Abstract
Cloning of complementary DNAs that encode either of two forms of the alpha subunit of the guanine nucleotide-binding regulatory protein (Gs) that stimulates adenylyl cyclase into appropriate plasmid vectors has allowed these proteins to be synthesized in Escherichia coli (Graziano, M.P., Casey, P.J., and Gilman, A.G. (1987) J. Biol. Chem. 262, 11375-11381). A rapid procedure for purification of milligram quantities of these proteins is described. As expressed in E. coli, both forms of Gs alpha (apparent molecular weights of 45,000 and 52,000) bind guanosine 5'-(3-O-thio)triphosphate stoichiometrically. The proteins also hydrolyze GTP, although at different rates (i.e. 0.13.min-1 and 0.34.min-1 at 20 degrees C for the 45- and the 52-kDa forms, respectively). These rates reflect differences in the rate of dissociation of GDP from the two proteins. Both forms of recombinant Gs alpha have essentially the same kcat for GTP hydrolysis, approximately 4.min-1. Recombinant Gs alpha interacts functionally with G protein beta gamma subunits and with beta-adrenergic receptors. The proteins can also be ADP-ribosylated stoichiometrically by cholera toxin. This reaction requires the addition of beta gamma subunits. Both forms of recombinant Gs alpha can reconstitute GTP-, isoproterenol + GTP-, guanosine 5'-(3-O-thio)triphosphate-, and fluoride-stimulated adenylyl cyclase activity in S49 cyc- membranes to maximal levels, although their specific activities for this reaction are lower than that observed for Gs purified from rabbit liver. Experiments with purified bovine brain adenylyl cyclase indicate that the affinity of recombinant Gs alpha for adenylyl cyclase is 5-10 times lower than that of liver Gs under these assay conditions; however, the intrinsic capacity of the recombinant protein to activate adenylyl cyclase is normal. These findings suggest that Gs alpha, when synthesized in E. coli, may fail to undergo a posttranslational modification that is crucial for high affinity interaction of the G protein with adenylyl cyclase.
Highlights
Cloning of complementary DNAs that encode either homologous guanine nucleotide-binding regulatory proteins of two formsof the (Y subunit of the guaninenucleotide- or G proteins’; Gproteins, in turn, control the activity of binding regulatory protein (G.) that stimulates ade- membrane-bound effector molecules such as adenylyl cyclase nylyl cyclase into appropriate plasmid vectors has allowed these proteins tobe synthesized in Escherichia coli
Can reconstitute GTP, isoproterenol GTP, guano- G, mediates the hormonal stimulation of adenylyl cyclase sine 5’-(3-O-thio)triphosphate,and fluoride-stimu- by a variety of hormones
Both proteins bind and hydrolyze GTP and can mediate hormonal stimulation of adenylyl cyclase, albeit that contained the peak of GTPyS-binding activity were pooled,and buffer was exchanged by sequential cycles of concentration and dilution into HED in a Centricon 30 microconcentrator (Amicon)
Summary
Purification of the protein indicated that therewere two major forms of the a subunit ofG, (Gs,), with apparent molecular weights of 45,000 and 52,000 [9]. The column was washed with one volume of HPHT buffer, and protein was eluted sessment of their activities and mechanistic evaluations of at a flow rate of0.75 ml/min with a linear gradient of potassium any differences that areobserved. In this report we present a method for the purification of a 45- and a 52-kDa form of G., followingtheir expression in E. coli As purified, both proteins bind and hydrolyze GTP and can mediate hormonal stimulation of adenylyl cyclase, albeit that contained the peak of GTPyS-binding activity were pooled,and buffer was exchanged by sequential cycles of concentration and dilution into HED in a Centricon 30 microconcentrator (Amicon).
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