Synthesis of a larger precursor for the subunit IV of rat liver cytochrome c oxidase in a cell-free wheat germ system.
Poly(A)+RNA from phenol-extracted rat liver polysomes was translated in a heterologous cell-free system derived from wheat germ. The RNA stimulated the incorporation of [35S]methionine into proteins 20- to 30-fold. The labeled translation products were incubated with an antiserum against cytochrome c oxidase. After binding of the antigen x immunoglobulin complex to and elution from protein A-Sepharose and sodium dodecyl sulfate (SDS)-polyacrylamide step gel electrophoresis, autoradiography was carried out. Mainly one major protein with an apparent molecular weight of 19,500 was visualized. When the unlabeled individual cytochrome c oxidase subunits IV, V, VI, or VII, isolated from preparative SDS-polyacrylamide gels, were added to the translation mixture, it was found that only subunit IV could compete with the in vitro-synthesized protein of 19.5 kilodaltons in respect to the binding to the cytochrome c oxidase antiserum. The in vitro-synthesized product was 3,000 daltons larger than the cytochrome c oxidase subunit polypeptide IV. It is concluded that the subunit IV is synthesized as a precursor. Evidence for the precursor form was obtained from translation experiments with [35S]methionine bound to a specific initiator tRNA which led to a radioactively labeled product of identical electrophoretic mobility as the 19.5 kilodalton protein. Furthermore, two dimensional tryptic fingerprints of subunit IV and its precursor show a high degree of similarity.
D000818:Animals; D002474:Cell-Free System; D003576:Electron Transport Complex IV / Q000096:biosynthesis*; D004792:Enzyme Precursors / Q000096:biosynthesis*; D008099:Liver / Q000201:enzymology*; D010944:Plants / Q000378:metabolism*; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger; D051381:Rats; D014908:Triticum / Q000378:metabolism
D004792:Enzyme Precursors; D012333:RNA, Messenger; D011061:Poly A; D012313:RNA; D003576:Electron Transport Complex IV