Purification and characterization of a GTP-binding protein serving as pertussis toxin substrate in starfish oocytes

Abstract

In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown. The 1-MA-initiated signal is, however, inhibited by prior microinjection of pertussis toxin into the oocytes (Shilling, F., Chiba, K., Hoshi, M., Kishimoto, T., and Jaffe, L. A. (1989) Dev. Biol. 133, 605–608), suggesting that a pertussis-toxin-sensitive guanine-nucleotide-binding protein (G protein) is involved in the 1-MA-induced signal transduction. Based on these findings, we purified a G protein serving as the substrate of pertussis toxin from the plasma membranes of starfish oocytes. The purified G protein had an αβγ-trimeric structure consisting of 39-kDa α, 37-kDa β, and 8-kDa γ subunits. The 39-kDa α subunit contained a site for ADP-ribosylation catalyzed by pertussis toxin. The α subunit was also recognized by antibodies specific for a common GTP-binding site of many mammalian α subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G-α. An antibody raised against mammalian 36-/35-kDa β subunits strongly reacted with the 37-kDa β subunit of starfish G protein. The purified starfish G protein had a GTP-binding activity with a high affinity and displayed a low GTPase activity. The activity of the G protein serving as the substrate for pertussis-toxin-catalyzed ADP-ribosylation was inhibited by its association with a non-hydrolyzable GTP analogue. Thus, the starfish G protein appeared to be similar to mammalian G proteins at least in terms of its structure and properties of nucleotide binding and the pertussis toxin substrate. A possible role of the starfish G protein is also discussed in the signal transduction between 1-MA receptors and reinitiation of meiosis with germinal vesicle breakdown.