Purification of unique α subunits of GTP-binding regulatory proteins (G proteins) by affinity chromatography with immobilized βγ subunits

Novel G protein α subunits were purified from rat brain by an affinity matrix containing immobilized βγ subunits (Pang, I.-H., and Sternweis, P.C. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7814-7818). They were unique based on the following criteria. These α subunits migrated differently through polyacrylamide gels with an apparent molecular mass of 42 kDa. They did not behave similarly to the other brain G proteins by conventional chromatographic techniques. Antisera raised against a common region of known a subunits failed to recognize these 42-kDa polypeptides. Finally, primary sequences of tryptic fragments of these proteins contain regions homologous to, yet unique from, the other α subunits. Sequences are identical with one or more members of a new family of α subunits recently identified by molecular genetic techniques (Strathmann, M., Wilke, T.M., and Simon, M.I. (1989) Proc. Natl. Acad. Sci. U. S. A. 86, 7407-7409); most of the primary sequence identifies an a subunit labeled α_q. These polypeptides were not substrates for ADP-ribosylation catalyzed by pertussis toxin. They bound GTP-γS only with slow rates and low stoichiometry. Antisera to peptides based on primary sequence were specific for the new α subunits and indicate that they are widely distributed at low levels in different tissues but more concentrated in brain and lung. This procedure provides a means of preparing native G proteins that have a potential role as modulators of pertussis toxin-insensitive regulatory pathways.