Isolation of two proteins with high affinity for guanine nucleotides from membranes of bovine brain.

The alpha subunit of GO was isolated without the use of ligands known to dissociate other G proteins, and should be of great utility in elucidating the mechanism of action of this family of GTP-binding proteins.

Abstract

Membranes from bovine brain bind relatively large quantities of guanosine 5'-(3-O-thio)triphosphate (GTP gamma S) with high affinity. The two proteins responsible for most of this activity were purified; they account for 1.5% of the membrane protein. The two proteins contain alpha subunits of either 39,000 or 41,000 Da, beta subunits of 36,000 or 35,000 Da, and a potential gamma subunit (11,000 Da). These structures are the same as a family of proteins that includes transducin and the regulatory proteins, GS and GI, of adenylate cyclase. The 41,000- and 39,000-Da polypeptides can be ADP-ribosylated with islet-activating protein from Bordetella pertussis, bind guanine nucleotides specifically, and migrate through polyacrylamide gels with rates similar to the alpha subunits of GI and transducin, respectively. The 36,000- and 35,000-Da polypeptides are similar to the beta subunits of GI and GS. The gamma subunit is found whenever beta subunits are present. The 41,000- and 39,000-Da polypeptides (with beta and gamma) are designated, respectively, GI and GO from brain. The alpha subunit of GO was isolated without the use of ligands known to dissociate other G proteins. GO alpha binds GTP gamma S reversibly in the absence of Mg2+ and is relatively stable in cholate. This isolated alpha subunit should be of great utility in elucidating the mechanism of action of this family of GTP-binding proteins.