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Biomedical subjects

T Fukui

Publications and source records attributed to T Fukui.

At least 397 records · Page 22Linked to original sources
PubMed1986

Amino acid sequence of mammalian elongation factor 2 deduced from the cDNA sequence: homology with GTP-binding proteins.

Complementary DNA clones, pHEW1 and pRE2, coding for hamster and rat polypeptide chain elongation factor 2 (EF-2), respectively, were isolated and sequenced. It was shown that the cDNA insert in pHEW1 contains a 2574-base-pair open reading frame coding for an 857-amino acid polypeptide with Mr 95,192, excluding the initiation methionine. Comparative studies of sequence homology among EF-2 and several GTP-binding proteins show that five regions in the amino-terminal position of EF-2, corresponding to about 160 amino acids, show homology with GTP-binding proteins, including protein synthesis elongation and initiation factors, mammalian ras proteins, and transducin. The carboxyl-terminal half of EF-2 contains several regions that have 34-75% homology with bacterial elongation factor G. These results suggest that the amino-terminal region of EF-2 participates in the GTP-binding and GTPase activity whereas the carboxyl-terminal region interacts with ribosomes. Finally, the sequence provides direct evidence that diphthamide (2-[3-carboxy-amido-3-(trimethylammonio)propyl]histidine), the site of ADP-ribosylation by diphtheria toxin, is produced by post-translational modification of a histidine residue in the primary translational product.

Amino Acid Sequence↗
PubMed1986

Inactivation of alcohol dehydrogenase in rice seedlings is related to a microsomal fatty acid alpha-oxidation system.

The selective inactivation of alcohol dehydrogenase by the inactivator found in the microsomal fraction of rice (Oryza sativa) seedlings growing in air (Shimomura, S. & Beevers, H. (1983) Plant Physiol. 71, 736-741; 742-746) was further studied. This inactivation was found to be essentially dependent on the presence of free fatty acids. The specificity for fatty acids and the inhibitory effects of imidazole, 2-hydroxyfatty acids and dithiothreitol on the inactivation were all consistent with the properties of the fatty acid alpha-oxidation system in plants. Both O2 consumption and decarboxylation of fatty acid due to alpha-oxidation were also demonstrated in rice microsomes. When purified rice alcohol dehydrogenase was added to the alpha-oxidation system in rice microsomes, the decarboxylation of fatty acid was inhibited, and the cysteinyl residues of alcohol dehydrogenase were oxidized. The oxidation of two cysteinyl residues per monomer resulted in the complete inactivation of the enzyme. The activity of the inactivator in the isolated microsomes was gradually lost during storage and was rapidly lost upon heating. The inactivation of alcohol dehydrogenase was observed even when the enzyme was separated from microsomes by a dialysis membrane. These results indicate that the inactivation of alcohol dehydrogenase is closely related to fatty acid alpha-oxidation. We postulate that an intermediate of alpha-oxidation is the inactivator.

Alcohol Dehydrogenase↗
PubMed1986

Purification and properties of an auxin-binding protein from maize shoot membranes.

An auxin-binding protein was purified from membranes of maize shoots including the coleoptiles, leaf rolls and mesocotyls. The method of Ca2+-promoted sedimentation of membrane particles was adopted for large-scale preparation. The auxin-binding protein was solubilized from the acetone-washed membranes, and purified by successive chromatographies on DEAE-Sephacel, 1-naphthylacetic acid-linked AH-Sepharose 4B, and Sephadex G-100 columns. The yield of the purified protein was about 0.2 mg from 1 kg of shoots. The binding protein exists as a dimer with a subunit molecular weight of 21,000, and possesses one auxin-binding site per dimer. The preparation also contains a minor molecular form with a subunit molecular weight of 20,000. The auxin-binding protein is not a hydrophobic protein, as judged from its amino acid composition and solubility. The circular dichroic (CD) spectrum of the binding protein resembles the spectrum anticipated from the beta-structures, and shows no alpha-helix characteristic in the secondary structure. The CD spectral changes induced on the binding of auxin and its antagonist seem to be related to the receptor function. The affinity of the binding protein for auxin is dependent on pH, with an optimum at pH 5.0, while the binding protein is unstable below pH 6. We discuss here the intracellular localization of the auxin-binding protein from the view point of the controversial pH-dependence of the binding affinity and stability.

