We have purified five different a subunits of guanine-nucleotide-binding proteins (G proteins) from bovine brain membranes as active forms bound to guanosine 5'-[y-thio]triphosphate (GTP[yS]). All the purified a subunits were interacted with py subunits and served as a substrate for pertussin-catalyzed ADP-ribosylation. Based on the findings of immunoblot analyses using specific antibodies raised against various CI subunits of G proteins, three of them were identified as M~-~, M~-~ and M~-~, and the other two were classified into a, type. One of the a,-type proteins was the most abundant in the brain membranes (termed CI,), and the other (a,2) appeared to differ from a, in its proteolytic digestion data. The physiological properties of these purified GTP[yS]-bound a subunits towards adenylate cyclase and atrial muscarinic K + channels were studied. The nucleotide-bound forms of ai-2, M~-~ and a,2 inhibited the adenylate cyclase activity of S49 cyc-membranes which had been reconstituted with GTP[yS]-treated G,; this inhibition appeared to be mainly competitive with the activated G,, ai-l having the most potent inhibitory activity among them. GTP[yS]-bound a,, however, could not inhibit the G,-stimulated activity at all. On the other hand, all the GTP[yS]-bound a subunits activated atrial muscarinic K + channels, accompanied by a lag time, at picomolar concentrations. The fly subunits resolved from G proteins also activated the K + channels without a lag time at nanomolar concentration. The maximum activation by the by subunits appeared to be more potent than that by any of the a subunits. These results suggest that CI and fly subunits might activate the K + channels by mechanisms different from each other.Guanine-nucleotide-binding proteins (G proteins) are a family of signal-coupling proteins that play key roles in many hormonal and sensory transduction processes in eukaryotes (for review see [l -31). G proteins, which have a common heterotrimetric structure consisting of an a, p and y subunit, carry signals from various membrane-bound receptors to effectors such as enzymes or ion channels. The ci subunits have a high-affinity binding site specific for GTP (or GDP) and a site for NAD-dependent ADP-ribosylation which is catalyzed by bacterial toxins such as pertussin (also termed isletactivating protein or IAP) and/or cholera toxin. Functions of G proteins as signal transducers are profoundly affected by these toxin-induced modifications of the M subunits. Thus, pertussin and cholera toxin have been widely used as tools to identify these G proteins and to analyze their functions. G proteins serving as the substrate of pertussin were initially classified into Gi and Go according to their distribution in the types of cells, besides two GI that were only present in retinal rod or cone. Further studies of the molecular cloning of a subunit gene [4] and cDNAs [5 -71 indicated the existence of at least three genes for the M subunit of Gi, namely the ai-z and ai-3. In contrast, only a single type of the a subunit of Go ...

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

Previously we have reported that there are at least four different forms of Goα in bovine brain membranes which can be distinguished by their elution profiles from Mono Q column and their immunological reactivities. The four α‐subunits are referred to as αo 1, αo 2, αo 3 and αo 4 in their elution orders from the column. Partial amino acid sequences of the purified αo 1 and αo 2 were determined and compared with the predicted sequences of two classes of Goα cDNAs, termed Goα‐1 and Goα‐2. There were at least two unique fragments corresponding with the predicted amino acid sequence of the Goα‐2 cDNA but different from that of the Goα‐1 cDNA upon tryptic digestion of αo 1‐ or αo 2‐subunit. The αo 3‐ and αo 4‐subunits, but not αo 1‐ and αo 2‐subunits, were recognized by an antibody raised against a unique amino acid sequence predicted from Goα‐1 cDNA. These results suggest that αo 1,2 subunits and αo 3,4 subunits are encoded by G.oα‐2 cDNA and Goα‐1 cDNA, respectively.

Recently we reported there were at least four types of G O or G0-1ike proteins in bovine brain membranes based on their elution profiles from Mono Q columns and their immunological reactivities; one of the proteins was purified as an ct-monomeric form, and the others as ~flT-trimers. The four proteins, of which ct-subunits were confirmed to be a family of Go-type by an immunoblot analysis, were thus referred to as %1, Go2, G03 and G04, respectively, in order of their elutions from the column. Immunostained peptide mappings arising from proteolytic digestions of the four ctsubunits, together with their fragmentation patterns containing radiolabeled ADP-ribose that had been incorporated by pertussis toxin-catalyzed ADP-ribosylation, suggested that the four G0-c~ were classified into either of two groups such as %1 and G02-c~ or Go3-C~ and Go4-C~. The kinetic parameters of their GTPase activities, however, revealed that there were different properties between %1 and Go2-c~ or Go3-c~ and Go4-a. Thus, the four G0-type proteins appeared to be different entities from one another.GTP-binding protein; Pertussis toxin (IAP); GTPase activity