A NEW METHOD FOR PREPARATION OF RAT LIVER LYSOSOMES - SEPARATION OF CELL ORGANELLES OF RAT LIVER BY CARRIER-FREE CONTINUOUS ELECTROPHORESIS
A NEW METHOD FOR PREPARATION OF RAT LIVER LYSOSOMES - SEPARATION OF CELL ORGANELLES OF RAT LIVER BY CARRIER-FREE CONTINUOUS ELECTROPHORESIS
复制标题
DOI：
10.1083/jcb.46.3.576
复制
发表时间：
1970-01-01
影响因子：
7.8
通讯作者：
HANNIG, K
中科院分区：
文献类型：
--
作者：
STAHN, R;MAIER, KP;HANNIG, K
差速离心与无载体连续电泳相结合是分离动物细胞器的一种新方法。系统地检查了各种缓冲液，以找到在自由流动电泳仪中保存细胞器并提供良好分离的系统。三乙醇胺-乙酸酯缓冲液（10 mM）， pH 7.4。根据标记酶和电子显微图，分离的溶酶体是纯的。在标记酶芳基硫酸酯酶和ß-葡萄糖醛酸酶方面，溶酶体在电泳迁移率方面表现出异质性。迁移率较高的溶酶体对芳基磺化酶的富集程度最高可达240倍。电泳迁移率较低的溶酶体对ß-葡萄糖醛酸酶表现出65倍的富集。在不同流动性溶酶体中，ß-葡萄糖醛酸酶与芳基硫酸酯酶的比例为2:1 ~ 1:2。每克肝蛋白的产量约为1毫克溶酶体蛋白。一次实验可获得5 - 8mg溶酶体。电泳分离被证明是获得纯净且保存良好的溶酶体的有效工具。
A combination of differential centrifugation and carrier-free continuous electrophoresis is introduced as a new method for the isolation of animal cell organelles. Various buffers were systematically checked in order to find the system which preserves the organelles and gives as well a good separation in the free-flow electrophoresis apparatus. Triethanolamine-acetate buffer (10 mM), pH 7.4 was used. The isolated lysosomes were pure according to marker enzymes and electron micrographs. A heterogeneity of the lysosomes in electrophoretic mobility was demonstrated with respect to the marker enzymes arylsulfatase and ß-glucuronidase. The lysosomes with higher mobility showed a maximum enrichment of 240-fold with respect to arylsulfatase. The lysosomes with lower electrophoretic mobility showed a 65-fold enrichment with respect to ß-glucuronidase. The ratio of ß-glucuronidase to arylsulfatase varied from 2:1 to 1:2 in lysosomes of different mobility. The yield amounted to approximately 1 mg of lysosomal protein per gram of liver protein. 5–8 mg of lysosomes can be obtained in one experiment. The electrophoretic separation proves to be an effective tool in obtaining pure and well preserved lysosomes.