Biomedical subjects

T Osumi

Publications and source records attributed to T Osumi.

At least 109 records · Page 6Linked to original sources

PubMed1989

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.

Amino Acid Sequence↗

PubMed1989

Molecular cloning and nucleotide sequence of cDNA encoding the entire precursor of rat mitochondrial acetoacetyl-CoA thiolase.

cDNA clones for rat mitochondrial acetoacetyl-CoA thiolase were isolated and sequenced. The most 5'-extended clone (RT2-6) consisted of 1,460 bases and contained a 1,272-base open reading frame encoding a polypeptide of 424 amino acid residues. A coupled in vitro transcription/translation analysis of RT2-6 revealed that RT2-6 encodes the entire precursor of this enzyme. The amino-terminal sequence and amino acid composition of the purified enzyme agreed with the primary structure deduced from the cDNA. The calculated molecular masses of the precursor and the subunit of the mature enzyme are 44,694 and 41,364 Da, respectively. The primary structure of this enzyme was compared with those of four other thiolases (rat mitochondrial and peroxisomal 3-ketoacyl-CoA thiolases, acetoacetyl-CoA thiolase of Zoogloea ramigera, and cytosolic acetoacetyl-CoA thiolase of Saccharomyces uvarum). Marked homology between any two of them (34-51% identity) indicates that the genes of thiolases have evolved from a common ancestral gene. It has been reported that this enzyme has two isoenzymes A and B. However, the purified isoenzymes were indistinguishable from each other in some analyses. Though 17 independent cDNA clones were isolated, no definite evidence indicating the presence of different cDNAs was found.

Acetyl-CoA C-Acetyltransferase↗

PubMed1989

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.

Acyl Coenzyme A↗

PubMed1988

Comparison of constitutive and inducible levels of expression of peroxisomal beta-oxidation and catalase genes in liver and extrahepatic tissues of rat.

Previous studies from our laboratories have shown that carcinogenic peroxisome proliferators significantly increase the mRNA levels of peroxisomal beta-oxidation genes in the rat liver by enhancing the transcriptional activity. Because of a good correlation between the inducibility of peroxisome proliferation and carcinogenicity of this class of xenobiotics, we proposed that sustained induction of peroxisomal beta-oxidation system and the resultant oxidative stress form the basis for carcinogenesis. Since this concept implies that tumors should develop only in tissues which display maximal peroxisome proliferation, we have now assessed the degree to which catalase and the three beta-oxidation genes are expressed in liver and 12 extrahepatic tissues of adult rats fed for 2 weeks a diet containing 0.025% ciprofibrate (w/w), a peroxisome proliferator. In the ciprofibrate-treated rats, the levels of catalase mRNA increased to less than 2-fold in liver, kidney, intestine, and heart, but no change was detected in other tissues. The mRNA levels of the three genes of beta-oxidation system in the liver of adult rats treated with ciprofibrate increased greater than 20-fold. In contrast, in the kidney, small intestine, and heart the increases in the mRNA levels of all three beta-oxidation genes were small and varied from 2- to 4-fold following ciprofibrate treatment. Ciprofibrate did not significantly increase the levels of these mRNAs in the other nine tissues. These results correlated well with the levels of peroxisomal beta-oxidation activity, peroxisome volume density, and the immunologically quantified proteins in various tissues. These results provide evidence for the presence of beta-oxidation enzymes in peroxisomes of many tissues of rat and for tissue (cell)-specific differences in the inducibility of mRNAs of these beta-oxidation genes. The marked inducibility of beta-oxidation genes in liver and subsequent development of liver tumors support the hypothesis that tumors develop in tissues that show inducibility of peroxisome proliferation vis a vis beta-oxidation system following exposure to peroxisome proliferators.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1988

Diminished synthesis of catalase due to the decrease in catalase mRNA in Japanese-type acatalasemia.

In order to characterize the genetic defect in Japanese-type acatalasemia, we examined catalase synthesis in the cultured fibroblasts isolated from a patient suffering from this disease. The transcript of the mutant catalase gene was also analyzed by Northern blotting. It was demonstrated that the severe reduction in catalase protein in the acatalasemic patient is due to the defective synthesis of the enzyme caused by the lack of catalase mRNA. Using Southern blotting, we did not detect large deletion, insertion or rearrangement in the mutant catalase gene as compared with the normal one. We conclude that Japanese-type acatalasemia in this study is resulted by a small mutation in the non-coding region of catalase gene which affects some step in the catalase mRNA metabolism.

