|Budget Amount *help
¥2,100,000 (Direct Cost : ¥2,100,000)
Fiscal Year 1993 : ¥700,000 (Direct Cost : ¥700,000)
Fiscal Year 1992 : ¥1,400,000 (Direct Cost : ¥1,400,000)
This study aimed at isolation and characterization of endogenous factors controlling molting in the crustacea. In the first year, to establish bioassay method for searching the factors. I tried some methods using the prawn, Penaeus japonicus, and the crayfish, Procambarus clarkii. But due to several reasons, the bioassay method could not be established. Therefore, in the second year, a molecule structurally similar to molt-inhibiting hormone (MIH) only isolated and characterized from the shore crab, Carcinus maenas, was searched by chemical assay. MIH is a member of CHH (crustacean hyperglycemic hormone) family of peptides, which share three characteristics of sequence homology, molecular weight (8500-9300) and intramolecular three disulfide bonds.
First, several candidate molecules for CHH-family peptides were purified from whole eyestalks each of the prawn and the crayfish by heat-treatment, acid extraction, pretreatment through a reverse phase mini-column, gel-filtration and repeated reverse phase HPLC.But sequence analyzes of these peptides revealed that they were not the peptides of CHH-family. Then, the extraction source was changed from eyestalks to sinus glands. Saline extracts of the sinus glands from both species were directly subjected to reverse phase HPLC.Some peptides, which satisfied the above chemical criteria for CHH-family peptides, were obtained from both species. Sequence analyzes of these peptides showed that the crayfish has two kinds of CHH family of peptides, CHH and MIH or vitellogenesis-inhibiting hormone (VIH). The former inhibited ecdysteroid production by Y-organ in vitro. On the other hand, the prawn was shown to have five molecular species of CHH, all of which were found to be produced in one individual.