Time-Resolved Imaging of Protein Kinase C Activation during Sea Urchin Egg Fertilization
| Autor: | Shigenori Kawahara, J. de Barry, Agnes Janoshazi, James L. Olds, Ko-Ichi Takamura, Yutaka Kirino, David S. Lester, Daniel L. Alkon, Tohru Yoshioka |
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| Rok vydání: | 1997 |
| Předmět: |
Boron Compounds
Male Proteases Indoles Time Factors Blotting Western Biology Human fertilization Western blot biology.animal Phorbol Esters medicine Animals Staurosporine Egtazic Acid Sea urchin Protein Kinase C Protein kinase C Chelating Agents Fluorescent Dyes chemistry.chemical_classification medicine.diagnostic_test Cell Biology Fluoresceins In vitro Cell biology Enzyme Activation Enzyme Microscopy Fluorescence Biochemistry chemistry Fertilization Sea Urchins Oocytes Calcium Female medicine.drug |
| Zdroj: | Experimental Cell Research. 234:115-124 |
| ISSN: | 0014-4827 |
| Popis: | To study protein kinase C (PKC) activation during sea urchin egg fertilization we used three different fluorescent probes specific for PKC, namely, fim-1, which recognizes the catalytic site of the enzyme, and BODIPY- and NBD-phorbol esters interacting with the PKC regulatory domain. We were able to follow PKC activation during the early steps of fertilization, the three different probes giving the same fluorescent pattern. Within 120 s following insemination, the fluorescent signal increased and clustered in the cortical zone of the cell. The process was Ca 2+ dependent and was inhibited in the presence of staurosporine, a PKC inhibitor. According to our in vitro probe characterization, this signal increase is due to PKC activation. These findings were further confirmed by Western blot analysis. This initial phase was followed by a rapid decrease which might be attributed to PKC hydrolysis by Ca 2+ -dependent proteases. The kinetics and the site distribution of PKC activation appear in complete agreement with the putative functions previously suggested for PKC during fertilization. |
| Databáze: | OpenAIRE |
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