On the attachment of ribosomes to microsomal membranes.
On the attachment of ribosomes to microsomal membranes.
复制标题
关于核糖体与微粒体膜的附着。
DOI：
10.1016/s0022-2836(66)80019-0
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发表时间：
1966
影响因子：
5.6
通讯作者：
G. Palade
中科院分区：
文献类型：
--
作者：
D. Sabatini;Y. Tashiro;G. Palade
用浓度递增的EDTA处理豚鼠肝微粒体，使其附着的核糖体解离成亚基。在20μmoles EDTA/0.5 g组织当量微粒体时，只有小的亚基（约32 s）被释放到蔗糖密度梯度中，根据RNA测定，这些亚基几乎占了微粒体制备物中最初存在的所有32 s亚基。在较高的EDTA浓度（20至200μ mol/0.5 g组织当量）下，释放的32 s亚基被降解，并且越来越多的大亚基（147 s）从微粒体膜上脱离。用[~ 3 H]亮氨酸标记后，分离的大亚基中含有新合成的蛋白质，超微结构分析证实，在EDTA浓度低于40μM时，大亚基优先释放，即使用500μ molEDTA/0.5g组织当量处理，仍有约30%的微粒体RNA结合在微粒体膜上。EDTA连续处理（500μ mol）和脱氧胆酸盐（0.5%）导致这种残留RNA的释放，通过分析离心，发现这种残留RNA与颗粒有关，主要是150 μ s颗粒，推测是大亚基。推测活性核糖体的大亚基更牢固地附着在微粒体膜上，小亚基的初始释放表明核糖体是通过其大亚基附着在微粒体膜上的。分离的负染色微粒体的电子显微镜结果支持这一假设。在有利的标本中，分离核糖体亚基的凹槽可以被识别，并被发现是定向一般平行于内质网或微粒体membrane.The功能的影响，这些发现进行了讨论，在有关的过程中分泌蛋白质的合成附着核糖体和随后释放到脑池空间。
Guinea pig hepatic microsomes were treated with increasing concentrations of EDTA to dissociate into subunits their attached ribosomes. The dissociation process was followed by zone centrifugation and its products characterized by analytical centrifugation.At 20μmoles EDTA per 0·5 g tissue equivalent of microsomes, only small subunits (∼ 32 s) were released to the sucrose density-gradients in amounts which, according to RNA determinations, accounted for practically all 32 s subunits originally present in the microsomal preparation. At higher EDTA concentrations (20 to 200μmoles/0·5 g tissue equivalent), the released 32 s subunits were degraded, and increasing amounts of large subunits (∼ 47 s) were detached from the microsomal membranes. Afterin vivolabeling with [3H]leucine, the detached large subunits contained newly synthesized proteins.Ultracentrifugal analysis confirmed the preferential release of small over large subunits at EDTA concentrations below 40μM.A fraction comprising ∼ 30% of the microsomal RNA remained bound to microsomal membranes even after treatment with 500μmoles EDTA/0·5 g tissue equivalent. Successive treatment with EDTA (500μmoles) and deoxy-cholate (0·5%) led to the release of this residual RNA which, by analytical centrifugation, was found to be associated with particles, primarily ∼ 50 s particles, presumably large subunits.After labelingin vivowith [3H]leucine, the particles resistant to detachment by EDTA contained ∼ 70% of the newly synthesized protein: presumably the large subunits of active ribosomes are more firmly attached to the microsomal membrane.The initial release of small subunits suggested that ribosomes are attached to the microsomal membrane by their large subunits. This assumption is supported by electron microscopy findings on isolated, negatively stained microsomes. In favorable specimens, the groove that separates the ribosomal subunits could be recognized and was found to be oriented generally parallel to the endoplasmic reticulum or microsomal membrane.The functional implications of these findings are discussed in relation to the processes of synthesis of secretory proteins on attached ribosomes and their subsequent release into the cisternal space.