Acatalasia↗

PubMed1987

Complete nucleotide sequence of cDNA and predicted amino acid sequence of rat acyl-CoA oxidase.

cDNA clones for rat acyl-CoA oxidase were isolated. The 3.8-kilobase mRNA sequence of the enzyme was completely covered by two overlapping clones. The composite cDNA sequence consisted of 3741 bases and contained a 1983-base open reading frame which encodes a polypeptide of 661 amino acid residues. Two species of acyl-CoA oxidase cDNA were identified. They differed in their coding nucleotide sequences, only within a small region. They contained the same number of nucleotides and can be translated in a common reading frame. They are 55% and 50% homologous in the above region at the nucleotide and the amino acid levels, respectively. Both types of cDNA were isolated from a library constructed from mRNA of a single rat, thereby suggesting the occurrence of two species of acyl-CoA oxidase in each rat. The amino terminus of the enzyme was determined to be N-acetylmethionine, which corresponds to the initiator methionine, thus confirming the absence of a terminal presequence. We reported previously that a purified preparation of the enzyme contained three polypeptide components, A, B, and C, and suggested that components B and C are produced by a proteolytic cleavage of component A (Osumi, T., Hashimoto, T., and Ui, N. (1980) J. Biochem. (Tokyo) 87, 1735-1746). We located components B and C on the amino- and the carboxyl-terminal sides of component A. Possible functional significances of several stretches of amino acids of the enzyme are discussed, based on the sequence comparison data between rat and yeast acyl-CoA oxidases.

Acyl-CoA Oxidase↗

PubMed1987

Isolation and structural characterization of the rat acyl-CoA oxidase gene.

Overlapping genomic clones containing the entire sequence of the rat acyl-CoA oxidase gene were isolated and characterized. This gene spans about 25 kilobases, and there are 14 exons and 13 introns. All of the exon-intron junction sequences agree with the GT/AG rule. The results of Southern blot analyses indicated that the gene occurs once per haploid genome. In a preceding paper (Miyazawa, S., Hayashi, H., Hijikata, M., Ishii, N., Furuta, S., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8131-8137), we described the presence of two species of acyl-CoA oxidase mRNA which have different sequences, only in a small region. We identified two exons corresponding to this region. The above two mRNA species are produced by alternative splicing of these exons. Several mRNA cap sites were detected by S1 mapping and by the primer extension method. The principal one was mapped at 75 nucleotides upstream of the initiator methionine codon. The 5'-flanking region of the acyl-CoA oxidase gene was sequenced up to about 1300 nucleotides, upstream of the cap sites. There was neither a "TATA" box nor a typical "CCAAT" box sequence, at least up to nucleotide -500, rather, this region is G + C-rich (65%). "GC" box hexanucleotides (GGGCGG or CCGCCC) repeat six times in this region. Two of them, located nearest the cap sites, are in a complete 11-base pair direct repeat, 5'-GGGCGGGGCCG-3'. The features of the 5'-flanking sequence of this gene resemble those of the gene for rat enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. The possible functional significance of the characteristic 5'-flanking sequence elements in the transcriptional regulation of acyl-CoA oxidase is given attention.

Acyl-CoA Oxidase↗

PubMed1987

Structural organization of the gene for rat enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme.

Enoyl-CoA hydratase:3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme is a monomeric protein which catalyzes the second and the third reactions of the peroxisomal beta-oxidation system. We cloned the gene for this enzyme from rat genomic libraries. The gene spans about 31 kilobases and consists of seven exons and six introns. The transcription initiation site was located 24 nucleotides upstream of the initiator methionine codon, ATG, determined by S1 nuclease mapping and primer extension analysis. The 5'-flanking region of the gene lacks typical TATA and CCAAT sequences, but contains G + C-rich sequences, including one CCGCCC ("GC" box) and two related GC hexanucleotides. Some of the structural features of the 5'-flanking region of this gene are shared by the 5'-upstream sequence of the gene for acyl-CoA oxidase, which catalyzes the first reaction of the peroxisomal beta-oxidation system and is induced coordinately with the bifunctional enzyme. Southern blot analysis of rat genomic DNA indicates that the bifunctional enzyme gene occurs once per haploid genome. The amino acid sequence of the carboxyl-terminal region of the bifunctional enzyme exhibits a significant level of homology to the sequence of pig mitochondrial 3-hydroxyacyl-CoA dehydrogenase. This region of the bifunctional enzyme is encoded mainly by the last exon (Exon VII) which codes for as much as 58% of the protein sequence. Based on the data plus our unpublished findings (N. Ishii, T. Osumi, and T. Hashimoto, unpublished data), we propose that the amino-terminal domain, which is principally encoded by Exons I-V, has enoyl-CoA hydratase activity, and the carboxyl-terminal one, which is mainly coded for by Exon VII, has a 3-hydroxyacyl-CoA dehydrogenase function.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1987

