Janski A M, Cornell N W
Biochem J. 1980 Feb 15;186(2):423-9. doi: 10.1042/bj1860423.
Conditions were determined for rapid separation of cytosolic and mitochondrial compartments by digitonin fractionation of rat hepatocytes. The minimum time required for separation of mitochondrial and cytosolic enzyme markers decreased rapidly with increasing temperature. Kyro EOB, a non-ionic detergent, increases the release of cytosolic enzymes, particularly at lower temperatures. Experimental procedures are described for greater than 90% release of cytosolic enzymes and less than 2% release of mitochondrial enzymes in 3s. By using appropriate concentrations of digitonin and Kyro EOB in a fractionation medium maintained at 1 degrees C and a minimum time of exposure to the medium, nearly separate patterns of release were obtained for enzyme markers for the cytosol, mitochondrial matrix and mitochondrial intermembrane space. The distribution of enzymes that exist in more than one of these compartments was quantified by comparing their rates of release with those of marker enzymes. The cytosol/mitochondrial-matrix distributions for such enzymes in hepatocytes from starved rats were 16%/84% for aspartate aminotransferase, 34%/66% for fumarase and 77%/23% for ATP citrate lyase. In hepatocytes from rats that were induced to synthesize ATP citrate lyase by starvation and re-feeding, the ratio had increased to 95%/5%. The maximum cytosol/intermembrane-space ratio for adenylate kinase was 8%/92%. A procedure is also described for treating commercial digitonin that increases its solubility in water from about 1mg/ml to more than 800mg/ml.
确定了通过用洋地黄皂苷分离大鼠肝细胞来快速分离胞质和线粒体区室的条件。分离线粒体和胞质酶标志物所需的最短时间随着温度升高而迅速减少。非离子去污剂Kyro EOB可增加胞质酶的释放，尤其是在较低温度下。描述了实验步骤，可在3秒内实现胞质酶释放率大于90%且线粒体酶释放率小于2%。通过在保持于1℃的分级分离培养基中使用适当浓度的洋地黄皂苷和Kyro EOB，并使细胞与培养基接触的时间最短，可获得胞质、线粒体基质和线粒体内膜间隙的酶标志物几乎分离的释放模式。通过比较存在于这些区室中不止一个区室的酶与标志物酶的释放速率，对其分布进行了定量。饥饿大鼠肝细胞中，天冬氨酸氨基转移酶的胞质/线粒体基质分布为16%/84%，延胡索酸酶为34%/66%，ATP柠檬酸裂解酶为77%/23%。在因饥饿和再喂食而被诱导合成ATP柠檬酸裂解酶的大鼠的肝细胞中，该比例已增至95%/5%。腺苷酸激酶的最大胞质/内膜间隙比例为8%/92%。还描述了一种处理市售洋地黄皂苷的方法，可使其在水中的溶解度从约1mg/ml增加至800mg/ml以上。
