AN EVALUATION OF THE MEASUREMENT OF THE ACTIVITIES OF COMPLEXES I-IV IN THE RESPIRATORY-CHAIN OF HUMAN SKELETAL-MUSCLE MITOCHONDRIA
AN EVALUATION OF THE MEASUREMENT OF THE ACTIVITIES OF COMPLEXES I-IV IN THE RESPIRATORY-CHAIN OF HUMAN SKELETAL-MUSCLE MITOCHONDRIA
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DOI：
10.1006/bmmb.1994.1004
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发表时间：
1994-02-01
期刊：
影响因子：
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通讯作者：
TURNBULL, DM
中科院分区：
文献类型：
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作者：
BIRCHMACHIN, MA;BRIGGS, HL;TURNBULL, DM
单个呼吸链复合物的测量是研究由于线粒体功能障碍引起的疾病的重要组成部分。我们已经评估了测定人骨骼肌线粒体中复合物I至IV的测定方法，并优化了这些测定方法，以提供灵敏可靠的诊断技术，特别是在需要确定单个复合物部分中断的情况下。使用几种已建立的膜破坏方法，我们已经发现，通过在低渗磷酸钾缓冲液中冻融线粒体获得复合物I和II的最佳活性，而通过加入洗涤剂n-十二烷基-β-D-麦芽糖苷，复合物III和IV的活性显著增加。在2.5（-1）牛血清白蛋白存在下测量复合物I活性，其增加鱼藤酮敏感性，并且我们已经表明NADH-细胞色素B（5）还原酶对鱼藤酮不敏感的NADH-泛醌氧化还原酶活性做出重要贡献。mg.ml在线粒体级分与琥珀酸盐预孵育以完全活化复合物后测量复合物II活性。复合物I和III的活性分别取决于泛醌和泛醇的类异戊二烯链的长度。这些试验已用于建立对照范围。(C)1994年出版社出版。
The measurement of individual respiratory chain complexes is an important component of the investigation of diseases due to mitochondrial dysfunction. We have evaluated assays which measure complexes I to IV in human skeletal muscle mitochondria and in addition optimized these assays to provide sensitive and reliable diagnostic techniques, particularly in situations where a partial interruption at a single complex needs to identified. Using several established methods of membrane disruption we have found that optimal activities of complexes I and II are obtained by freeze-thawing the mitochondria in hypotonic potassium phosphate buffer, whereas complex III and IV activities are markedly increased by the addition of the detergent n-dodecyl-beta-D-maltoside. Complex I activity is measured in the presence of 2.5 mg.ml(-1) bovine serum albumin, which increases rotenone sensitivity, and we have shown that NADH-cytochrome b(5) reductase makes an important contribution to the rotenone-insensitive NADH-ubiquinone oxidoreductase activity. Complex II activity is measured after preincubation of the mitochondrial fraction with succinate to fully activate the complex. Complex I and III activities are dependent upon the length of the isoprenoid chain of the ubiquinone and ubiquinol, respectively. These assays have been used to establish a control range. (C) 1994 Academic Press, Inc.