Purification and characterization of five different α subunits of guanine-nucleotide-binding proteins in bovine brain membranes

Their physiological properties concerning the activities of adenylate cyclase and atrial muscarinic K+ channels


Correspondence to T. Katada, Department of Life Science, Faculty of Science, Tokyo Institute of Technology, Yokohama 227, Japan


We have purified five different α subunits of guanine-nucleotide-binding proteins (G proteins) from bovine brain membranes as active forms bound to guanosine 5′-[γ-thio]triphosphate (GTP[γS]). All the purified α subunits were interacted with βγ subunits and served as a substrate for pertussin-catalyzed ADP-ribosylation. Based on the findings of immunoblot analyses using specific antibodies raised against various α subunits of G proteins, three of them were identified as αi-1i-2 and αi-33, and the other two were classified into α0 type. One of the α0-type proteins was the most abundant in the brain membranes (termed α0), and the other (α02) appeared to differ from α0 in its proteolytic digestion data. The physiological properties of these purified GTP[γS]-bound α subunits towards adenylate cyclase and atrial muscarinic K+ channels were studied. The nucleotide-bound forms of αi-1, αi-2. αi-3 and α02 inhibited the adenylate cyclase activity of S49 cyc membranes which had been reconstituted with GTP[γS]-treated Gs; this inhibition appeared to be mainly competitive with the activated Gs, αi-1 having the most potent inhibitory activity among them. GTP[γS]-bound α0, however, could not inhibit the Gs-stimulated activity at all. On the other hand, all the GTP[γS]-bound α subunits activated atrial muscarinic K+ channels, accompanied by a lag time, at picomolar concentrations. The βγ subunits resolved from G proteins also activated the K+ channels without a lag time at nanomolar concentration. The maximum activation by the βγ subunits appeared to be more potent than that by any of the a subunits. These results suggest that α and βγ subunits might activate the K+ channels by mechanisms different from each other.

G protein

guanine-nucleotide-binding protein

Gs and GJ

G proteins of adenylate cyclase that mediate stimulation and inhibition, respectively


a G protein of unknown function purified from brain


the G protein from rod outer segment


guanosine 5′-[y-thio]triphosphate


Adenylate cyclase (EC


lysylendopeptidase, an endoproteinase from Lysobacter enzymogens (EC


Staphylococus aureus V8 protease (EC


trypsin (EC


chymotrypsin A4 (EC