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Biomedical subjects

T Osumi

Publications and source records attributed to T Osumi.

At least 91 records · Page 5Linked to original sources
PubMed1991

Evidence for a structural mutation (347Ala to Thr) in a German family with 3-ketothiolase deficiency.

The molecular basis of 3-ketothiolase deficiency (3KTD) was examined in a 3KTD family. Immunochemical analyses showed that mitochondrial acetoacetyl-CoA thiolase (T2) biosynthesized in the patient's fibroblasts (GK06) was unstable and that the parents and brother were obligatory carriers of 3KTD. When sequencing the PCR-amplified patient's T2 cDNA, we noted a G to A replacement which caused 347Ala to Thr substitution of the mature T2 subunit. Transfection analysis revealed that this substitution resulted in an instability of the T2 protein. Analyses of the T2 cDNA and gene of the family indicated that the patient was a compound heterozygote; the allele that derived from the mother had a point mutation (347Ala to Thr) and the other allele from the father has a mutation which would abolish the T2 gene expression. This report is apparently the first definition of a mutant allele for 3KTD, at the gene level.

Acetyl-CoA C-Acyltransferase↗
PubMed1991

Two cis-acting regulatory sequences in the peroxisome proliferator-responsive enhancer region of rat acyl-CoA oxidase gene.

To clarify the mechanism of transcriptional induction of the rat liver acyl-CoA oxidase gene by hypolipidemic agents, we searched for cis-acting regulatory sequences in the 5' upstream region of the gene by transfection studies. We found that the sequence between -639 and -472 acts as a peroxisome proliferator-responsive, tissue-specific enhancer. Footprint analysis revealed two protein binding sites in this region. One of these sequences exhibited a positive, whereas the other a negative, regulatory activity in transcription assays.

Acyl-CoA Oxidase↗
PubMed1990

Molecular cloning, sequencing, and expression of mouse ferrochelatase.

The cDNA encoding mouse ferrochelatase (protoheme ferrolyase, EC 4.99.1.1) was isolated from a mouse erythroleukemia (MEL) cell cDNA library in lambda gt11 expression vector, by immunoscreening with a polyclonal antibody. Two full-length clones containing cDNA inserts of 2.2 and 2.90 kilobases were obtained. These clones have the same entire enzyme coding region, but alternative putative polyadenylation sites in the 3'-noncoding regions. From the deduced primary structure, a putative leader sequence of 53 amino acid residues resulted in a precursor protein of 420 amino acid residues (Mr 47,130) and a mature protein of 367 residues (Mr 41,692). The cDNA allows for the expression of active ferrochelatase by transfected culture cells. RNA blot analysis showed two species of ferrochelatase mRNA consistent with findings of two polyadenylation sites. Both the mRNAs increased by treatment of the MEL cells with dimethyl sulfoxide. The band pattern of the RNA of the mouse liver was the same as that of the MEL cells. Based on these results, we deduce that ferrochelatase in erythroid and hepatic cells can be only of one type.

Amino Acid Sequence↗
PubMed1990

cDNA cloning of rat liver 2,4-dienoyl-CoA reductase.

cDNA clones of 2,4-dienoyl-CoA reductase were isolated from rat liver cDNA libraries constructed in phages lambda gt11 and lambda gt10. Hybrid selected translation analysis revealed that 2,4-dienoyl-CoA reductase was translated as a polypeptide with a molecular weight of about 36,000, which was about 3,000 molecular weight units larger than mature reductase. Sequencing analysis revealed that the open reading frame encoded a polypeptide consisting of 335 amino acid residues (predicted molecular weight = 36,132), which contained an N-terminal extension peptide of 34 amino acid residues (presequence) in addition to the mature enzyme. Thus, 2,4-dienoyl-CoA reductase is synthesized as a larger precursor polypeptide, and the N-terminal extension peptide may be acting as the mitochondrial import signal.

Amino Acid Sequence↗
PubMed1990

Human c-erb A protein expressed in Escherichia coli: changes in hydrophobicity upon thyroid hormone binding.

