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M Takiguchi

Publications and source records attributed to M Takiguchi.

At least 289 records · Page 16Linked to original sources
PubMed1984

Cell-free synthesis of ornithine decarboxylase. Changes in mRNA activity in the liver of thioacetamide-treated rats.

Ornithine decarboxylase (ODC)mRNA associated with free polysomes of rat liver was translated in a reticulocyte lysate cell-free system. Newly synthesized ODC protein was identified by specific immunoprecipitation, molecular size as determined by polyacrylamide gel electrophoresis with sodium dodecyl sulfate, and competition by excess unlabeled ODC in the immunoprecipitation. A single injection of thioacetamide was found to cause several fold increases in both immunotitratable ODC protein and polysomal ODC-mRNA activity, while it provoked a much larger increase in ODC activity in rat liver. The results indicate that the induction of hepatic ODC activity by thioacetamide treatment is due not only to an increase in the activity of polysomal ODC-mRNA but also to a translational and/or posttranslational control.

Acetamides↗
PubMed1984

Biosynthesis and intracellular transport of enzymes of peroxisomal beta-oxidation.

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.

3-Hydroxyacyl CoA Dehydrogenases↗
PubMed1984

Molecular cloning and nucleotide sequence of cDNA for rat ornithine carbamoyltransferase precursor.

Messenger RNA of rat ornithine carbamoyltransferase (EC 2.1.3.3), a mitochondrial matrix enzyme, was enriched by immunoprecipitation of rat liver free polysomes, and recombinant plasmids were prepared from the enriched mRNA by a vector-primer method. The cDNA clones for ornithine carbamoyltransferase were identified by hybrid-arrested translation and hybrid-selected translation. One of the clones, designated pOTC-1, contained a 1.6-kilobase insert and hybridized to a mRNA of approximately equal to 1.8 kilobases in rat liver. The cDNA clone was subjected to nucleotide sequence analysis. The deduced amino acid sequence indicates that the ornithine carbamoyltransferase precursor consists of the mature enzyme of 322 amino acid residues and an NH2-terminal peptide extension (presequence) of 32 amino acid residues. The presequence contains 8 basic amino acid residues, no acidic residues, and no hydrophobic amino acid stretch. The amino acid sequence of the rat ornithine carbamoyltransferase was compared with the recently reported sequence of the human enzyme [Horwich, A. L., Fenton, W. A., Williams, K. R., Kalousek, F., Kraus, J. P., Doolittle, R. F., Konigsberg, W. & Rosenberg, L. E. (1984) Science 224, 1068-1074]. The sequences of the mature enzyme portion are 93% identical, whereas those of the presequences are 69% identical. There are two highly conserved segments in the presequences of the rat and human enzymes. One of the two conserved segments is significantly similar to a segment of the presequence of yeast mitochondrial elongation factor EF-Tu. These results suggest that the homologous segments are important for the proteins that are synthesized in the cytosol to be transported into the mitochondrial matrix.

Amino Acid Sequence↗
PubMed1984

Immunological studies on Kawasaki disease. I. Appearance of Hanganutziu-Deicher antibodies.

Sera of patients with Kawasaki disease were studied for heterophile antibodies by means of enzyme immunoassay (EIA) with enzyme conjugated antisera to human IgM, IgG, IgA and IgE. Antibodies of IgM (43%), IgG (3%), IgA (11%) and IgE (49%) classes were demonstrated that combined with high molecular weight glycoprotein (HMWGP) of bovine red blood cells (BRBC) one of the antigenic preparations of the Hanganutziu-Deicher (H-D) heterophile system. Studies on sequential sera of the patients revealed that HMWGP antibodies of IgM and IgE classes began to appear in the second week, reached their peaks in the third week of the disease and declined gradually thereafter. Absorption studies on the positive sera showed that the HMWGP antibody activities were abolished by BRBC, sheep red blood cells and guinea-pig kidney tissues, confirming H-D specificity of these antibodies. EIA inhibition studies showed that the antibody activity was inhibited by HMWGP and partially by asialo-HMWGP and NGNA ganglioside rich preparation of BRBC but not by purified Paul-Bunnell or Forssman antigens. These results indicate that the H-D antibodies under investigation consist of antibodies of two different specificities; one directed against asialo-HMWGP and the other NGNA ganglioside of BRBC. Circulating immune complexes (IC) were demonstrated in 23% of the patients by means of anti-antibody inhibition test. Evidence was presented that IC in the sera of five patients were composed of H-D (HMWGP) antigen and its corresponding antibodies.

Antibodies, Heterophile↗
PubMed1983

Synthesis, intracellular transport and processing of mitochondrial urea cycle enzymes.

