PREPARATION OF ACTIVATED ALPHA SUBUNITS OF GS AND GIS : FROM ERYTHROCYTE TO ACTIVATED SUBUNIT

Publisher Summary This chapter discusses procedure for the purification of G s and G i (mixture of G i2 and G i3 ) from human erythrocytes, followed by an example of separate purification of G i2 from G i3 . A general protocol for activation and isolation of the activated α subunits of the three G proteins is given. The activation and isolation procedure are applied for α subunits to purified G i1 and G 0 proteins as well. The procedures for G-protein purification avoid the use of stabilizing ligands such as NaF/Mg and nonhydrolyzable GTP analogs [e.g., guanylyl imidodiphosphate (GMP-P(NH)P) or GTPγS] which are known to affect functionally the behavior and activity of G proteins. While eliminating many possible alterations of the subunit composition of these proteins as might result from the effect of these ligands to induce subunit dissociation. The γ subunits of G s and G i comigrate on urea and polyacrylamide gel electrophoresis with an apparent Mr of about 5000, and using two-dimensional peptide mapping they appear to be the same for G s and a pool of G i2 plus G i3.