DEFECTIVE PATTERN OF MITOCHONDRIAL RESPIRATORY ENZYMES IN MITOCHONDRIAL MYOPATHY
DEFECTIVE PATTERN OF MITOCHONDRIAL RESPIRATORY ENZYMES IN MITOCHONDRIAL MYOPATHY
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DOI：
10.1007/bf01799245
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发表时间：
1989-01-01
影响因子：
4.2
通讯作者：
TADA, K
中科院分区：
文献类型：
--
作者：
MIYABAYASHI, S;HAGINOYA, K;TADA, K
材料与方法取肌肉组织，用0.25mol/L蔗糖、2 mmol/L EDTA、10 mmol/L TrisHCl（pH7.4）和14 μ g/ml胶原酶（Sigma，Type VII）9倍体积的缓冲液冲洗30 min，用Potter匀浆器离心后，差速离心分离线粒体。用3 mg/ml毛地黄皂苷处理成纤维细胞，释放胞浆酶用于线粒体酶测定。NADH-细胞色素c氧化还原酶、琥珀酸-细胞色素c氧化还原酶和琥珀酸脱氢酶（EC 1.3. 99.1）根据Sanadi et al.（1967），Kennaway et al.（1984）和彭宁顿（1961）。根据Wharton和Tzagoloff（1967）的描述，通过还原型细胞色素c的氰化物敏感性氧化速率测定细胞色素c氧化酶活性。Mg 2+依赖性ATP酶（EC 3.6. 1.4)用香川（1974）的方法测定活性。用Bookelman埃塔尔的方法测定细胞色素的浓度。（1978年）。对于免疫印迹，基本上根据Kadenbach等人（1983）的方法进行SDS-PAGE。将SDS-PAGE分离的线粒体蛋白按方法转移至Durapore GVHP过滤器中
MATERIALS AND METHODSFor biochemical analysis, biopsied muscle was cut in small pieces and rinsed in 9 volumes of 0.25 mol/L sucrose, 2mmol/L EDTA, 10mmol/L Tris HC1, pH 7.4 and 14~ g/ml collagenase (Sigma, type VII) for 30 min. A mitochondrial fraction was isolated via differential centrifugation after homogenizing in Potter homogenizer. Fibroblasts were treated with 3 mg/ml digitonin to release cytoplasmic enzymes for m~ tochondrial enzyme assay. Activities of NADH-cytochrome c oxidoreductase, succinate-cytochrome c oxidoreductase and succinate dehydrogenase (EC 1.3. 99.1) were determined according to the methods of Sanadi et al.(1967), Kennaway et al.(1984) and Pennington (1961), respectively. Cytochrome c oxidase activity was measured by the rate of cyanide-sensitive oxidation of reduced eytochrome c, essentially as described by Wharton and Tzagoloff (1967). Mg2+-dependent ATPase (EC 3.6. 1.4) activity was measured by the method of Kagawa (1974). The concentrations of cytochromes were determined by the method of Bookelman etal.(1978). For immunoblotting, SDS-PAGE was made essentially according to the method of Kadenbach et aL (1983). Mitochondrial proteins separated by SDS-PAGE were electrophoretically transferred to Durapore GVHP filter according to the method