博士 / 國立臺灣大學 / 動物學研究所 / 87 / Crustacean hyperglycemic hormone (CHH), a physiologically important neurohormone in crustaceans, primarily regulates carbohydrate metabolism and are also involved roles in reproduction, molting and other physiological processes. The eyestalk ablated freshwater giant prawn, Macrobrachium rosenbergii, were used for bioassay on the hyperglycemic activity of eyestalk extract, and the hyperglycemic response was peaked at 1 hr after the extract being administered. The injection dosage of 0.5 eyestalk equivalent produced the highest hyperglycemic response. CHH fractionated from RP-HPLC and confirmed by its hyperglycemic activity and partial amino acid sequences, named as Mar-CHH. The molecular weight of Mar-CHH is 8534.5 Da, determined by matrix-assisted laser desorption ionization mass spectrometry-time of flight analysis (MALDI-TOF). The amino acid sequences of the first 25 residues of CHH showed 72% homology to the partial sequences from CHH A and CHH B of the American lobster Homarus americanus.
Nested oligo-nucleotide primer sets were designed from Mar-CHH N-terminal partial amino acid sequence and highly conserved sequences of CHH from other species. Total RNA prepared from eyestalk tissue was used for rapid amplification of cDNA End (RACE), and with skewed anchored primer concentration, a 614 bp DNA fragment was amplified. The DNA fragment was cloned into pGEM-T easy vector, which contained a putative CHH coding region in 222 bps, translated into 72 residues and 2 amidated peptide residues. The molecular mass derived from deduced amino acid sequence is 8534 Da.
Phylogenic relationships among deduced Mar-CHH amino acid sequences and others in CHH-family showed that Mar-CHH is closer to the CHH sequences of Astacidea and Brachyura species, then those of Penaeid. Two peptides designated as MIH were clustered into CHH sub-family of the phyletic tree being build. Further survey suggested the mis-match between the biological function and the sequence derived from molecular cloning.
The two dimensional structure was attempted by using simulation programs available, but the three dimensional structure of Mar-CHH could not be solved to date, due to the lack of any proper 3D model pre-existed in protein database. It is however, speculated that 3D structure of Mar-CHH should be scoped in a hydrophobic core in β-sheet structure, surrounding with two α-helix and four coil domains, based on the results from primary sequence analysis, 2D structure prediction, disulfide bond assignment and hydrophobicity plotting. The highly conserved sequence formed the β-sheet core would be important to the biological activity to the members of CHH family.
| Identifer | oai:union.ndltd.org:TW/087NTU00312003 |
| Date | January 1999 |
| Creators | Chung-Yen Lin, 林仲彥 |
| Contributors | Ching-Ming Kuo, G. H. Kou, 郭欽明, 郭光雄 |
| Source Sets | National Digital Library of Theses and Dissertations in Taiwan |
| Language | zh-TW |
| Detected Language | English |
| Type | 學位論文 ; thesis |
| Format | 123 |
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