Structural analysis of cDNA for rat peroxisomal 3-ketoacyl-CoA thiolase.

cDNA clones of rat peroxisomal 3-ketoacyl-CoA thiolase were isolated. By blotting analysis using the cDNAs as probes, the mRNA for this enzyme was estimated to be about 1.9-kilobase pairs. Elevation of mRNA levels in the liver with administration of di(2-ethylhexyl)phthalate was also evident. Sequencing analysis revealed 1,272 bases of the open reading frame which encoded 424 amino acid residues. Amino acid sequence data on six tryptic peptides and the amino terminus of the purified enzyme confirmed the cDNA sequence. The precursor of peroxisomal thiolase contains at its amino terminus a peptide extension of 26 residues. The mature enzyme is composed of 398 amino acids and the molecular weight is 41,074. The presequence has a net positive charge, lacks a long stretch of hydrophobic residues, and contains a cluster of serine residues. When the primary structure of the precursor was compared to structure of known peroxisomal proteins, there was no common homologous sequence. Peroxisomal thiolase exhibits a significant sequence homology with the mitochondrial thiolase. Possible location of the transport signal of the peroxisomal thiolase is discussed. Acyl-CoA binding sites were also located on primary structures of the two thiolases. The occurrence of interrupting sequences in several clones likely originates from intron sequences.

Acetyl-CoA C-Acyltransferase↗

PubMed1987

Peroxisomal beta-oxidation enzyme proteins in adrenoleukodystrophy: distinction between X-linked adrenoleukodystrophy and neonatal adrenoleukodystrophy.

Very long chain fatty acids, which accumulate in plasma and tissues in X-linked adrenoleukodystrophy (ALD), neonatal ALD, and the Zellweger cerebrohepatorenal syndrome, are degraded by the peroxisomal beta-oxidation pathway, consisting of acyl-CoA oxidase, the bifunctional enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase, and beta-ketothiolase. A marked deficiency of all three enzyme proteins was reported in livers from patients with the Zellweger syndrome, a disorder in which peroxisomes are decreased or absent. Peroxisomes are not as markedly decreased in neonatal ALD and appear normal in X-linked ALD. Immunoblot analysis of the peroxisomal beta-oxidation enzymes revealed an almost complete lack of the bifunctional enzyme in neonatal ALD liver, similar to the finding in Zellweger tissue. In contrast, acyl-CoA oxidase and beta-ketothiolase were present in neonatal ALD liver, although the thiolase appeared to be in precursor form (2-3 kDa larger than the mature enzyme) in neonatal ALD. Unlike either neonatal ALD or Zellweger syndrome, all three peroxisomal beta-oxidation enzymes were present in X-linked ALD liver. Despite the absence in neonatal ALD liver of bifunctional enzyme protein, its mRNA was detected by RNA blot analysis in fibroblasts from these patients. These observations suggest that lack of bifunctional enzyme protein in neonatal ALD results from either abnormal translation of the mRNA or degradation of the enzyme prior to its entry into peroxisomes.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1986

Molecular cloning of cDNA for rat mitochondrial 3-oxoacyl-CoA thiolase.

Messenger RNA of rat 3-oxoacyl-CoA thiolase (acetyl-CoA acyltransferase), a mitochondrial matrix enzyme involved in fatty acid beta-oxidation, was enriched by immunoprecipitation of rat liver free polysomes and recombinant plasmids were prepared from the enriched mRNA by a modification of the vector-primer method of Okayama and Berg. The transformants were initially screened for 3-oxoacyl-CoA thiolase cDNA sequences by differential colony hybridization with [32P]cDNAs, synthesized from the immunopurified and unpurified mRNAs. The cDNA clones for 3-oxoacyl-CoA thiolase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pT1-1, contained a 700-base insert and hybridized to a mRNA species of 1.6 X 10(3) bases in rat liver. The transformants were rescreened using the cDNA insert of pT1-1 as a hybridization probe and a clone (pT1-19) with a 1.5 X 10(3)-base insert was obtained. Activity and concentration of 3-oxoacyl-CoA thiolase mRNA were quantified by in vitro translation and dot-blot analysis using the cDNA insert as a hybridization probe. The level of translatable and hybridizable mRNA in rat liver was increased about 5.1-fold and 4.6-fold, respectively, after administration of di-(2-ethylhexyl)phthalate, a potent inducer of the enzyme. The 3-oxoacyl-CoA thiolase mRNA levels thus determined correlated closely with levels of the activity and amount of this enzyme.

