Symons L J, Jonas A J
Department of Pediatrics, University of Texas Health Science Center at Houston 77030.
Anal Biochem. 1987 Aug 1;164(2):382-90. doi: 10.1016/0003-2697(87)90508-2.
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 beta-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.
已使用Percoll密度梯度离心法纯化了呈膜泡形式的正常大鼠肝脏溶酶体膜。使用与蔗糖等渗并缓冲至pH 7.0的Percoll梯度，溶酶体（密度 = 1.111）被纯化了约63±12倍（平均值±标准差，n = 5）。然后将这些溶酶体暴露于pH 7.0的10 mM蛋氨酸甲酯中，其摄取导致溶酶体肿胀和破裂，随后形成囊泡。使用第二个Percoll密度梯度进一步将这些囊泡（密度 = 1.056）与残留的线粒体和质膜酶活性分离。标记酶分析和电子显微镜表明，溶酶体膜基本不含可溶性溶酶体酶β-己糖胺酶和污染的细胞器。溶酶体膜中溶酶体ATP酶的比活性比完整溶酶体中的高四倍。
