ISOLATION OF HIGHLY PURIFIED LYSOSOMES FROM RAT-LIVER - IDENTIFICATION OF ELECTRON CARRIER COMPONENTS ON LYSOSOMAL MEMBRANES
ISOLATION OF HIGHLY PURIFIED LYSOSOMES FROM RAT-LIVER - IDENTIFICATION OF ELECTRON CARRIER COMPONENTS ON LYSOSOMAL MEMBRANES
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DOI：
10.1093/oxfordjournals.jbchem.a123616
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发表时间：
1991-10-01
影响因子：
2.7
通讯作者：
OHKUMA, S
中科院分区：
文献类型：
--
作者：
ARAI, K;KANASEKI, T;OHKUMA, S
用 1 mM Ca2+ 处理核后上清液 (PNS) 使线粒体膨胀并变亮后，使用 Percoll 密度梯度离心，从给予右旋糖酐的大鼠肝脏中高产率 (25%) 分离出高度纯化的溶酶体（右旋糖体）。通过该方法获得的溶酶体级分中的 N-乙酰基-β-氨基葡萄糖苷酶和芳基硫酸酯酶富集了 100 倍以上，酸性磷酸酶和 β-葡萄糖苷酶富集了 40 倍以上。电镜检查和各种亚细胞器标记酶活性的测量表明，溶酶体部分基本上没有受到其他细胞器的污染。在溶酶体膜上发现并研究了黄素、泛醌和血色素。纯化的溶酶体膜的FAD和泛醌-9含量分别为0.118和6.93 nmol/mg蛋白质。溶酶体中的血色素显示出与 b 型细胞色素相似的光谱，其中 α 峰位于 562 nm，γ 峰位于 436 nm。
Using Percoll density gradient centrifugation after treatment of the postnuclear supernatant (PNS) with 1 mM Ca2+ to swell and lighten mitochondria, we isolated highly purified lysosomes (dextranosomes) in high yield (25%) from the livers of rats to which dextran had been administered. The lysosomal fraction obtained by this method was enriched more than 100-fold in N-acetyl-beta-glucosaminidase and arylsulfatase and 40-fold in acid phosphatase and beta-glucosidase. Electron microscopic examination and measurement of marker enzyme activity for various subcellular organella indicated that the lysosomal fraction was essentially free from contamination by other organella. Flavins, ubiquinones, and hemochromes were found on lysosomal membranes and investigated. The FAD and ubiquinone-9 contents of the purified lysosomal membranes were 0.118 and 6.93 nmol/mg of protein, respectively. Hemochromes in lysosomes showed spectra similar to that of a b-type cytochrome, with the alpha-peak at 562 nm and the gamma-peak at 436 nm.