1992 Fiscal Year Final Research Report Summary
GTP-binding Proteins in Starfish Oocyte
| Project/Area Number |
03044063
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| Research Category |
Grant-in-Aid for international Scientific Research
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| Allocation Type | Single-year Grants |
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| Section | Joint Research |
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| Research Institution | Tokyo Instiute of Technology |
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Principal Investigator |
KATADA Toshiaki Department of Life Science, Tokyo Institute of Technology, 生命理工学部, 教授 (10088859)
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| Co-Investigator(Kenkyū-buntansha) |
SHILLING Fraser Department of Physiology, The University of Connecticut Health Center, コネチカット大学・医学部, 研究員
JAFFE Laurinda Department of Physiology, The University of Connecticut Health Center, コネチカット大学・医学部, 教授
CHIBA Kazuyoshi Department of Life Science, Tokyo Institute of Technology, 生命理工学部, 助手 (70222130)
NISHINA Hiroshi Department of Life Science, Tokyo Institute of Technology, 生命理工学部, 助手 (60212122)
TAKAHASHI Katsunobu Department of Life Science, Tokyo Institute of Technology, 生命理工学部, 助手 (40183850)
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| Project Period (FY) |
1991 – 1992
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| Keywords | GTP-binding proteins / pertussis toxin / 1-methyladenine / Oocyte maturation / Fertilization / Membrae receptors |
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| Research Abstract |
In response to a meiosis-inducing hormone, 1-methyladenine (1-MA), starfish oocytes undergo reinitiation of meiosis with germinal vesicle breakdown (GVBD). The 1-MA-initiated GVBD is, however, inhibited by prior microinjection of pertussis toxin into the oocytes, suggesting that a pertussis toxin-sensitive GTP-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 alphabetagamma-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 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 ass
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ociation 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 pertussis toxin substrate. The microinjection of the purified betagamma-subunits into the oocytes induced GVBD as had been observed with 1-MA. There were apparently two forms of 1-MA receptors with a high and a low affinities in the membranes. The high-affinity form was converted into the low-affinity one in the presence of a non-hydrolyzable analogue of GTP. The 39-kDa alpha-subunit of the starfish G protein was also ADP-ribosylated by cholera toxin only when 1-MA was added to the membranes. The ADP-ribosylated 39-kDa alpha-subunit could be immunoprecipitated with an antibodies raised against the carboxy-terminal site of mammalian inhibitory G-alpha. These results indicate that 1-MA receptors are functionally coupled with the pertussis toxin-substrate G protein and that the betagamma-complex resolved from alpha-subunit is an active molecule for the initiation of GVBD in starfish oocytes. Less
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