This title appears in the Scientific Report :
2005
Please use the identifier:
http://hdl.handle.net/2128/453 in citations.
Biochemische und biophysikalische Untersuchungen zur Chemotaxis von Spermien des Seeigels Arbacia punctulata
Biochemische und biophysikalische Untersuchungen zur Chemotaxis von Spermien des Seeigels Arbacia punctulata
In order to fertilize the egg, sperm cells orient in a chemical gradient of factors that are secreted by the egg. This process is called chemotaxis. The chemotactic factor of sperm cells of the sea urchin Arbacia punctulata is the peptide resact. Resact binds to a receptorguanylyl cyclase (GC) which...
Saved in:
Personal Name(s): | Helbig, Annika (Corresponding author) |
---|---|
Contributing Institute: |
Zelluläre Signalverarbeitung; IBI-1 |
Imprint: |
Jülich
Forschungszentrum Jülich GmbH Zentralbibliothek, Verlag
2005
|
Physical Description: |
VI, 142 Seiten |
Dissertation Note: |
Köln, Univ., Diss., 2005 |
Document Type: |
Book Dissertation / PhD Thesis |
Research Program: |
Neurowissenschaften |
Series Title: |
Berichte des Forschungszentrums Jülich
4174 |
Subject (ZB): | |
Link: |
OpenAccess |
Publikationsportal JuSER |
In order to fertilize the egg, sperm cells orient in a chemical gradient of factors that are secreted by the egg. This process is called chemotaxis. The chemotactic factor of sperm cells of the sea urchin Arbacia punctulata is the peptide resact. Resact binds to a receptorguanylyl cyclase (GC) which synthesizes cGMP from GTP. The increase in the cGMP-concentration leads to the opening of Ca$^{2+}$-channels - either directly or indirectly. The present study includes the biochemical and functional characterization of Arbacia-GC as well as first experiments concerning the adaptation of sperm cells. The GC density in the flagellum of A.punctulata sperm was calculated to be ~ 400 GC molecules/$\mu$m$^{2}$. With two different experimental approaches, I found a 12 - 24-fold higher GC density. Densitometric analysis of Coomassie-stained protein gels and resact binding assays gave values of ~ 4.700 and ~ 11.000 GC-molecules/$\mu$m$^{2}$, respectively. In Cross-link experiments I could identify the resact-receptor as a trimer formed from GC-subunits. The binding assay revealed that the receptor down-regulates its binding affinity by negative cooperativity. The receptor senses resact-concentrations varying by four orders of magnitude. By means of GC-density, resact affinity, and cGMP-measurements in quenchflow experiments I determined a rate of cGMP synthesis of 4 - 5 cGMP/GC* s$^{-1}$. The enzymatic activity of the Arbacia-GC is fairly similar to that of GC-E from vertebrate photoreceptor cells. The synthesis of 4 to 5 cGMP/GC s$^{-1}$ is sufficient to evoke a Ca$^{2+}$-signal upon binding of a single resact-molecule. After a signaling pathways has been activated it also has to be turned off. This can be achieved at different levels. My experiments show that the resact-receptor is inactivated by dephosphorylation with a half-life of ~ 200 ms. Pioneering experiments concerning the adaptation of sperm cells showed unexpected and interesting results: Sperm cells that have been preincubated with a background concentration of resact (0,005 to 5 nM) showed a larger Ca$^{2+}$-signal in response to a given resact concentration (testpuls) than sperm cells that have not been preincubated with resact: So interestingly, sperm cells show an increase in sensitivity over a wide range of resact-background concentrations. Sensitization of sperm cells increases with the concentration of the testpuls. The Ca$^{2+}$-signal becomes smaller – sperm cells desensitize - when testpulses are given on a high resact-background concentration. |