In vitro synthesis of laminin and entactin polypeptides.
Total RNA and poly(A+) RNA, isolated from 13.5-day-old mouse embryo parietal endoderm cells and from differentiated F9 teratocarcinoma cells that synthesize laminin and entactin, were translated in the reticulocyte lysate. Antiserum raised against purified and denatured laminin B chains specifically immunoprecipitated from the translation reaction polypeptides of Mr = 205,000, 200,000, and 185,000. Antiserum against the native complex of laminin and entactin also immunoprecipitated these polypeptides, although less efficiently. In addition, this antiserum immunoprecipitated polypeptides of Mr = 300,000, 270,000, and 140,000. Antiserum against purified and denatured entactin immunoprecipitated only the Mr = 140,000 polypeptide. In contrast, no polypeptides were immunoprecipitated from translation reactions programmed with RNA from undifferentiated F9 cells that produce only small amounts of laminin and entactin. The in vitro synthesized polypeptides migrate on NaDodSO4-polyacrylamide gel electrophoresis slower than the respective unglycosylated laminin and entactin chains isolated from cells treated with tunicamycin. Supplementing the reticulocyte lysate with dog pancreas microsomal membranes yields in vitro translation products which co-migrate with the respective glycosylated laminin and entactin chains of control cells. Taken together, these results suggest that the polypeptides described represent in vitro synthesized laminin and entactin chains.
D000818:Animals; D002454:Cell Differentiation; D002460:Cell Line; D004707:Endoderm / Q000378:metabolism; D006023:Glycoproteins / Q000096:biosynthesis*; D007163:Immunosorbent Techniques; D007797:Laminin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins / Q000096:biosynthesis; D051379:Mice; D008970:Molecular Weight; D011061:Poly A / Q000378:metabolism; D014176:Protein Biosynthesis; D012313:RNA / Q000378:metabolism; D012333:RNA, Messenger / Q000378:metabolism; D011817:Rabbits; D012156:Reticulocytes / Q000378:metabolism; D013724:Teratoma
D006023:Glycoproteins; D007797:Laminin; D008562:Membrane Glycoproteins; D008565:Membrane Proteins; D012333:RNA, Messenger; C029967:nidogen; D011061:Poly A; D012313:RNA