The Gα(z) gene product in human erythrocytes. Identification as a 41-kilodalton protein

A cDNA encoding a previously unknown G protein α-subunit lacking the site for pertussis toxin-catalyzed ADP-ribosylation was recently cloned and its putative protein product named G(z) (Fong, H.K.W., Yoshimoto, K.K., Eversole-Cire, P., and Simon, M.I. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 3066-3070) or G(x) (Matsuoka, M., Itoh, H. Kozasa, T., and Kaziro, Y. (1988) Proc. Natl. Acad. Sci. U.S.A. 85, 5384-5388). A synthetic peptide corresponding to the deduced carboxyl-terminal decapeptide of this putative protein (α(z)) has been synthesized and used to prepare a polyclonal rabbit antiserum directed against the protein. The specificity and cross-reactivity of this antiserum was assessed using bacterially expressed recombinant G protein α-subunit fusion proteins (rα). The crude antiserum strongly recognizes rα(z) in immunoblots. Pretreatment of antiserum with antigen peptide greatly reduces the interaction of the antiserum with rα(z). Affinity purified antiserum strongly recognizes expressed rα(z), does not recognize rα(s1), rα(s), rα(o), or rα(i3), and very weakly interacts with rα(i1) and rα(i2). In contrast, the α-subunits of purified bovine brain G(i1) and human erythrocyte G(i2) and G(i3) did not react with the α(z)-antiserum. Partially purified mixtures of human erythrocyte G proteins contain a 41-kDa protein that reacts specifically in immunoblots with both crude and affinity purified α(z)-specific antiserum. Quantitative immunoblotting using rα(z) as a standard indicates that there is 60-100 ng of α(z)/μg of 40/41-kDa α-subunit protein in partially purified human erythrocyte G protein preparations. We conclude that we have identified the α(z) gene product as a 41-kDa trace protein in human erythrocytes.