Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.
Isolation of the alpha subunits of GTP-binding regulatory proteins by affinity chromatography with immobilized beta gamma subunits.
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通过固定化 β γ 亚基的亲和层析分离 GTP 结合调节蛋白的 α 亚基。
DOI：
10.1073/pnas.86.20.7814
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发表时间：
1989
影响因子：
11.1
通讯作者：
Sternweis,PC
中科院分区：
文献类型：
--
作者：
Pang,IH;Sternweis,PC
固定化的GTP结合调节蛋白（G蛋白）的β-γ亚基被用来从牛组织的溶解膜中分离α亚基，并根据它们对β-γ亚基的不同亲和力来分离特定的α亚基。β γ亚基与ω-氨基丁基琼脂糖交联。高达7 nmol的α亚基可以结合到每毫升的β γ-琼脂糖，并通过用AIF 4-洗脱回收。这种亲和树脂通过利用Go的α亚基对β γ亚基的明显较低的亲和力，有效地将Gi 1和Gi 2的α亚基与Go的“污染”α亚基分离，Go是牛脑中最丰富的G蛋白。β-γ-琼脂糖也被用来从来自不同牛组织的膜的胆酸盐提取物中分离α亚基的混合物。纯化39-41 kDa的α亚基（以各种比例）以及Gs的α亚基。提取物的产率超过60%的所有α亚基检查，显然代表了组织中的α亚基的相对含量。该技术可从少量样品中快速分离鉴定各种组织细胞中的内源性G蛋白。到目前为止，用这种方法仅检测到39-52 kDa范围内的多肽。如果其他GTP结合蛋白与这些β-γ亚基相互作用，则相互作用要么是低亲和力的，要么是本研究中分离的α亚基的独特机制。
Immobilized beta gamma subunits of GTP-binding regulatory proteins (G proteins) were used to isolate alpha subunits from solubilized membranes of bovine tissues and to separate specific alpha subunits based on their differential affinities for beta gamma subunits. The beta gamma subunits were cross-linked to omega-aminobutyl agarose. Up to 7 nmol of alpha subunit could bind to each milliliter of beta gamma-agarose and be recovered by elution with AIF4-. This affinity resin effectively separated the alpha subunits of Gi1 and Gi2 from "contaminating" alpha subunits of Go, the most abundant G protein in bovine brain, by taking advantage of the apparent lower affinity of the alpha subunits of Go for beta gamma subunits. The beta gamma-agarose was also used to isolate mixtures of alpha subunits from cholate extracts of membranes from different bovine tissues. alpha subunits of 39-41 kDa (in various ratios) as well as the alpha subunits of Gs were purified. The yields from extracts exceeded 60% for all alpha subunits examined and apparently represented the relative content of alpha subunits in the tissues. This technique can rapidly isolate and identify, from a small amount of sample, the endogenous G proteins in various tissues and cells. So far, only polypeptides in the range of 39-52 kDa have been detected with this approach. If other GTP-binding proteins interact with these beta gamma subunits, the interaction is either of low affinity or mechanistically unique from the alpha subunits isolated in this study.