Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate

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Abstract
The site of synthesis and induction by clofibrate of peroxisomal 3-ketoacyl-CoA thiolase was investigated. Free and membrane-bound polyribosomal RNA species from the livers of normal rats and rats treated with clofibrate, a hypolipidemic drug that causes marked proliferation of peroxisomes, were translated in a nuclease-treated rabbit reticulocyte-lysate cell-free protein-synthesizing system with [35S]methionine as label. The cell-free translation products were immunoprecipitated with monospecific rabbit anti-thiolase serum and analyzed by sodium dodecyl sulfate/polyacrylamide-gel electrophoresis [SDS-PAGE] and fluorography. Thiolase mRNA was found predominantly in free polyribosomes, in both normal and clofibrate-treated rats. Clofibrate treatment increased mRNA activity for thiolase .apprx. 20-fold. The translation product of clofibrate-induced thiolase mRNA migrated slightly faster in SDS-PAGE than did the translation product of normal thiolase mRNA. Both the normal and the clofibrate-induced translation products were .apprx. 6000 Da [daltons] larger than 41,000-Da subunit of the purified enzyme. Immunoblot analysis of liver homogenates, isolated peroxisomes and purified enzyme indicated that the thiolase subunit was .apprx. 41,000 Da in all samples, ruling out proteolysis during the purification of thiolase. Thiolase biogenesis thus differs from that of rat liver peroxisomal proteins studied previously in that it is synthesized as a larger precursor, implying post-translational import of thiolase into peroxisomes with proteolytic processing. Clofibrate apparently alters the size as well as the amount of the translation product.