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A cholera toxin-sensitive G-protein stimulates exocytosis in sea urchin eggs
Abstract:
To identify guanine nucleotide binding proteins (G-proteins) in sea urchin eggs and to investigate their role in signal transduction at fertilization, we used cholera toxin (CTX) and pertussis toxin (PTX), which catalyze the specific ADP-ribosylation of G-proteins. Cell surface complex, consisting of plasma membranes and adhering cortical vesicles, was prepared from eggs of Lytechinus variegatus and incubated with 32P-labeled NAD in the presence of CTX or PTX. CTX catalyzed the ADP-ribosylation of a 47-kDa polypeptide, whereas PTX catalyzed the ADP-ribosylation of a 40-kDa polypeptide. Microinjection of approximately 30 micrograms/ml whole CTX or approximately 20 micrograms/ml CTX subunit A into intact eggs caused exocytosis of cortical vesicles. However, if the eggs were first injected with EGTA (0.6-1.4 mM), injection of CTX did not cause exocytosis. Eggs injected with 0.8-2.8 mM cAMP or 1.0-4.0 mM adenosine 3':5'-monophosphotioate cyclic Sp-isomer (cAMP-S), a hydrolysis-resistant analog of cAMP, did not undergo exocytosis. These results suggest that a CTX-sensitive G-protein is involved in regulating Ca2+ release and exocytosis of cortical vesicles in sea urchin eggs.
Insights
Cholera toxin (CTX) sensitive G-proteins in sea urchin eggs regulate calcium release and cortical vesicle exocytosis during fertilization. This study identifies specific G-proteins involved in this crucial signal transduction pathway.
Area of Science:
- Marine Biology
- Cell Biology
- Biochemistry
Background:
- Guanine nucleotide-binding proteins (G-proteins) are key regulators of signal transduction pathways in eukaryotic cells.
- Understanding G-protein involvement in fertilization is crucial for reproductive biology and developmental processes.
- Sea urchin eggs serve as a model system for studying fertilization due to their accessibility and well-characterized responses.
Purpose of the Study:
- To identify G-proteins in sea urchin eggs.
- To investigate the role of G-proteins in signal transduction during fertilization.
- To elucidate the mechanism of cortical vesicle exocytosis.
Main Methods:
- Use of cholera toxin (CTX) and pertussis toxin (PTX) to ADP-ribosylate G-proteins.
- Preparation of cell surface complexes from Lytechinus variegatus eggs.
- Incubation with 32P-labeled NAD in the presence of toxins.
- Microinjection of CTX, CTX subunit A, EGTA, cAMP, and cAMP-S into sea urchin eggs.
Main Results:
- CTX catalyzed ADP-ribosylation of a 47-kDa polypeptide.
- PTX catalyzed ADP-ribosylation of a 40-kDa polypeptide.
- Microinjection of CTX induced cortical vesicle exocytosis, which was blocked by EGTA.
- cAMP or cAMP-S injection did not induce exocytosis.
Conclusions:
- A CTX-sensitive G-protein is involved in regulating Ca2+ release in sea urchin eggs.
- This G-protein plays a role in the exocytosis of cortical vesicles during fertilization.
- The findings provide insights into the molecular mechanisms of fertilization signal transduction.