Protein synthesis by polysomes from the epidermis of blowfly larvae: Dependence of formation of DOPA-decarboxylase on developmental stage

Polysomes were isolated from the epidermis of six day old larvae and of untanned pupae of Calliphora vicina R.-D. A prerequisite for polysome isolation is that the epidermis be scraped off the overlying cuticle and homogenized in a buffer containing heparin sulfate. A discontinuous sucrose gradient was used to avoid contaminating the preparation with the protein Calliphorin. The incorporation of amino acids into protein by the polysomal preparations has been optimized in respect to monovalent and divalent cations, and the various parameters influencing protein synthesis have been examined. Synthesis is dependent on the presence of pH 5 factors, of ATP and of an energy regenerating system, but not on GTP or an amino acid mixture. Cycloheximide, puromycin, streptovitacin and chloromycetin inhibit protein synthesis. The synthesized polypeptides are not released from the polysomes. One of the proteins synthesized in vitro is DOPA-decarboxylase, as shown by affinity chromatography on a DOPA-decarboxylase IgG-Sepharose column and SDS-acrylamide gel electrophoresis. The polysomes from untanned pupae synthesize three to four times more DOPA-decarboxylase than do those from six day old larvae.