The human c-erb A beta gene sequence was inserted in an Escherichia coli expression vector plasmid. The E. coli cells transformed with this plasmid produced proteins with molecular masses of 52 and 50 kDa. These products bound 3,5,3'-triiodo-L-thyronine (T3) with an affinity constant of 4.3 x 10(9) liter/mol. The order of affinity for iodothyronine analogs was triiodothyroacetic acid greater than T3 greater than 3,5,3'-triiodo-D-thyronine greater than L-thyroxine. Affinity labeling experiments showed that the 50 kDa protein was covalently labeled with [125I]T3, and this was competed by triiodothyroacetic acid, T3, and L-thyroxine (from potent to weaker competitor). The c-erb A protein bound to calf thymus DNA-cellulose and the binding was inhibited by 0.3 M KCl or 10 mM pyridoxal 5'-phosphate. Aqueous two-phase partitioning studies showed that the c-erb A product became less hydrophobic upon T3 or triiodothyroacetic acid binding. The same finding was obtained when T3 bound to partially purified rat liver nuclear thyroid hormone receptor. However, thyroxine binding globulin became more hydrophobic upon T3 binding. Since the T3 molecule partitioned preferentially into the upper polyethylene glycol-rich phase, the alteration of partitioning behavior of thyroxine binding globulin was explained by a simple additive effect of T3. In contrast, the alteration of partitioning behavior of the c-erb A product or receptor reflected a conformational transition upon T3 binding. The c-erb A protein expressed in E. coli showed various characteristics similar to classical thyroid hormone receptor and may be useful in studying the structure and function of the thyroid hormone receptor.

Base Sequence↗
PubMed1990

The 70-kDa peroxisomal membrane protein is a member of the Mdr (P-glycoprotein)-related ATP-binding protein superfamily.

The 70-kDa peroxisomal membrane protein (PMP70) is one of the major integral membrane proteins of rat liver peroxisomes. cDNA clones for PMP70 were isolated and sequenced. The predicted amino acid sequence (659 amino acid residues) revealed that the carboxyl-terminal region of PMP70 has strong sequence similarities to a group of ATP-binding proteins such as MalK and Mdr. These proteins form a superfamily and are involved in various biological processes including membrane transport. Limited protease treatment of peroxisomes showed that the ATP-binding domain of PMP70 is exposed to the cytosol. The hydropathy profile, in comparison with those of several other members of the ATP-binding protein superfamily, suggests that PMP70 is a transmembrane protein possibly forming a channel. Based on these results, we propose that PMP70 is involved in active transport across the peroxisomal membrane.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

Rat peroxisomal 3-ketoacyl-CoA thiolase gene. Occurrence of two closely related but differentially regulated genes.

We isolated and analyzed the genes for rat peroxisomal 3-ketoacyl-CoA thiolase. Two active genes (termed A and B), both likely to be coding for the peroxisomal thiolase, were identified per haploid rat genome. These genes span approximately 9 and 11 kilobases, respectively, and both contain 12 exons and 11 introns, with overall similar organizations. The nucleotide sequences are highly conserved in the coding regions and in many introns. The A and B genes code for slightly different amino acid sequences, and the known cDNA sequence (Hijikata, M., Ishii, N., Kagamiyama, H., Osumi, T., and Hashimoto, T. (1987) J. Biol. Chem. 262, 8151-8158) corresponds to the B gene product. By in vitro transcription/translation, the A gene was shown to produce a thiolase precursor having a 10-residue longer amino-terminal presequence than that of the B gene product. Primer extension analysis revealed that the A gene is constitutively expressed and is not influenced by the administration of a peroxisome proliferator, whereas the B gene is markedly activated by the same treatment. Several common sequences were noted in the 5'-flanking regions of the B gene and in the genes of two other peroxisomal beta-oxidation enzymes, induced in parallel by peroxisome proliferators.

Acetyl-CoA C-Acyltransferase↗
PubMed1990

Molecular analysis of human acatalasemia. Identification of a splicing mutation.

To search for the molecular defect of Japanese-type acatalasemia, we cloned the mutant catalase gene from a person with this deficiency. The nucleotide sequence of the mutant gene was determined for all exons, exon/intron junctions, and 5' and 3' flanking regions, and the findings were compared with the sequence from the normal gene. Seven base differences were found between the two genes. Among them, a G to A substitution at the fifth position of intron 4 (a splicing mutation) seemed most likely to be responsible for the defective catalase synthesis in the subject. To obtain suggestive evidence, we constructed chimeric genes that contained a segment of either the normal or mutant catalase gene, encompassing a 3' part of exon 4, the entire intron 4 and a 5' portion of exon 5, within the third exon of the human alpha-globin gene. When this chimeric gene construct was introduced into simian virus 40-transformed simian cells (COS-7), the transcript of the normal catalase/alpha-globin chimeric gene was spliced correctly, as revealed by Northern blotting and RNase mapping techniques. In contrast, the splicing of the mutant chimeric pre-mRNA occurred between the 5' donor site of the preceding intron and the 3' acceptor site of the intron containing the substitution, thereby skipping one entire exon sequence. Thus, the G to A transition at the fifth position of intron 4 of the catalase gene indeed severely limits the correct splicing of the RNA product. The same splice site mutation was found in the genomic DNA of another acatalasemic individual from an unrelated family. We suggest that this base substitution is the causal mutation of these cases of Japanese-type acatalasemia.

