IN-VIVO, IN-VITRO AND CELL-FREE SYNTHESIS OF HEMOCYANIN IN THE SHRIMPPENAEUS-EMISULCATUS (DEHAAN)

Hemocyanin (Hc) synthesis was compared at three different levels: in v ivo, after injection of [S-35]methionine and ill vitro, by incubating hepatopancreas (HEP) tissue fragments or by translating RNA extracted from HEP. The HEP of male Penaeus semisulcatus contains mRNA specific for Hc. Poly(A(+))RNA was used as a template for cell-free translation assays. Two polypeptides of 64 and 73 kDa were immunoisolated from th ese in vitro translation lysates using antiserum prepared against puri fied Hc. The two polypeptides exhibited similar mobility to the polype ptides of Hc purified from the hemolymph. Hc was not precipitated from translation products when immunoisolation was performed using the pol yclonal antisera that were prepared against two crustacean lipoprotein s (LP1 from the hemolymph and vitellin from the ovary). The same polyp eptides were immunoisolated from HEP fragments that were incubated in vitro with [S-35]methionine. In order to verify that the immunoisolate d proteins from the ill vitro labelled HEP were indeed Hc, immunoisola tion was conducted in the presence of purified Hc. The labelled Hc was displaced by the unlabelled Hc. In contrast, no precipitate was isola ted after using non-immunized rabbit serum. The method of Hc isolation determined the number of protein bands that were visualized after imm unoisolation from hemolymph samples removed from males injected in viv o with [S-35]methionine. After coagulation, two of the visualized band s were found to have an electrophoretic mobility similar to that of pu rified Hc.