title: Biosynthesis and intracellular transport of enzymes of peroxisomal beta-oxidation.
abstract: Three peroxisomal enzymes of beta-oxidation from rat liver were synthesized in a cell-free protein-synthesizing system derived from a lysate of rabbit reticulocytes. The in vitro products of acyl-CoA oxidase (EC 1.3.99.3) and a bifunctional protein containing enoyl-CoA hydratase (EC 4.2.1.17) and 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) activities were apparently the same in size and charge as the subunit of the respective mature enzymes; that of 3-ketoacyl-CoA thiolase (EC 2.3.1.16) was about 3,000 Da larger and more basic than its mature subunit. The free polysome fraction of rat liver was 3.1-5.7 times more active than the membrane-bound polysome fraction in the synthesis of the three peroxisomal enzymes; these values were similar to those for cytosolic enzymes and differed from that for serum albumin. In isolated rat hepatocytes, radiolabeled acyl-CoA oxidase and bifunctional protein increased with time with no appreciable change in the subunit size. On the other hand, the labeled putative precursor of 3-ketoacyl-CoA thiolase, as well as the mature form of the enzyme, was detected in the hepatocytes. The radioactivity of the putative precursor reached a plateau in 30 min; that of the mature subunit appeared after a lag time of about 5 min and increased with time up to 90 min. In pulse-chase experiments, the putative precursor disappeared with an apparent half-life of several minutes. When the hepatocytes were fractionated into the cytosolic and the particulate fractions, one half of labeled acyl-CoA oxidase and 60% of the bifunctional protein were recovered in the cytosolic fraction after 10 min of labeling, whereas 70-80% of the labeled enzymes were recovered in the particulate fraction after 40-60 min of labeling. These results indicate that the three enzymes of peroxisomal beta-oxidation are synthesized on free polysomes, released into the cytosol, and then transported into peroxisomes. Our findings also indicate that 3-ketoacyl-CoA thiolase undergoes proteolytic processing during maturation. The temporal sequence of the proteolytic cleavage and intracellular transport of the thiolase remains to be determined.
mesh_terms: D015094:3-Hydroxyacyl CoA Dehydrogenases / Q000235:genetics*; D042962:Acyl-CoA Oxidase; D000818:Animals; D004746:Enoyl-CoA Hydratase / Q000235:genetics*; D006836:Hydro-Lyases / Q000235:genetics*; D007700:Kinetics; D008099:Liver / Q000201:enzymology*; D046911:Macromolecular Substances; D008297:Male; D008830:Microbodies / Q000201:enzymology*; D008970:Molecular Weight; D009097:Multienzyme Complexes / Q000378:metabolism; D010088:Oxidoreductases / Q000235:genetics*; D010446:Peptide Fragments / Q000032:analysis; D014176:Protein Biosynthesis; D011817:Rabbits; D051381:Rats; D011919:Rats, Inbred Strains; D012156:Reticulocytes / Q000378:metabolism; D013347:Subcellular Fractions / Q000201:enzymology
chemical_list: D046911:Macromolecular Substances; D009097:Multienzyme Complexes; D010446:Peptide Fragments; D010088:Oxidoreductases; D015094:3-Hydroxyacyl CoA Dehydrogenases; D042962:Acyl-CoA Oxidase; D006836:Hydro-Lyases; D004746:Enoyl-CoA Hydratase