Adelman M R, Sabatini D D, Blobel G
J Cell Biol. 1973 Jan;56(1):206-29. doi: 10.1083/jcb.56.1.206.
In a medium of high ionic strength, rat liver rough microsomes can be nondestructively disassembled into ribosomes and stripped membranes if nascent polypeptides are discharged from the bound ribosomes by reaction with puromycin. At 750 mM KCl, 5 mM MgCl(2), 50 mM Tris.HCl, pH 7 5, up to 85% of all bound ribosomes are released from the membranes after incubation at room temperature with 1 mM puromycin. The ribosomes are released as subunits which are active in peptide synthesis if programmed with polyuridylic acid. The ribosome-denuded, or stripped, rough microsomes (RM) can be recovered as intact, essentially unaltered membranous vesicles Judging from the incorporation of [(3)H]puromycin into hot acid-insoluble material and from the release of [(3)H]leucine-labeled nascent polypeptide chains from bound ribosomes, puromycin coupling occurs almost as well at low (25-100 mM) as at high (500-1000 mM) KCl concentrations. Since puromycin-dependent ribosome release only occurs at high ionic strength, it appears that ribosomes are bound to membranes via two types of interactions: a direct one between the membrane and the large ribosomal subunit (labile at high KCl concentration) and an indirect one in which the nascent chain anchors the ribosome to the membrane (puromycin labile). The nascent chains of ribosomes specifically released by puromycin remain tightly associated with the stripped membranes. Some membrane-bound ribosomes (up to 40%) can be nondestructively released in high ionic strength media without puromycin; these appear to consist of a mixture of inactive ribosomes and ribosomes containing relatively short nascent chains. A fraction ( approximately 15%) of the bound ribosomes can only be released from membranes by exposure of RM to ionic conditions which cause extensive unfolding of ribosomal subunits, the nature and significance of these ribosomes is not clear.
在高离子强度介质中，如果新生多肽通过与嘌呤霉素反应从结合核糖体上释放出来，大鼠肝脏粗面微粒体可以无损地分解为核糖体和脱膜。在750 mM KCl、5 mM MgCl₂、50 mM Tris·HCl（pH 7.5）条件下，室温下用1 mM嘌呤霉素孵育后，高达85%的结合核糖体从膜上释放出来。核糖体以亚基形式释放，如果用聚尿苷酸编程，这些亚基在肽合成中具有活性。核糖体剥脱的粗面微粒体（RM）可以作为完整的、基本未改变的膜泡回收。从[³H]嘌呤霉素掺入热酸不溶性物质以及从结合核糖体上释放[³H]亮氨酸标记的新生多肽链来看，在低（25 - 100 mM）和高（500 - 1000 mM）KCl浓度下，嘌呤霉素偶联几乎同样良好。由于依赖嘌呤霉素的核糖体释放仅在高离子强度下发生，似乎核糖体通过两种类型的相互作用与膜结合：一种是膜与核糖体大亚基之间的直接相互作用（在高KCl浓度下不稳定），另一种是新生链将核糖体锚定到膜上的间接相互作用（对嘌呤霉素敏感）。由嘌呤霉素特异性释放的核糖体的新生链与脱膜紧密结合。一些膜结合核糖体（高达40%）在无嘌呤霉素的高离子强度介质中可以无损释放；这些似乎由无活性核糖体和含有相对短新生链的核糖体组成。一部分（约15%）结合核糖体只有通过将RM暴露于导致核糖体亚基广泛展开的离子条件下才能从膜上释放，这些核糖体的性质和意义尚不清楚。
