine, proline and valine were increased at urea added group. 4. In general, concentration of amino acid in rumen vein plasma was lower than in rumen mucous membrane and muscular layer. Absorption ratio of amino acid is decreased due to methionine deficiency, and tripling of methionine or urea adding caused increment of amino acid. Absorption pattern is thus varied depending on the composition of amino acid. 5. At the urea added group, content of ammonia-N, amino-N and urea were increased in rumen muscular layer. As the inside of goat's rumen was unable to clean thoroughly, investigation was made on remaining bacteria, however, variation of ammonia-N was affected by these bacterial content. 6. Variation in rumen structure by differential absorption of amino acid was observed by general microscope and fluorescent microscope. According to the result of observation in the methionine 3 times group, single cylinder epithelium of mucous membrane showed rather thin, and it was thick at urea added group though no significant differences existed among test groups in submucous membrane and muscular layer.