| Budget Amount *help |
¥14,000,000 (Direct Cost: ¥14,000,000)
Fiscal Year 2001: ¥3,100,000 (Direct Cost: ¥3,100,000)
Fiscal Year 2000: ¥2,900,000 (Direct Cost: ¥2,900,000)
Fiscal Year 1999: ¥8,000,000 (Direct Cost: ¥8,000,000)
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| Research Abstract |
In crustaceans, neuropeptides produced in the X-organ and stored in the sinus glands are responsible for many physiological phenomena such as glucose metabolism, molting, vitellogenesis, body color changes and so on. Among them, a group of peptides called crustacean hyperglycemic hormone (CHH) family involves hyperglycemic hormone, molt-inhibiting hormone (MIH) and vitellogenesis-inhibiting hormone. We previously isolated and characterized CHH-family peptides from the kuruma prawn, and then cloned their cDNAs, thereby clarifying the structures of hormone precursors. The aim of this study is to clarify structure-activity relationship of these peptides at a tertiary structural level. First, a recombinant MIH was produced in the Escherichia colt expression system using the cDNA. The recombinant peptide was not hormonally active just after expression, but a refolding reaction according to the conventional method afforded an active recombinant MIH. The recombinant MIH was as active as the natural MIH. The arrangement of the three disulfide bonds in the recombinant MIH was determined by analyzing its tryptic peptides and was found to be identical to that of the natural MIH. The CD spectrum of the recombinant MIH was very close to that of the natural MIH. Which suggested that they were rich in a-helix. Next, we prepared a double-labled recombinant MIH with ^<13>C and ^<15>N, and attempted to analyze its tertiary structure by nuclear magnetic rezonance techniques. Until now, we have finished to assign about 80 % of proton signals and have also obtained much NOE information. The analyzes are in progress and I hope the tertiary structure will be obtained in the near future. By the same method, we are producing one of CHHs and will analyze its tertiary structure and compare it with that of MIH. Such analyzes will give us important information on the molecular evolution of these peptides.
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