PURIFICATION OF RECOMBINANT G-PROTEINS FROM SF9 CELLS BY HEXAHISTIDINE TAGGING OF ASSOCIATED SUBUNITS - CHARACTERIZATION OF ALPHA(12), AND INHIBITION OF ADENYLYL-CYCLASE BY ALPHA(Z)
PURIFICATION OF RECOMBINANT G-PROTEINS FROM SF9 CELLS BY HEXAHISTIDINE TAGGING OF ASSOCIATED SUBUNITS - CHARACTERIZATION OF ALPHA(12), AND INHIBITION OF ADENYLYL-CYCLASE BY ALPHA(Z)
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DOI：
10.1074/jbc.270.4.1734
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发表时间：
1995-01-27
影响因子：
4.8
通讯作者：
GILMAN, AG
中科院分区：
文献类型：
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作者：
KOZASA, T;GILMAN, AG
描述了从感染重组杆状病毒的 Sf9 细胞中纯化 G 蛋白 α 和 β γ 亚基的方法。待纯化的亚基与带有六组氨酸标签的相关亚基共表达。低聚物吸附到含有Ni2+的柱上后，通过用AlF4-促进亚基解离，特异性地洗脱待纯化的亚基。通过该方法可以轻松有效地纯化 G(12)、G(q)、G(z) 和 G(i1) 的 a 亚基以及 beta(1) gamma(2) 亚基复合物，在所有情况下结果均优于既定程序。首次对纯化的 alpha(12) 进行了表征。该蛋白的鸟嘌呤核苷酸交换速率较慢（k(on,GTP gamma S) = 0.01 min(-1)），并且 GTP 水解的 k(cat) 非常慢（0.1-0.2 min(-1)）。 GTP gamma S（鸟苷 5'-3-O-（硫代）三磷酸）.alpha(12) 不影响多种腺苷酸环化酶或磷脂酶的活性。活化的 α(z) 抑制 I 型和 V 型腺苷酸环化酶的活性。它是比活化的 α(i1) 更有效的 V 型腺苷酸环化酶抑制剂。
A method is described for purification of G protein alpha and beta gamma subunits from Sf9 cells infected with recombinant baculoviruses. The subunit to be purified is coexpressed with an associated subunit bearing a hexahistidine tag. After adsorption of the oligomer to a Ni2+-containing column, the subunit to be purified is eluted specifically by promoting subunit dissociation with AlF4-. The a subunits of G(12), G(q), G(z), and G(i1) and the beta(1) gamma(2) subunit complex were easily and efficiently purified by this method, Results were superior to established procedures in all cases.Purified alpha(12) was characterized for the first time. The protein has a slow rate of guanine nucleotide exchange (k(on,GTP gamma S) = 0.01 min(-1)) and a very slow k(cat) for hydrolysis of GTP (0.1-0.2 min(-1)). GTP gamma S (guanosine 5'-3-O-(thio)triphosphate).alpha(12), does not influence the activity of several adenylyl cyclases or phospholipases. Activated alpha(z) inhibits the activity of type I and type V adenylyl cyclases. It is a somewhat more potent inhibitor of type V adenylyl cyclase than is activated alpha(i1).