碩士 / 國立彰化師範大學 / 生物學系 / 96 / Sco-CHH and Sco-CHH-L (CHH-like peptide), two structural variants of the crustacean hyperglycemic hormone family identified in the mud crab (Scylla olivacea), are identical up to the 40th residue, but different from each other in the remaining sequence. In this study, recombinant proteins (rSco-CHH and rSco-CHH-L) were produced by an E. coil expression system, refolded, purified, and confirmed by Western blotting and mass spectrometric analyses. Purified rSco-CHH was C-terminally amidated (rSco-CHHamide) in accordance with its native counterpart. Circular dichromatic spectra of rSco-CHHamide and rSco-CHH-L indicate they are rich in -helixes (47 % and 46 %, respectively); mass spectrometric analyses of peptide fragments of rSco-CHH and rSco-CHH-L reveal a common disulfide bond pattern typical of CHH family peptides. Functionally, rSco-CHHamide at 10 or 100 pmoles/animal elicited significant hyperglycemic responses, whereas rSco-CHH-L had no effect at the same dosage. On the other hand, rSco-CHH-L, but not rSco-CHHamide, dose-dependently (0-400 nM) increased the gill Na+/K+-ATPase activity. Finally, gene expression assays showed that the levels of Sco-CHH-L, but not Sco-CHH, were significantly increased in acclimated animals 12 h after exposing to an osmotic stress. In summary, recombinant Sco-CHH and Sco-CHH-L were successfully produced and characterized. Studies using the recombinant proteins provide evidence revealing that the 2 CHH structural variants diverge functionally.
| Identifer | oai:union.ndltd.org:TW/096NCUE5112033 |
| Date | January 2008 |
| Creators | Chih-Chun Chang, 張智鈞 |
| Contributors | Chi-Ying Lee, 李奇英 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 119 |
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