Abstract
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 Ni(2+)-containing column, the subunit to be purified is eluted specifically by promoting subunit dissociation with AIF4-. The alpha subunits of G12, Gq, Gz, and Gi1 and the beta 1 gamma 2 subunit complex were easily and efficiently purified by this method. Results was 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 (kon, GTP gamma S = 0.01 min-1) and a very slow kcat 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.
Highlights
A method is described for purification of G protein a and f3y subunits from 8f9 cells infected with recombinant baculoviruses
The protein has a slow rate of guanine nucleotide exchange and a very slow keat for hydrolysis of GTP (0.1-0.2 min-I)
Activated a z inhibits the activity of type I and type V adenylyl cyclases
Summary
Vol 270, No., Issue of January 27, pp. 1734-1741, 1995 Printed in U.S.A. Tohru Kozasa and Alfred G. Each G protein oligomer contains a guanine nucleotide-binding a subunit, which can be palmitoylated and, in some cases, myristoylated, and a high affinity dimer of f3 and "y subunits; "y is prenylated. The oligomer is adsorbed to a Ni 2 + -containing column, and the desired protein is eluted by subunit dissociation (activation with AlFi). This combination of specific adsorption and elution yields a highly enriched product that can be brought to essential homogeneity by one step of ion exchange chromatography. We have applied this method to permit characterization of a l2 and further investigation of az
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.