Abstract
Rat liver polysomal RNA was translated in the rabbit reticulocyte lysate and in the wheat germ cell-free protein-synthesizing systems, using [(35)S]methionine as label. The catalase (hydrogen-peroxide:hydrogen-peroxide oxidoreductase, EC 1.11.1.6) that was synthesized was isolated by immunoprecipitation and characterized by electrophoresis in sodium dodecyl sulfate/polyacrylamide gels followed by fluorography. The catalase made in both systems migrated more slowly during electrophoresis than did purified peroxisomal catalase. By comparison with standards of known molecular mass, the cell-free products were estimated to be about 4000 daltons larger than the purified enzyme. We also investigated the biosynthesis of catalase in vivo by injecting [(35)S]methionine into rats. The precursor of catalase known to be synthesized in liver and found in the high-speed supernatant 8 min later [Lazarow, P. B. & de Duve, C. (1973) J. Cell Biol. 59, 491-506] was isolated immunochemically. For comparison, 1-day-old completed catalase was immunoprecipitated from peroxisomes. The migrations in sodium dodecyl sulfate gels of the 8-min-old precursor and the subunit of the day-old enzyme were indistinguishable and approximately the same as the migration of the cell-free products. These results indicate that catalase's apparent size does not change when it enters peroxisomes but rather decreases during the chemical purification procedure.