Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients.
Isolation of highly purified rat liver lysosomal membranes using two Percoll gradients.
复制标题
使用两种 Percoll 梯度分离高度纯化的大鼠肝溶酶体膜。
DOI：
10.1016/0003-2697(87)90508-2
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发表时间：
1987
影响因子：
2.9
通讯作者：
Jonas,AJ
中科院分区：
文献类型：
--
作者：
Symons,LJ;Jonas,AJ
用Percoll密度梯度离心法对正常大鼠肝脏溶酶体膜进行了纯化。用Percoll与蔗糖等渗梯度纯化溶酶体(密度=1.111)约63±12倍(平均值±标准差，n=5)，并缓冲至pH 7.0。然后将这些溶酶体暴露在10 mM甲硫氨酸甲酯中，pH 7.0，摄取甲硫氨酸甲酯导致溶酶体肿胀和破裂，随后形成囊泡。使用第二个Percoll密度梯度将这些囊泡(密度=1.056)与剩余的线粒体和质膜酶活性进一步分离。标记酶分析和电子显微镜分析表明，溶酶体膜基本上不含可溶性溶酶体酶β-氨基己糖苷酶，也不含污染细胞器。溶酶体膜中溶酶体ATPase的比活力是完整溶酶体膜中的4倍。
Normal rat liver lysosomal membranes in the form of membrane vesicles have been purified using Percoll density gradient centrifugation. Lysosomes (density = 1.111) were purified approximately 63 ± 12-fold (mean ± standard deviation, n = 5) using a gradient of Percoll made isotonic with sucrose and buffered to pH 7.0. These lysosomes were then exposed to 10 mm methionine methyl ester, pH 7.0, the uptake of which resulted in swelling and breakage of the lysosomes with subsequent vesicle formation. These vesicles (density = 1.056) were further separated from residual mitochondrial and plasma membrane enzyme activities using a second Percoll density gradient. Marker enzyme analysis and electron microscopy indicated that the lysosomal membranes were essentially free of both β-hexosaminidase, a soluble lysosomal enzyme, and contaminating organelles. The specific activity of lysosomal ATPase in the lysosomal membranes was fourfold greater than in the intact lysosomes.