Takashi Osumi

Amino-terminal presequence of the precursor of peroxisomal 3-ketoacyl-CoA thiolase is a cleavable signal peptide for peroxisomal targeting

To examine the function of the amino-terminal presequence of rat peroxisomal 3-ketoacyl-CoA thiolase precursor, fusion proteins of various amino-terminal regions of the precursor with non-peroxisomal enzymes were expressed in cultured mammalian cells. On immunofluorescence microscopy, all constructs carrying the presequence part exhibited punctate patterns of distribution, identical with that of catalase, a peroxisomal marker. Proteins lacking all or a part of the prepiece were found in the cytosol. These results indicate that the presequence of the thiolase has sufficient information for peroxisomal targeting.

Acyl-CoA oxidase of rat liver: A new enzyme for fatty acid oxidation

Abstract Acyl-CoA oxidase was purified from rat liver based on the palmitoyl-CoA-dependent H2O2-forming activity. Enoyl-CoA formation from palmitoyl-CoA by this enzyme was shown by the following observations; first, palmitoyl-CoA-dependent NAD+ reduction in the presence of this enzyme, crotonase, and 3-hydroxyacyl-CoA dehydrogenase, and, second, palmitoyl-CoA-dependent increase in absorbance at 263 nm. Same amounts of enoyl-CoA and H2O2 were formed during the reaction. It is concluded that this enzyme catalyzes the following reaction: Palmitoyl-CoA + O2 → transm -2-Hexadecenoyl-CoA + H2O2. It was most active toward C12-C18 acyl-CoAs. C20 and C22 acyl-CoAs were also oxidized, but C4 and C6 acyl-CoAs were hardly oxidized at all.

Peroxisome targeting signal of rat liver acyl-coenzyme A oxidase resides at the carboxy terminus.

To identify the topogenic signal of peroxisomal acyl-coenzyme A oxidase (AOX) of rat liver, we carried out in vitro import experiments with mutant polypeptides of the enzyme. Full-length AOX and polypeptides that were truncated at the N-terminal region were efficiently imported into peroxisomes, as determined by resistance to externally added proteinase K. Polypeptides carrying internal deletions in the C-terminal region exhibited much lower import activities. Polypeptides that were truncated or mutated at the extreme C terminus were totally import negative. When the five amino acid residues at the extreme C terminus were attached to some of the import-negative polypeptides, the import activities were rescued. Moreover, the C-terminal 199 and 70 amino acid residues of AOX directed fusion proteins with two bacterial enzymes to peroxisomes. These results are interpreted to mean that the peroxisome targeting signal of AOX residues at the C terminus and the five or fewer residues at the extreme terminus have an obligatory function in targeting. The C-terminal internal region also has an important role for efficient import, possibly through a conformational effect.

Peroxisomal βoxidation system of rat liver. Copurification of enoyl-CoA hydratase and 3-hydroxyacyl-CoA dehydrogenase

Abstract Activity of enoyl-CoA hydratase in rat liver was elevated about 6-fold by the administration of di-(2-ethylhexyl)phthalate, a hepatic peroxisome proliferator. Almost all of the increased activity was the peroxisomal enzyme, which was distinguished by its heat-lability from mitochondrial one. Heat-labile enoyl-CoA hydratase was copurified with peroxisomal 3-hydroxyacyl-CoA dehydrogenase. The purified enzyme corresponded to a peroxisome specific peptide with a molecular weight of 80,000.

Purification and properties of mitochondrial and peroxisomal 3-hydroxyacyl-CoA dehydrogenase from rat liver☆

Abstract There are two 3-hydroxyacyl-CoA dehydrogenase (EC 1.1.1.35) in rat liver, one in mitochondria (type I enzyme), and another in peroxisomes (type II enzyme). In a series of the studies on the properties and the physiological roles of fatty acid oxidation systems in both organelles, the two enzymes were purified and compared for their properties. The final preparations obtained were judged to be homogeneous based on the results of sodium dodecyl sulfate-polyacrylamide gel electrophoresis and sedimentation velocity analysis. Type I enzyme was composed of two identical subunits of molecular weight of 32,000, whereas type II enzyme was a monomeric enzyme having a molecular weight of 70,000–77,000. These subunit structures were confirmed by the results of fluorescence studies. Both enzymes were different in amino acid compositions, especially in the contents of tryptophan and half-cystine. Antibodies against them formed single precipitin lines for the corresponding enzymes, but not for the others when subjected to an Ouchterlony double-diffusion test. The K m values of type II enzyme for various substrates were lower than those of type I enzyme except those for acetoacetyl-CoA. As for 3-hydroxyacyl-CoA substrates, both enzymes had lower K m s for longer-chain substrates. The V for the substrates of C 4 C 10 were similar for each enzyme, though the value of type II enzyme for C 10 substrate was rather lower. The results of fluorescence studies suggested that their dissociation constants for NADH were lower and those for NAD + were higher at lower pH. Both enzymes were specific to l -form of 3-hydroxyacyl-CoA substrate. The optimal pH of the forward reaction of type I and type II enzymes was 9.6 and 9.8, and of the reverse reaction, 4.5 and 6.2, respectively. From these results they were concluded to be completely different enzymes.

