In vivo, in vitro and cell-free synthesis of hemocyanin in the shrimp Penaeus semisulcatus (de Haan)
1995
Khayat, M. | Funkenstein, B. | Tietz, A. | Lubzens, E.
Hemocyanin (Hc) synthesis was compared at three different levels: in vivo, after injection of [35S]methionine and in 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 these in vitro translation lysates using antiserum prepared against purified Hc. The two polypeptides exhibited similar mobility to the polypeptides of Hc purified from the hemolymph. Hc was not precipitated from translation products when immunoisolation was performed using the polyclonal antisera that were prepared against two crustacean lipoproteins (LP1 from the hemolymph and vitellin from the ovary). The same polypeptides were immunoisolated from HEP fragments that were incubated in vitro with [35S]methionine. In order to verify that the immunoisolated proteins from the in vitro labelled HEP were indeed Hc, immunoisolation was conducted in the presence of purified Hc. The labelled Hc was displaced by the unlabelled Hc. In contrast, no precipitate was isolated after using non-immunized rabbit serum. The method of Hc isolation determined the number of protein bands that were visualized after immunoisolation from hemolymph samples removed from males injected in vivo with [35S]methionine. After coagulation, two of the visualized bands were found to have an electrophoretic mobility similar to that of purified Hc.
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