Purification of GTP‐binding proteins from bovine brain membranes

Abstract

Using high-resolution Mono Q column chromatography, we purified 6 distinct peaks of GTP-binding proteins from bovine brain membranes. Five of them consisted of 3 polypeptides with a/+subunits and served as the substrate of islet-activating protein (IAP), pertussis toxin. The other one was purified as a-subunit alone and was also ADP-ribosylated by IAP in the presence of &subunits. When each a-subunit was characterized by immunoblot analysis using various antibodies with defined specificity, the two of them were identified as G,., and Gi.2, and other 4 appeared to be G, or G,-like G proteins. The a-subunits of immunologically Go-like proteins were apparently distinguishable from one another on elution profiles from the Mono Q column. Thus, there was a heterogeneity of the a-subunit of G, in the brain membranes.ADP-ribosylation; GTP-binding protein; Islet-activating protein

Characterization of four GO‐type proteins purified from bovine brain membranes

“…We have recently reported that there are at least six distinct G proteins serving as the substrate for IAPcatalyzed ADP-ribosylation in bovine brain membranes [10]. Two of them were identified as Gi-1 and Gi-2 by immunoblot analysis with specific anti-c~i_l and anti-c~i_2 antisera, and the other four cross-reacting strongly with specific anti-c~o IgG (A) were attributed to Go-type proteins.…”

“…Although Bysubunits associated with each o~-subunit were different from one another, the distinct elution profiles of the four Go-type proteins from the Mono Q column were due to the properties of their c~-subunits. The four c~-subunits resolved from their fly-subunits had been eluted separately from the column in manners similar to those observed with their trimeric forms [10].…”

“…Lang suggested that there were two distinct Go proteins based on the findings of their peptide mappings [9]. In the previous studies, we also reported there were four types of Go (or Go-like) proteins cross-reacting with a polyclonal anti-ceo antibody in bovine brain membranes [10]. However, it has not been clear that those ce-subunits of Go-type proteins differ with respect to their primary sequences or posttranslational modifications.…”

“…Four Go proteins were purified from bovine brain membranes as described in [10]. The purified proteins were quantitated by staining with Amido black with bovine serum albumin as a standard protein [13].…”

“…The films were incubated with various antibodies at 30°C for 12 h and treated with biotinylated anti-rabbit IgG and streptavidine HRP. The four purified Go proteins were also ADP-ribosylated by IAP in the presence of 32p-labeled NAD as in [10][11][12]14] and followed by digestions with lysylendopeptidase as described above. The samples were electrophoresed, and the radiolabeled polypeptides were detected by autoradiography.…”