G(z), a guanine nucleotide-binding protein with unique biochemical properties

Cloning of a complementary DNA (cDNA) for G(zα), a newly apprecitated member of the family of guanine nucleotide-binding regulatory proteins (G proteins), has allowed preparation of specific antisera to identify the protein in tissues and to assay it during purification from bovine brain. Additionally, expression of the cDNA in Escherichia coli has resulted in the production and purification of the recombinant protein. Purification of G(z) from bovine brain is tedious, and only small quantities of protein have been obtained. The protein copurifies with the βγ subunit complex common to other G proteins; another 26-kDa GTP-binding protein is also present in these preparations. The purified protein could not serve as a substrate for NAD-dependent ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. Purification of recombinant G(zα) (rG(zα)) from E. coli is simple, and quantities of homogeneous protein sufficient for biochemical analysis are obtained. Purified rG(zα) has several properties that distinguish it from other G protein α subunit polypeptides. These include a very slow rate of guanine nucleotide exchange (k = 0.02 min^-1), which is reduced > 20-fold in the presence of mM concentrations of Mg²⁺. In addition, the rate of the intrinsic GTPase activity of G(zα) is extremely slow. The hydrolysis rate (k(cat)) for rG(zα) at 30°C is 0.05 min^-1, or 200-fold slower than that determined for other G protein α subunits. rG(zα) can interact with bovine brain βγ but does not serve as a substrate for ADP-ribosylation catalyzed by either pertussis toxin or cholera toxin. These studies suggest that G(z) may play a role in signal transduction pathways that are mechanistically distinct from those controlled by the other members of the G protein family.