RESUMO
To design a homologous bioassay for the molt-inhibiting hormone and the crustacean hyperglycemic hormone of the shrimp Penaeus vannamei, the effect of sinus gland homogenate (SGh), in vitro, on ecdysteroid production by Y-organs (YOs), and the effect of the injection of SGh, in vivo, on the glycemia of shrimps have been investigated. Addition of SGh to incubation medium of shrimp YOs dose dependently reduced, within a few hours, ecdysteroid release into the medium. Moreover, inhibition by SGh decreases drastically in YOs from animals in late premolt stages, when there is maximal ecdysteroid production. Injection of SGh into shrimps evokes a hyperglycemic response maximal after 2 hr. Immunoadsorption of SGh with an anti-Homarus americanus cHHA antiserum inhibited both biological activities of the homogenate. After fractionation of acidic sinus gland extract by RP-HPLC, the maximal response in both bioassays was associated with the major UV absorbent peak, which was also the major immunoreactive peak when tested by ELISA with the anti-lobster cHHA. After a further purification step, the molecular mass of the bioactive and immunoreactive peptide was found to be 8627 +/- 0.3 Da by electrospray ionization mass spectrometry. The amino acid sequence of the first 38 residues of this peptide was established by gas-phase microsequencing. This sequence shows 55% homology with the first 38 residues of the lobster cHHA.
