Synthesis of 3-ketoacyl-CoA thiolase of rat liver peroxisomes on free polyribosomes as a larger precursor. Induction of thiolase mRNA activity by clofibrate.
The site of synthesis and induction by clofibrate of peroxisomal 3-ketoacyl-CoA thiolase (acetyl-CoA acyltransferase; EC 2.3.1.16) was investigated. Free and membrane-bound polyribosomal RNA species from the livers of normal rats and rats treated with clofibrate, a hypolipidaemic 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 X rabbit anti-thiolase serum and analysed by sodium dodecyl sulphate/polyacrylamide-gel electrophoresis and fluorography. Thiolase mRNA was found predominantly in free polyribosomes, in both normal and clofibrate-treated rats. Clofibrate treatment increased mRNA activity for thiolase approx. 20-fold. The translation product of clofibrate-induced thiolase mRNA migrated slightly faster in sodium dodecyl sulphate/polyacrylamide-gel electrophoresis than did the translation product of normal thiolase mRNA. Both the normal and the clofibrate-induced translation products were approx. 6000 Da larger than the 41000-Da subunit of the purified enzyme. Immunoblot analysis of liver homogenates, isolated peroxisomes and the purified enzyme indicated that the thiolase subunit was approx. 41000 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.
D000102:Acetyl-CoA C-Acyltransferase / Q000096:biosynthesis*; D000217:Acyltransferases / Q000096:biosynthesis*; D000818:Animals; D002474:Cell-Free System; D002994:Clofibrate / Q000494:pharmacology*; D004591:Electrophoresis, Polyacrylamide Gel; D004790:Enzyme Induction / Q000187:drug effects; D007122:Immunoelectrophoresis; D066298:In Vitro Techniques; D008099:Liver / Q000187:drug effects / Q000201:enzymology*; D008830:Microbodies / Q000201:enzymology; D011132:Polyribosomes / Q000201:enzymology; D014176:Protein Biosynthesis; D012333:RNA, Messenger / Q000378:metabolism*; D051381:Rats
D012333:RNA, Messenger; D000217:Acyltransferases; D000102:Acetyl-CoA C-Acyltransferase; D002994:Clofibrate