A method is described for purification of G protein α and βγ 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 AlF- 4. The α subunits of G12, Gq, Gz, and Gi1 and the β1γ2 subunit complex were easily and efficiently purified by this method. Results were superior to established procedures in all cases. Purified α12 was characterized for the first time. The protein has a slow rate of guanine nucleotide exchange (kon,GTPγS = 0.01 min-1) and a very slow kcat for hydrolysis of GTP (0.1-0.2 min-1). GTPγS (guanosine 5′-3-O-(thio)triphosphate)·α12 does not influence the activity of several adenylyl cyclases or phospholipases. Activated αz 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 αi1.
|Original language||English (US)|
|Number of pages||8|
|Journal||Journal of Biological Chemistry|
|Publication status||Published - Jan 27 1995|
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