EXPRESSION OF GS-ALPHA IN ESCHERICHIA-COLI - PURIFICATION AND PROPERTIES OF 2 FORMS OF THE PROTEIN

  • 5 January 1989
    • journal article
    • research article
    • Vol. 264  (1) , 409-418
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 protein also hydrolyze GTP, although at different rates (i.e. 0.13 .cntdot. min-1 and 0.34 .cntdot. min-1 at 20.degree. 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, .apprx. 4 .cntdot. 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 indicates 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.

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