Smith W P, Tai P C, Davis B D
Proc Natl Acad Sci U S A. 1978 Dec;75(12):5922-5. doi: 10.1073/pnas.75.12.5922.
To determine the length of secreted nascent polypeptide chain that is surrounded by membrane, we digested labeled nascent chains protruding from protoplasts of Bacillus subtilis with Pronase and isolated the residual ribosome-attached chains from the membrane-polysome fraction. Gel chromatography revealed a sharp major peak that had been protected by membrane plus bound ribosomes. The ribosomes themselves protected half as great a length. Because no free chain between the ribosome and the membrane was detected by Pronase treatment, the difference between the two protected lengths should measure the length protected by the membrane. More accurate measurements of these lengths, obtained by dansylation of the exposed NH2 terminus of the isolated fragments, yielded a difference of 21 amino acids. This value corresponds to an extended chain of 75 A, which is approximately the thickness of the bacterial cell membrane. We earlier presented evidence that bacterial ribosomes are attached to membrane solely by their secreted chain. The present results further show that after loss of the extracellular segment of the chain its attachment persists, at 37 degrees as well as 0 degrees C. These findings suggest that the chain does not slip through a passive membrane but is actively held within a channel.
为了确定被膜包围的分泌新生多肽链的长度，我们用链霉蛋白酶消化从枯草芽孢杆菌原生质体伸出的标记新生链，并从膜 - 多核糖体组分中分离出残留的核糖体附着链。凝胶色谱显示出一个尖锐的主峰，该峰受到膜加结合核糖体的保护。核糖体本身保护的长度只有其一半。由于用链霉蛋白酶处理未检测到核糖体与膜之间的游离链，所以这两个受保护长度的差异应能测量出膜保护的长度。通过对分离片段暴露的NH2末端进行丹磺酰化得到的这些长度的更精确测量结果显示，差异为21个氨基酸。该值对应于75埃的伸展链，这大约是细菌细胞膜的厚度。我们之前提供的证据表明，细菌核糖体仅通过其分泌链附着于膜。目前的结果进一步表明，在链的细胞外区段丢失后，其附着在37℃以及0℃时仍然存在。这些发现表明，链不是被动地穿过膜，而是被主动地保持在一个通道内。
