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.
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.
Kozasa
T
T
Department of Pharmacology, University of Texas Southwestern Medical Center, Dallas 75235.
Gilman
A G
AG
D000067956:Adenylyl Cyclase Inhibitors; D000595:Amino Acid Sequence; D000818:Animals; D016367:Baculoviridae; D001483:Base Sequence; D001667:Binding, Competitive; D002417:Cattle; D002460:Cell Line; D002462:Cell Membrane / Q000378:metabolism; D002846:Chromatography, Affinity; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D020558:GTP Phosphohydrolases / Q000378:metabolism*; D019204:GTP-Binding Proteins / Q000096:biosynthesis / Q000302:isolation & purification* / Q000378:metabolism*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000378:metabolism; D006639:Histidine; D007700:Kinetics; D046911:Macromolecular Substances; D008969:Molecular Sequence Data; D008970:Molecular Weight; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins / Q000096:biosynthesis / Q000302:isolation & purification / Q000378:metabolism; D012265:Ribonucleotides / Q000494:pharmacology; D018411:Spodoptera
D000067956:Adenylyl Cyclase Inhibitors; D046911:Macromolecular Substances; D009838:Oligodeoxyribonucleotides; D011994:Recombinant Proteins; D012265:Ribonucleotides; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D006639:Histidine; D020558:GTP Phosphohydrolases; D019204:GTP-Binding Proteins