Kawashima A, Sato A, Kawashima M, Nitta K, Yumura W, Sugino N, Nihei H, Natori Y
Department of Medicine, Tokyo Women's Medical College, Japan.
Kidney Int. 1998 Jul;54(1):275-8. doi: 10.1046/j.1523-1755.1998.00958.x.
A procedure for the isolation of highly purified lysosomes from normal rat kidney is described.
The method depends on the swelling of mitochondria when the postnuclear supernatant fraction is incubated with 2 mM Ca2+. The lysosomes can then be separated from the swollen mitochondria by Percoll density gradient centrifugation.
The lysosomal fraction obtained by our method was enriched more than 30-fold in terms of marker enzymes with a yield of about 11%. Electron microscopic examination and the measurement of the activities of marker enzymes for various subcellular organelles indicated that our lysosomal preparation was essentially free from contamination by other organelles.
We believe that this procedure for isolating kidney lysosome will be useful in the study of the mechanisms of specific modification, processing and catabolism of proteins.
描述了一种从正常大鼠肾脏中分离高度纯化溶酶体的方法。
该方法基于当核后上清液部分与2 mM Ca2+孵育时线粒体的肿胀。然后通过Percoll密度梯度离心将溶酶体与肿胀的线粒体分离。
通过我们的方法获得的溶酶体部分在标记酶方面富集了30多倍，产率约为11%。电子显微镜检查和各种亚细胞器标记酶活性的测量表明，我们制备的溶酶体基本上没有其他细胞器的污染。
我们认为这种分离肾脏溶酶体的方法将有助于研究蛋白质的特异性修饰、加工和分解代谢机制。
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