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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作者:
KOZASA, T
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机构:Department of Pharmacology, Univ. Texas Southwestern Med. Ctr., Dallas
KOZASA, T
GILMAN, AG
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机构:Department of Pharmacology, Univ. Texas Southwestern Med. Ctr., Dallas
GILMAN, AG
机构:
[1] Department of Pharmacology, Univ. Texas Southwestern Med. Ctr., Dallas
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).