Purification of Heterotrimeric GTP-Binding Proteins from Brain: Identification of a Novel Form of G

Using high-resolution Mono-Q anion-exchange chromatography, we purified four distinct GTP-binding proteins from bovine brain. Each consists of a and associated β/γ subunits, and each is a substrate for pertussis toxin catalyzed ADP-ribosylation. We defined the relationship between the a subunits of the purified proteins and cloned cDNAs encoding putative a subunits (1) by performing immunoblots with peptide antisera with defined specificity and (2) by comparing the migration on two-dimensional gel electrophoresis of the purified proteins, and of the in vitro translated products of cDNAs encoding a subunits. Purified G proteins with a subunits of 39, 41, and 40 kDa (G₃₉, G₄₁, and G₄₀in order of abundance) correspond to the products of G_o, G_i1, and G_i2cDNAs. We purified a novel G protein with an α subunit slightly above 39 kDa (G₃₉*). G₃₉* is less abundant than G₃₉, elutes earlier than G₃₉on Mono-Q chromatography, and has a more basic pI (6.0 vs 5.6) than G₃₉. G₃₉and G₃₉*, however, are indistinguishable on immunoblots with a large number of specific antisera. The data suggest that G₃₉* may represent a novel form of G_o, differing in posttranslational modification rather than primary sequence.