Purification and characterization of a GTP-binding protein serving as pertussis toxin substrate in starfish oocytes.
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 alpha beta gamma-trimeric structure consisting of 39-kDa alpha, 37-kDa beta, and 8-kDa gamma subunits. The 39-kDa alpha subunit contained a site for ADP-ribosylation catalyzed by pertussis toxin. The alpha subunit was also recognized by antibodies specific for a common GTP-binding site of many mammalian alpha subunits or a carboxy-terminal ADP-ribosylation site of mammalian inhibitory G-alpha. An antibody raised against mammalian 36-/35-kDa beta subunits strongly reacted with the 37-kDa beta 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.
Tadenuma
H
H
Department of Life Science, Tokyo Institute of Technology, Yokohama, Japan.
Chiba
K
K
Takahashi
K
K
Hoshi
M
M
Katada
T
T
D000595:Amino Acid Sequence; D000818:Animals; D002384:Catalysis; D019204:GTP-Binding Proteins / Q000737:chemistry / Q000302:isolation & purification*; D016244:Guanosine 5'-O-(3-Thiotriphosphate) / Q000737:chemistry; D006868:Hydrolysis; D015151:Immunoblotting; D008969:Molecular Sequence Data; D009865:Oocytes / Q000737:chemistry*; D037342:Pertussis Toxin; D011485:Protein Binding; D012995:Solubility; D013215:Starfish; D010566:Virulence Factors, Bordetella / Q000737:chemistry*
D010566:Virulence Factors, Bordetella; D016244:Guanosine 5'-O-(3-Thiotriphosphate); D037342:Pertussis Toxin; D019204:GTP-Binding Proteins