Pang I H, Sternweis P C
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Proc Natl Acad Sci U S A. 1989 Oct;86(20):7814-8. doi: 10.1073/pnas.86.20.7814.
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.
固定化的GTP结合调节蛋白（G蛋白）的βγ亚基被用于从牛组织的可溶性膜中分离α亚基，并根据它们对βγ亚基的不同亲和力来分离特定的α亚基。βγ亚基被交联到ω-氨基丁基琼脂糖上。每毫升βγ-琼脂糖最多可结合7 nmol的α亚基，并通过用AlF4-洗脱来回收。这种亲和树脂利用Go的α亚基对βγ亚基明显较低的亲和力，有效地将Gi1和Gi2的α亚基与牛脑中最丰富的G蛋白Go的“污染”α亚基分开。βγ-琼脂糖还被用于从不同牛组织的膜的胆酸盐提取物中分离α亚基混合物。纯化了39 - 41 kDa的α亚基（以各种比例）以及Gs的α亚基。对于所有检测的α亚基，提取物的产率超过60%，并且显然代表了组织中α亚基的相对含量。该技术可以从少量样品中快速分离和鉴定各种组织和细胞中的内源性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βγ亚基相互作用，这种相互作用要么亲和力低，要么在机制上与本研究中分离的α亚基不同。
