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)

被引:282
作者
KOZASA, T
GILMAN, AG
机构
[1] Department of Pharmacology, Univ. Texas Southwestern Med. Ctr., Dallas
关键词
D O I
10.1074/jbc.270.4.1734
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
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).
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页码:1734 / 1741
页数:8
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