Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.
Isolation of autophagic vacuoles from rat liver: morphological and biochemical characterization.
复制标题
从大鼠肝脏中分离自噬液泡：形态学和生化表征。
DOI：
10.1083/jcb.93.1.144
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发表时间：
1982-04
影响因子：
7.8
通讯作者：
Glaumann, H
中科院分区：
文献类型：
--
作者：
Marzella, L;Ahlberg, J;Glaumann, H
已发现由长春碱（VBL）引起的自噬的诱导伴随着来自大鼠肝脏的线粒体-溶酶体（ML）级分中的蛋白水解的刺激（Marzella和Glaumann，1980，Lab.投资，42：8-17。Marzella和Glaumann，1980年，实验室。投资，42：18-27）。在这一部分中，增强的蛋白水解与自噬泡（AV）的相对体积分数增加三倍相关。在试图分离AV，我们亚分级的ML悬浮液在不同的时间间隔诱导自噬VBL通过离心的不连续Metrizamide梯度从50%到15%。收集24 - 20%和20 - 15%界面处的材料条带。形态学分析表明，诱导自噬后3小时，这些馏分主要由（约90%）完整的自噬空泡。这些自噬泡含有细胞质、线粒体、部分内质网，偶尔还含有极低密度脂蛋白、游离或高尔基体衍生物中的颗粒，特别是分泌颗粒。被隔离的物质显示出持续降解的超微结构迹象。除了含有典型的自噬空泡外，分离的组分由缺乏形态学上可识别的细胞组分的溶酶体组成。根据形态测定分析判断，非溶酶体物质的污染仅占百分之几。典型的溶酶体“标记”酶富集15倍，而与匀浆相比，分离的AV级分中的蛋白水解活性富集10至20倍。最初，非溶酶体的线粒体和微粒体酶活性的产量增加与诱导的自噬平行，但后来，减少与先进的降解的隔离的细胞器。因此，在AV的情况下，非溶酶体标记酶的存在不能用于计算级分纯度，因为新隔离的细胞器具有酶活性。分离的自噬泡在体外孵育时显示蛋白水解活性。AV级分的相对高的磷脂/蛋白质比率（0.5）表明磷脂比蛋白质降解得更慢。结论是AVs可以被分离成纯的组分，并且是诱导自噬后大鼠肝脏中蛋白质降解增强的亚细胞位点。
The induction of autophagy caused by vinblastine (VBL) has been found to be concomitant with a stimulation of proteolysis in a mitochondrial- lysosomal (ML) fraction from the rat liver (Marzella and Glaumann, 1980, Lab. Invest., 42: 8-17. Marzella and Glaumann, 1980, Lab. Invest., 42:18-27). In this fraction the enhanced proteolysis is associated with a threefold increase in the relative fractional volume of autophagic vacuoles (AVs). In an attempt to isolate the AVs, we subfractionated the ML suspension at different intervals after the induction of autophagy by VBL by centrifugation on a discontinuous Metrizamide gradient ranging from 50% to 15%. The material banding at the 24 to 20% and the 20 to 15% interphases was collected. Morphological analysis reveals that 3 h after induction of autophagy these fractions consist predominantly (approximately 90%) of intact autophagic vacuoles. These autophagic vacuoles contain cytosol, mitochondria, portions of endoplasmic reticulum, and occasional very low density lipoprotein, particles either free or in Golgi apparatus derivatives, in particular secretory granules. The sequestered materials show ultrastructural signs of ongoing degradation. In addition to containing typical autophagic vacuoles, the isolated fractions consist of lysosomes lacking morphologically recognizable cellular components. Contamination from nonlysosomal material is only a few percent as judged from morphometric analysis. Typical lysosomal "marker" enzymes are enriched 15-fold, whereas the proteolytic activity is enriched 10- to 20-fold in the isolated AV fraction as compared to the homogenate. Initially, the yield of nonlysosomal mitochondrial and microsomal enzyme activities increases in parallel with the induction of autophagy but, later on, decreases with advanced degradation of the sequestered cell organelles. Therefore, in the case of AVs the presence of nonlysosomal marker enzymes cannot be used for calculation of fraction purity, since newly sequestered organelles are enzymatically active. Isolated autophagic vacuoles show proteolytic activity when incubated in vitro. The comparatively high phospholipid/protein ratio (0.5) of the AV fraction suggests that phospholipids are degraded more slow than proteins. Is it concluded that AVs can be isolated into a pure fraction and are the subcellular site of enhanced protein degradation in the rat liver after induction of autophagy.