Acetyl-CoA C-Acyltransferase↗

PubMed1986

Complete nucleotide sequence of cDNA and deduced amino acid sequence of rat liver catalase.

We have isolated five cDNA clones for rat liver catalase (hydrogen peroxide:hydrogen peroxide oxidoreductase, EC 1.11.1.6). These clones overlapped with each other and covered the entire length of the mRNA, which had been estimated to be 2.4 kilobases long by blot hybridization analysis of electrophoretically fractionated RNA. Nucleotide sequencing was carried out on these five clones and the composite nucleotide sequence of catalase cDNA was determined. The 5' noncoding region contained 83 bases and was followed by 1581 bases of an open reading frame that encoded 527 amino acids. The 3' noncoding region was 831 bases long and contained long repeats of the unit AC. The amino acid sequence deduced from the nucleotide sequence of the cDNAs showed about 90% homology with the reported primary structure of bovine liver catalase. The molecular weight of rat liver catalase was calculated to be 59,758 from the predicted amino acid sequence. The amino acid residues in contact with the heme group are completely identical for bovine liver and rat liver catalases. The amino acid sequence at the COOH terminus was confirmed by the results of carboxypeptidase P treatment of the protein purified from rat liver in the presence of leupeptin. Rat liver catalase has no cleavable signal peptide for translocation of the enzyme into peroxisomes.

Amino Acid Sequence↗

PubMed1986

Transcription regulation of peroxisomal fatty acyl-CoA oxidase and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase in rat liver by peroxisome proliferators.

The structurally diverse peroxisome proliferators ciprofibrate, clofibrate, and bis(2-ethylhexyl) phthalate [(EtHx)2 greater than Pht] increase the activities of hepatic catalase and peroxisomal fatty acid beta-oxidation enzymes in conjunction with profound proliferation of peroxisomes in hepatocytes. In order to delineate the level at which these enzymes are induced in the liver, the transcriptional activity of specific genes for fatty acyl-CoA oxidase (FAOxase) and enoyl-CoA hydratase/3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme (PBE), the first two enzymes of the peroxisomal beta-oxidation system, and for catalase were measured in isolated hepatocyte nuclei obtained from male rats following a single intragastric dose of ciprofibrate, clofibrate, or (EtHx)2 greater than Pht. All three peroxisome proliferators rapidly increased the rate of FAOxase and PBE gene transcription in liver, with near maximal rates (9-15 times control) reached by 1 hr and persisting until at least 16 hr after administration of the compound. FAOxase and PBE mRNA levels, measured by blot-hybridization analysis and FAOxase and PBE protein content, analyzed by immunoblotting, increased concurrently up to at least 16 hr following a single dose of peroxisome proliferator. The catalase mRNA level increased about 1.4-fold, but the transcription rate of the catalase gene was not significantly affected. The results show that the peroxisome proliferators clofibrate, ciprofibrate, and (EtHx)2 greater than Pht selectively increase the rate of transcription of peroxisomal fatty acid beta-oxidation enzyme genes. Whether the transcriptional effects are mediated by peroxisome proliferator-receptor complexes remains to be elucidated.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1985

Molecular cloning and nucleotide sequence of the cDNA for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme.

For the studies on the mechanism of induction of peroxisomal beta-oxidation enzymes and biogenesis of the organelle, we have isolated cDNA clones for rat peroxisomal enoyl-CoA: hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. On blotting experiments with liver RNA, the cDNAs hybridized to a 3.0-kilobase RNA which was increased 5-7-fold by the administration of di-(2-ethylhexyl)phthalate to rats. Nucleotide sequencing was carried out for four cloned cDNAs and one obtained by a primer extension method. By overlapping these sequences with each other, we identified 20 nucleotides of 5'-noncoding, 2,166 nucleotides of coding, and 910 nucleotides of 3'-noncoding regions. The deduced amino acid sequence of the enzyme is composed of 722 residues, and the composition agrees with that of the protein data. The sequence was confirmed by the amino acid compositions and sequence analyses of some of the tryptic peptides. The molecular weight of the mature enzyme is calculated to be 78,511 from the predicted amino acid sequence. The enzyme has no terminal peptide extension as a signal for translocation into peroxisomes.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1985

Induction of peroxisomal beta-oxidation enzymes in primary cultured rat hepatocytes by clofibric acid.

