PURIFICATION OF A MEMBRANE-ASSOCIATED PROTEIN COMPLEX REQUIRED FOR PROTEIN TRANSLOCATION ACROSS THE ENDOPLASMIC-RETICULUM
PURIFICATION OF A MEMBRANE-ASSOCIATED PROTEIN COMPLEX REQUIRED FOR PROTEIN TRANSLOCATION ACROSS THE ENDOPLASMIC-RETICULUM
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DOI：
10.1073/pnas.77.12.7112
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发表时间：
1980-01-01
期刊：
影响因子：
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通讯作者：
BLOBEL, G
中科院分区：
文献类型：
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作者：
WALTER, P;BLOBEL, G
[狗胰腺]微粒体膜转运新生的前分泌蛋白跨越其脂质双层的能力，可以在很大程度上废除提取它们与高离子强度缓冲液。可以通过将盐提取物添加回耗尽的膜来重建它。利用疏水色谱法，将盐提取物的蛋白质组分纯化至均质，所述蛋白质组分重建所提取的膜的易位活性。该组分在凝胶过滤、离子交换色谱、吸附色谱和蔗糖梯度离心后表现为均质物质。当在[十二烷基硫酸钠]中通过聚丙烯酰胺凝胶电泳检查时，观察到表观MW为72，000、68，000、54，000、19，000、14，000和9000的6种多肽的化学计量约相等且恒定，表明它们是复合物的亚基。11 S的沉降系数与各亚基分子量之和基本一致。MW 68，000和9000亚基强烈标记N-[3 H]乙基马来酰亚胺。因此，报道的未分级提取物中易位活性的巯基要求可以定位于纯化复合物的MW 68，000和9000亚基中的一个或两个。
The capacity of [dog pancreas] microsomal membranes to translocate nascent presecretory proteins across their lipid bilayer can be largely abolished by extracting them with high ionic strength buffers. It can be reconstituted by adding the salt extract back to the depleted membranes. Utilizing hydrophobic chromatography a protein component of the salt extract that reconstitutes the translocation activity of the extracted membranes was purified to homogenicity. This component behaves as a homogeneous species upon gel filtration, ion-exchange chromatography, adsorption chromatography and sucrose-gradient centrifugation. When examined by polyacrylamide gel electrophoresis in [sodium dodecylsufate], 6 polypeptides with apparent MW of 72,000, 68,000, 54,000, 19,000, 14,000 and 9000 are observed in about equal and constant stoichiometry, suggesting that they are subunits of a complex. The sedimentation coefficient of 11S is in good agreement with the sum of the MW of the subunits. The MW 68,000 and 9000 subunits label intensely with N-[3H]ethylmaleimide. Thus, the reported sulfhydryl group requirement of the translocation activity in the unfractionated extract may be localized to either or both the MW 68,000 and 9000 subunits of the purified complex.