Acatalasia↗
PubMed1990

Topogenesis of peroxisomal proteins.

Molecular and biochemical analysis of the biogenesis of peroxisomes has made rapid progress in recent years. Research on the mechanism of targeting of peroxisomal proteins has revealed that many, but not all, peroxisomal proteins have a conserved tripeptide motif in their carboxy-terminal portions which is required for entry into peroxisomes; the topogenic signal mechanism thus differs in these instances from those employed in mitochondria and endoplasmic reticulum. Other factors involved in peroxisome biogenesis are also coming to light.

Amino Acid Sequence↗
PubMed1990

Proliferation of peroxisomes and induction of peroxisomal beta-oxidation enzymes in rat hepatoma H4IIEC3 by ciprofibrate.

For the analysis of the molecular mechanism of the action of peroxisome proliferators, we attempted to establish the optimal conditions for obtaining the effects of the chemicals in vitro, employing an established cell line, Reuber rat hepatoma H4IIEC3. Histochemical analyses revealed a marked increase in the number, size, and catalase content of peroxisomes in the cells cultured on a medium containing 0.5 mM ciprofibrate, a peroxisome proliferator. The activity of acyl-CoA oxidase, the initial enzyme of the peroxisomal beta-oxidation system, was increased by more than 10-fold by the same treatment. Catalase was also induced significantly, whereas the activities of glutamate dehydrogenase and lactate dehydrogenase, mitochondrial and cytosolic marker enzymes, did not change upon the treatment. Immunoblotting and RNA-blotting analyses confirmed the increases in the amount of protein and mRNA for all the three enzymes of the peroxisomal beta-oxidation system. Cell fractionation experiments gave a partial separation of peroxisomes from other organelles for the induced culture. Thus, H4IIEC3 cells offer a good in vitro model system of the induction of peroxisomes and peroxisomal beta-oxidation enzymes by peroxisome proliferators.

Animals↗
PubMed1990

Molecular cloning and sequence of the complementary DNA encoding human mitochondrial acetoacetyl-coenzyme A thiolase and study of the variant enzymes in cultured fibroblasts from patients with 3-ketothiolase deficiency.

Complementary DNAs encoding the precursor of human hepatic mitochondrial acetoacetyl-CoA thiolase (T2) (EC 2.3.1.9) were cloned and sequenced. The cDNA inserts in these clones were 1,518 bases in length when overlapped, and encoded the 427-amino acid precursor of this enzyme (45,199 mol wt). This amino acid sequence included a 33-residue leader peptide moiety and a 394-amino acid subunit of the mature enzyme (41,385 mol wt). The T2 gene expression in fibroblasts from four patients with 3-ketothiolase deficiency was analyzed by Northern blotting. The T2 mRNA in all four cell lines had the same 1.7 kb as that of the control. However, the amounts of T2 mRNA differed: the content was reduced in two cell lines (cases 1 and 3), whereas it was within a normal range in others (cases 2 and 4). Pulse labeling followed by subcellular fractionation revealed that the T2 proteins in the fibroblasts from these patients are present in the mitochondria. These results suggest that different mechanisms are involved in the enzyme defects in the four patients.

Acetyl-CoA C-Acetyltransferase↗
PubMed1989

Molecular cloning and sequence analysis of the cDNA for rat mitochondrial enoyl-CoA hydratase. Structural and evolutionary relationships linked to the bifunctional enzyme of the peroxisomal beta-oxidation system.

To elucidate structural relationships between the mitochondrial and peroxisomal isozymes of beta-oxidation systems, cDNA of the mitochondrial enoyl-CoA hydratase was cloned and sequenced. The 1454-bp cDNA sequence contained a 870 bp of open reading frame, encoding a polypeptide of 290 amino acid residues. When compared with the amino-terminal sequence of the mature enzyme, the predicted sequence contained a 29-residue presequence at the amino terminus. This presequence had characteristics typical of a mitochondrial signal peptide. The primary structure of this enzyme showed significant similarity with the amino-terminal portion of sequence of the peroxisomal enoyl-CoA hydratase: 3-hydroxyacyl-CoA dehydrogenase bifunctional enzyme. The carboxy-terminal part of the latter enzyme has sequence similarity with mitochondrial 3-hydroxyacyl-CoA dehydrogenase [Ishii, N., Hijikata, M., Osumi, T. & Hashimoto, T. (1987) J. Biol. Chem. 262, 8144-8150]. These findings suggest that the peroxisomal bifunctional enzyme has the hydratase and dehydrogenase functions on the amino- and carboxy-terminal sides, respectively. The mitochondrial beta-oxidation enzymes and the peroxisomal bifunctional enzyme may have common evolutionary origins.

Amino Acid Sequence↗