Rat hepatocytes were cultured for 72 h with or without the addition of 0.5 mM clofibric acid. The activities of individual enzymes of the peroxisomal beta-oxidation pathway (acyl-CoA oxidase, enoyl-CoA hydratase-3-hydroxyacyl-CoA dehydrogenase bifunctional protein, and 3-ketoacyl-CoA thiolase) decreased in the control culture, but markedly increased synchronously in the clofibric acid-treated culture. The levels of mRNAs coding for these enzymes and the rates of synthesis of the enzymes were also elevated in the clofibric acid-treated culture, although no proportional relationship was observed between the time-dependent changes of these parameters. The increase in mRNAs was much higher than the increase in the rate of synthesis of the enzymes. The activity of catalase, its mRNA level and the rate of its synthesis were slightly affected. The effects of clofibric acid on the peroxisomal beta-oxidation enzymes and catalase in primary cultured hepatocytes were very similar to those observed in vivo. These results, therefore, suggest that primary culture of hepatocytes should provide a useful means for investigating the mechanism of induction of peroxisomal enzymes and the mechanism of action of peroxisome proliferators.

3-Hydroxyacyl CoA Dehydrogenases↗

PubMed1985

Physico-chemical properties and stability of cilostazol.

The physico-chemical properties of cilostazol (6-[4-(1-cyclohexyl-1H-tetrazol-5-yl)butoxy]-3,4-dihydro-2(1H)-qui nolinone, OPC-13013), a new potential antithrombotic and vasodilating drug, were studied by clarifying its elemental composition, melting point, spectra (UV, IR, NMR and mass), X-ray diffraction pattern, thermal properties, solubilities and partition coefficient. Analytical test methods such as HPLC and TLC were established for use in stability tests, and the stability study of cilostazol in an aqueous solution (acid and base) and in a solid form was carried out. Cilostazol was shown to be stable with no changes from the initial values.

Azoles↗

PubMed1984

Biosynthesis of enzymes of rat-liver mitochondrial beta-oxidation.

The biogenesis of seven enzymes involved in the mitochondrial fatty acid beta-oxidation of rat liver was studied. Hepatic RNA was translated in vitro in a rabbit reticulocyte lysate cell-free system and the translation products were immunoprecipitated, subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by fluorography. The translation products obtained in vitro of medium-chain and/or long-chain acyl-CoA dehydrogenase (these enzymes were immunochemically cross-reactive), enoyl-CoA hydratase, 3-hydroxyacyl-CoA dehydrogenase, and acetoacetyl-CoA thiolase and probably also short-chain acyl-CoA dehydrogenase were larger than the subunits of the corresponding mature enzymes by 2-4.5 kDa, whereas the 3-oxoacyl-CoA thiolase obtained in vitro was approximately the same size as the mature subunit. The free polysome fraction of rat liver was 4.3-9.0-times more active than the membrane-bound polysome fraction in the synthesis of these seven enzymes. The enzyme activities were increased after administration of di(2-ethylhexyl)phthalate; the extent of the increase varied from one enzyme to another. The increase in the cell-free translation activity of total hepatic RNA for these enzymes after administration of the chemical was markedly different among individual enzymes and higher than that in the rates of synthesis of the corresponding enzymes which were determined by the experiment in vivo.

Animals↗

PubMed1984

Molecular cloning of cDNA for rat liver catalase.

For the studies on the induction of peroxisomal enzymes by hypolipidemic agents, we have tried to isolate a cDNA clone for rat liver catalase. A recombinant clone, pMJ501, was isolated, of which cDNA insert specifically hybridized to catalase mRNA in hybridization-selected translation. On RNA blot hybridization, it hybridized to 2.4-kilobases RNA which was increased about 1.5-fold by the administration of di-(2-ethylhexyl)phthalate to the rats. The nucleotide sequence of the cDNA contains a reading frame for 109 amino acid residues which match the reported amino acid sequence of bovine liver catalase at the carboxyl end with 82% homology. It is concluded that pMJ501 contains a cDNA sequence for rat liver catalase.

Amino Acid Sequence↗