Post-translational import of fatty acyl-CoA oxidase and catalase into peroxisomes of rat liver in vitro.
Total polysomal RNA of rat liver was translated in vitro in a rabbit reticulocyte lysate system. The translation products were mixed with a postnuclear supernatant fraction of rat liver and incubated post-translationally at 26 degrees C for 15-60 min. The import assay mixture was separated into a particulate fraction and supernatant by centrifugation, both of which were analyzed by immunoprecipitation with a goat antibody against rat liver peroxisomal proteins, sodium dodecyl sulfate-polyacrylamide gel electrophoresis, and fluorography. One peroxisomal translation product (Mr 72,000) appeared in the particulate fraction, was partly proteinase K-resistant, and addition of detergents prior to proteolysis abolished this resistance. In isopycnic centrifugation of the uptake assay mixture, the protease-resistant 35S-polypeptide of Mr 72,000 cosedimented with the peroxisomes. This translation product was identified immunochemically as fatty acyl-CoA oxidase; both before and after import it was indistinguishable in size from subunit A of the purified enzyme by prolonged sodium dodecyl sulfate-polyacrylamide gel electrophoresis. When the cell-free translation products were incubated with highly purified peroxisomes, 35S-catalase entered peroxisomes (by the criterion of protease resistance), and its entry was stimulated by the addition of a high speed supernatant (cytosolic) fraction of rat liver. These results demonstrate the post-translational import into peroxisomes in vitro of at least two cell-free translation products.
D042962:Acyl-CoA Oxidase; D000818:Animals; D002374:Catalase / Q000378:metabolism*; D002500:Centrifugation, Isopycnic; D007700:Kinetics; D008099:Liver / Q000648:ultrastructure*; D008830:Microbodies / Q000201:enzymology*; D008970:Molecular Weight; D010088:Oxidoreductases / Q000378:metabolism*; D011499:Protein Processing, Post-Translational; D051381:Rats
D010088:Oxidoreductases; D002374:Catalase; D042962:Acyl-CoA Oxidase