利用洋地黄皂苷提取法区分肝细胞中自噬溶酶体对[14C]蔗糖的隔离与线粒体对[14C]蔗糖的摄取。
Use of digitonin extraction to distinguish between autophagic-lysosomal sequestration and mitochondrial uptake of [14C]sucrose in hepatocytes.
作者信息
Gordon P B, Tolleshaug H, Seglen P O
出版信息
Biochem J. 1985 Dec 15;232(3):773-80. doi: 10.1042/bj2320773.
Abstract
In isolated rat hepatocytes, electroinjected [14C]sucrose is sequestered both by mitochondria and by autophagosomes/lysosomes. Radioactivity can be selectively extracted from the latter organelles by low concentrations of digitonin, thereby providing a specific bioassay for autophagic sequestration. By including a digitonin extraction step in the assay procedure, autophagic [14C]sucrose sequestration could be shown to be virtually completely (greater than 90%) suppressed by the autophagy inhibitor 3-methyladenine (10 mM), whereas mitochondrial sugar uptake was unaffected. An amino acid mixture likewise suppressed autophagic sequestration very strongly, while having no detectable effect on the mitochondria.
摘要
在分离的大鼠肝细胞中，电注射的[14C]蔗糖被线粒体以及自噬体/溶酶体隔离。低浓度的洋地黄皂苷可从后一种细胞器中选择性地提取放射性，从而为自噬隔离提供一种特异性生物测定法。通过在测定程序中加入洋地黄皂苷提取步骤，可以发现自噬性[14C]蔗糖隔离几乎完全（超过90%）被自噬抑制剂3-甲基腺嘌呤（10 mM）抑制，而线粒体对糖的摄取不受影响。氨基酸混合物同样非常强烈地抑制自噬隔离，而对线粒体没有可检测到的影响。
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