The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions.
The large-scale separation of peroxisomes, mitochondria, and lysosomes from the livers of rats injected with triton WR-1339. Improved isolation procedures, automated analysis, biochemical and morphological properties of fractions.
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从注射Triton WR-1339的大鼠肝脏的过氧化物酶体，线粒体和溶酶体的大规模分离。改进的隔离程序，自动分析，分数的生化和形态特性。
DOI：
10.1083/jcb.37.2.482
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发表时间：
1968-05
影响因子：
7.8
通讯作者：
De Duve, C
中科院分区：
文献类型：
--
作者：
Leighton, F;Poole, B;Beaufay, H;Baudhuin, P;Coffey, J W;Fowler, S;De Duve, C
本文描述了从注射了Triton WR-1339的大鼠的肝脏中提纯和分析过氧化物酶、溶酶体和线粒体的改进的、基本上自动化的方法。利用这些新方法，可以在不到6小时的时间内获得几乎没有污染的线粒体，以及数量更少(约100毫克蛋白质)、纯度(95%)的稀有细胞质颗粒，使它们适合于详细的生化研究。到目前为止，在这些制剂上获得的结果对这三组细胞质颗粒的生化和形态特性做出了更确凿和准确的先前估计。此外，发现过氧化体含有肝脏的所有L-α-羟基酸氧化酶，以及一小部分NADP连接的异柠檬酸脱氢酶活性。后一种酶的另一小部分存在于线粒体中，其余的与细胞汁液有关。具有代谢活性的细胞质DNA的线粒体定位可以得到验证。线粒体、整个过氧化物体、过氧化物体核心、溶酶体和内质网中各组分的相对含量是通过直接测量电子显微镜和酶分析结果的线性规划(基于颗粒的生物化学均一性)独立地估计的。除了细胞色素氧化酶活性低与线粒体损伤有关的一部分外，这两种类型的估计非常吻合。
Improved, largely automated methods are described for the purification and analysis o peroxisomes, lysosomes, and mitochondria from the livers of rats injected with Triton WR-1339. With these new methods, it has become possible to obtain, in less than 6 hr and with reliable reproducibility, mitochondria practically free of contaminants, as well as the rarer cytoplasmic particles in amounts (about 100 mg of protein) and in a state of purity (95%) that make them suitable for detailed biochemical studies. The results obtained so far on these preparations have made more conclusive and precise previous estimates of the biochemical and morphological properties of the three groups of cytoplasmic particles. In addition, peroxisomes were found to contain essentially all the L-α-hydroxy acid oxidase of the liver, as well as a small, but significant fraction of its NADP-linked isocitrate dehydrogenase activity. Another small fraction of the latter enzyme is present in the mitochondria, the remainder being associated with the cell sap. The mitochondrial localization of the metabolically active cytoplasmic DNA could be verified. The relative content of the fractions in mitochondria, whole peroxisomes, peroxisome cores, lysosomes, and endoplasmic reticulum was estimated independently by direct measurements on electron micrographs, and by linear programming (based on the assumption that the particles are biochemically homogeneous) of the results of enzyme assays. The two types of estimates agreed very well, except for one fraction in which low cytochrome oxidase activity was associated with mitochondrial damage.