Nigam S K
Laboratory of Cell Biology, Howard Hughes Medical Institute, Rockefeller University, New York, NY 10021.
Proc Natl Acad Sci U S A. 1990 Feb;87(4):1296-9. doi: 10.1073/pnas.87.4.1296.
Subfractionation of a canine pancreatic homogenate was performed by several differential centrifugation steps, which gave rise to fractions with distinct marker profiles. Specific binding of guanosine 5'-[gamma-[35S]thio]triphosphate (GTP[gamma-35S]) was assayed in each fraction. Enrichment of GTP[gamma-35S] binding was greatest in the interfacial "smooth" microsomal fraction, expected to contain Golgi and other smooth vesicles. There was also marked enrichment in the rough microsomal fraction. Electron microscopy and marker protein analysis revealed the rough microsomes (RMs) to be highly purified rough endoplasmic reticulum (RER). The distribution of small (low molecular weight) GTP binding proteins was examined by a [alpha-32P]GTP blot-overlay assay. Several apparent GTP binding proteins of molecular masses 22-25 kDa were detected in various subcellular fractions. In particular, at least two such proteins were found in the Golgi-enriched and RM fractions, suggesting that these small GTP binding proteins were localized to the Golgi and RER. To more precisely localize these proteins to the RER, native RMs and RMs stripped of ribosomes by puromycin/high salt were subjected to isopycnic centrifugation. The total GTP[gamma-35S] binding, as well as the small GTP binding proteins detected by the [alpha-32P]GTP blot overlay, distributed into fractions of high sucrose density, as did the RER marker ribophorin I. Consistent with a RER localization, when the RMs were stripped of ribosomes and subjected to isopycnic centrifugation, the total GTP[gamma-35S] binding and the small GTP binding proteins detected in the blot-overlay assay shifted to fractions of lighter sucrose density along with the RER marker.
通过几个差速离心步骤对犬胰腺匀浆进行亚分级分离，从而得到具有不同标志物谱的分级分离物。对每个分级分离物中鸟苷5'-[γ-[35S]硫代]三磷酸（GTP[γ-35S]）的特异性结合进行了测定。GTP[γ-35S]结合的富集在界面“光滑”微粒体分级分离物中最为显著，预期该分级分离物包含高尔基体和其他光滑囊泡。粗面微粒体分级分离物中也有明显的富集。电子显微镜和标志物蛋白分析显示粗面微粒体（RMs）为高度纯化的粗面内质网（RER）。通过[α-32P]GTP印迹覆盖分析检测小（低分子量）GTP结合蛋白的分布。在各种亚细胞分级分离物中检测到几种表观分子量为22 - 25 kDa的GTP结合蛋白。特别地，在富含高尔基体的分级分离物和RM分级分离物中发现了至少两种这样的蛋白，这表明这些小GTP结合蛋白定位于高尔基体和RER。为了更精确地将这些蛋白定位于RER，将天然RMs和用嘌呤霉素/高盐去除核糖体的RMs进行等密度离心。总GTP[γ-35S]结合以及通过[α-32P]GTP印迹覆盖检测到的小GTP结合蛋白，与RER标志物核糖体结合糖蛋白I一样，分布到高蔗糖密度的分级分离物中。与RER定位一致，当RMs去除核糖体并进行等密度离心时，印迹覆盖分析中检测到的总GTP[γ-35S]结合和小GTP结合蛋白与RER标志物一起转移到蔗糖密度较低（较轻）的分级分离物中。