Carbamyl phosphate synthetase I and ornithine transcarbamylase are matrix enzymes synthesized outside the mitochondria in the form of larger precursors and are transported rapidly into mitochondria, in association with post-translational proteolytic processing to the mature enzymes. Treatment of isolated rat hepatocytes with 40 micrograms/ml of rhodamine 123 resulted both in a potent inhibition of the processing of the enzyme precursors and in accumulation of the precursors. In pulse-chase experiments, the labeled precursor disappeared much more slowly in the presence of the dye. Rhodamine 123 strongly inhibited the uptake and processing of the ornithine transcarbamylase precursor by isolated rat liver mitochondria. Other positively charged rhodamines such as rhodamines 6G and 6GX were also strongly inhibitory. On the other hand, rhodamine B which has no net charge was much less inhibitory. These results suggest that the positively charged rhodamines inhibit the binding of the positively charged enzyme precursors to a negatively charged protein(s) or to phospholipids of the mitochondrial outer membrane. Potassium and magnesium ions, and probably a cytosolic protein(s), were required for the maximal uptake and processing of the ornithine transcarbamylase precursor by the isolated mitochondria. The concentrations of potassium and magnesium ions required for the maximal transport and processing were about 120 mM and 0.8-1.6 mM, respectively. Dialyzed postribosomal supernatant of rabbit reticulocyte lysate (36-72 mg protein/ml), in combination with potassium and magnesium ions, stimulated the precursor transport and processing 3- to 4-fold. The stimulatory activity of the dialyzed lysate was inactivated by trypsin treatment or heat treatment. No significant amount of the enzyme precursor was associated with the mitochondria when incubation was performed in the absence of these compounds. All these results indicate that potassium and magnesium ions, and probably a cytosolic protein(s), are required for the binding of the ornithine transcarbamylase precursor to the mitochondria or its transport into the organelle.

Animals↗
PubMed1983

Transport of proteins into mitochondria: a high conservation of precursor uptake and processing system.

Ornithine transcarbamylase (EC 2.1.3.3) of rat (Rattus norvegicus var. albus) liver, a urea cycle enzyme, is synthesized extramitochondrially as a larger precursor which is transported posttranslationally into mitochondria and processed to the mature enzyme. The precursor synthesized in vitro was taken up and processed to the mature enzyme by isolated pigeon (Columba livia var. domestica) liver and frog (Rana catesbeiana) liver mitochondria. Carp (Cyprinus carpio) liver mitochondria could also process the precursor. These results indicate that the mitochondrial transport and processing activities are conserved between mammalian and bird, amphibian or fish systems. However, attempts to demonstrate the precursor uptake and processing by Saccharomyces cerevisiae mitochondria were unsuccessful.

Animals↗
PubMed1982

Ornithine transcarbamylase in liver mitochondria.

Ornithine transcarbamylase (ornithine carbamoyltransferase, EC 2.1.3.3), the second enzyme of urea synthesis, is localized in the matrix of liver mitochondria of ureotelic animals. The enzyme is encoded by a nuclear gene, synthesized outside the mitochondria, and must then be transported into the organelle. The rat liver enzyme is initially synthesized on membrane-free polysomes in the form of a larger precursor with an amino-terminal extension of 3 400-4 000 daltons. In rat liver slices and isolated rat hepatocytes, the pulse-labeled precursor is first released into the cytosol and is then transported with a half life of 1-2 min into the mitochondria where it is proteolytically processed to the mature form of the enzyme. The precursor synthesized in vitro exists in a highly aggregated form and has a conformation different from that of the mature enzyme. The precursor has an isoelectric point (pI = 7.9) higher than that of the mature enzyme (pI = 7.2). The precursor synthesized in vitro can be taken up and processed to the mature enzyme by isolated rat liver mitochondria. The mitochondrial transport and processing system requires membrane potential and a high integrity of the mitochondria. The transport and processing activities are conserved between mammals and birds or amphibians and is presumably common to more than one precursor. Potassium ion, magnesium ion, and probably a cytosolic protein(s), in addition to the transcarbamylase precursor and the mitochondria, are required for the maximal transport and processing of the precursor. A mitochondrial matrix protease which converts the precursor to a product intermediate in size between the precursor and the mature subunit has been highly purified. The protease has an estimated molecular weight of 108 000 and an optimal pH of 7.5-8.0, and appears to be a metal protease. The protease does not cleave several of the protein and peptide substrates tested. The role of this protease in the precursor processing remains to be elucidated. Rats subjected to different levels of protein intake and to fasting show significant changes in the level of enzyme protein and activity of ornithine transcarbamylase. The dietary-dependent changes in the enzyme level are due mainly to an altered level of functional mRNA for the enzyme. In contrast, during fasting, the increase in the enzyme level is associated with a decreased level of translatable mRNA for the enzyme. Pathological aspects of ornithine transcarbamylase including the enzyme deficiency and reduced activities of the enzyme in Reye's syndrome are also described. A possibility that impaired transport of the enzyme precursor into the mitochondria leads to a reduced enzyme activity, is proposed.

Animals↗