Complementation study of peroxisome-deficient disorders by immunofluorescence staining and characterization of fused cells

SummaryGenetic heterogeneity in peroxisome-deficient disorders, including Zellwegers cerebrohepatorenal syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease, was investigated. Fibroblasts from 17 patients were fused using polyethylene glycol, cultivated on cover slips, and the formation of peroxisomes in the fused cells was visualized by immunofluorescence staining, using anti-human catalase IgG. Two distinct staining patterns were observed: (1) peroxisomes appeared in the majority of multinucleated cells, and (2) practically no peroxisomes were identified. Single step 12-(1′-pyrene) dodecanoic acid/ultraviolet (P12/UV)-selection confirmed that the former groups were resistant to this selection, most of the surviving cells contained abundant peroxisomes, and the latter cells died. In the complementary matching, [1-14C]lignoceric acid oxidation and the biosynthesis of peroxisomal proteins were also normalized. Five complementation groups were identified. Group A: Zellweger syndrome and infantile Refsum disease; Groups B, C and D: Zellweger syndrome; Group E: Zellweger syndrome, neonatal adrenoleukodystrophy and infantile Refsum disease. We compared these groupings with those of Roscher and identified eight complementation groups. There was no obvious relation between complementation groups and clinical phenotypes. These results indicate that the transport, intracellular processing and function of peroxisomal proteins were normalized in the complementary matching and that at least eight different genes are involved in the formation of normal peroxisomes and in the transport of peroxisomal enzymes.

Nucleotide sequence of the human 70 kDa peroxisomal membrane protein: a member of ATP-binding cassette transporters☆

The cDNA sequence of human liver 70 kDa peroxisomal membrane protein (hPMP70) was determined. The nucleotide sequence contains an open reading frame of 1977 base pairs and encodes an amino acid sequence of 659 residues which exhibits 95.0% identity with that of rat liver PMP70. hPMP70 shares close similarity to the members of a superfamily of ATP-binding transport proteins.

The inducible fatty acid oxidation system in mammalian peroxisomes

Abstract There are two β-oxidation systems in animal cells: one in mitochondria and the other in peroxisomes. These two systems consist of different sets of enzymes. Since these two β-oxidation systems are located in different organelles, it is possible for them to respond differently to various physiological and biochemical conditions.

Isolation and characterization of the rat catalase-encoding gene

Using cDNA clones coding for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6), overlapping genomic clones were isolated. By Southern blotting analysis and nucleotide sequencing, the gene was characterized to be about 33 kb in length and to have 13 exons and twelve introns. S1 mapping and primer extension analyses showed the presence of multiple transcription start points (tsp) located between 66 bp and 105 bp upstream from the translational start codon. Nucleotide sequence immediately upstream from the tsp lacks the TATA box but contains multiple CCAAT boxes and GC-like boxes.

Turnover of enzymes of peroxisomal β-oxidation in rat liver

Male Wistar rats were given a diet containing 2% (w/w) di-ethylhexyl)-phthalate (DEHP), a peroxisomal proliferator, for 4 weeks. The activities of enzymes of peroxisomal beta-oxidation and of catalase were markedly increased by the DEHP administration. The time required to reach halfway to the maximal induction for enzymes of peroxisomal beta-oxidation was 5--7 days, whereas that for catalase was 3 days. A separate DEHP group was placed on the control diet after 14 days of feeding with the DEHP diet. On the withdrawal of DEHP, activities of enzymes of the beta-oxidation system and of catalase decreased to the control levels with a half-life of 2--3 days. Responses of some mitochondrial enzymes involved in fatty acid oxidation are also described.