Amino Acids↗
PubMed1985

Cloning and characterization of the cDNAs for human and rabbit interleukin-1 precursor.

DNA sequence complementary to the mRNA for rabbit interleukin-1 precursor (preIL-1) has been cloned from the cDNA library constructed using partially purified poly(A)+RNA from induced rabbit alveolar macrophages by mRNA hybridization-translation assay. By using this cDNA as a probe, human IL-1 cDNA was isolated from the cDNA library prepared using poly(A)+RNA from induced HL-60 cells, a human monocyte-like cell line. The amino acid sequences of the human and rabbit preIL-1 deduced from the cDNA sequences reveal their primary structures which consists of 271 and 267 amino acid residues, respectively. The amino acid sequence is 64% conserved between human and rabbit. The difference in number of amino acid residues results from the carboxy-terminal extention of 4 amino acid residues in human preIL-1. Expression of the cloned human cDNA in E. coli yielded biologically active IL-1.

Amino Acid Sequence↗
PubMed1985

A new affinity labeling reagent for the active site of glycogen synthase. Uridine diphosphopyridoxal.

A new affinity labeling reagent for glycogen synthase a from rabbit muscle, uridine diphosphopyridoxal, has been prepared. Incubation of the enzyme with this reagent resulted in a time-dependent, almost complete loss of activity. The inactivation was pseudo-first order, and the results of the kinetic analysis suggested the formation of a noncovalent enzyme-reagent complex prior to the covalent reaction, with a Kinact of 25 microM and a maximal rate constant of 0.22 min-1. The inactivation was pronouncedly protected by UDP-Glc and UDP, but not by the allosteric activator glucose 6-phosphate. The increase in a spectral peak at 425 nm and the decrease in enzymatic activity were well correlated, suggesting that the reagent causes the inactivation of the enzyme by the formation of a Schiff base. The rate of inactivation increased as the pH was raised, giving a pK of 8.85. Almost all the original activity was recovered by the treatment of the inactivated enzyme with cysteamine or any other aminothiol compound. No recovery of the activity, however, was observed with inactivated enzyme which had been treated with NaBH4. A peptide containing the labeled amino acid was isolated for inactivated enzyme after reduction with NaBH4, carboxymethylation, and chymotryptic digestion by fractionation on a Bio-Gel P-6 column and high performance liquid chromatographies. Manual Edman degradation established the sequence as Glu-Val-Ala-Asn-labeled Lys-Val-Gly-Gly-Ile-(Tyr). The introduction of an active site-directing moiety to pyridoxal 5'-phosphate makes the resultant reagent an effective probe for the active site of glycogen synthase.

Affinity Labels↗
PubMed1985

Purification and properties of extracellular poly(3-hydroxybutyrate) depolymerases from Pseudomonas lemoignei.

Extracellular poly(3-hydroxybutyrate) depolymerase was purified from the culture medium of Peudomonas lemoignei and separated into four isozymes (A1, A2, B1 and B2) by CM-Sepharose CL-6B chromatography. The molecular weights of A1 and A2 and those of B1 and B2 were estimated to be 54 000 and 58 000, respectively, by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The isoelectric points of A1, A2, B1 and B2 were found to be approximately pH 9.7, 10.0, 10.0 and 10.6, respectively, by isoelectric focusing. All four enzymes hydrolyzed poly(3-hydroxybutyrate) and oligomeric esters of D-(-)-3-hydroxybutyrate, but showed no activity toward the dimeric ester. Analysis of hydrolytic products of the oligomeric esters with A1 and B2 suggested that the enzymes hydrolyzed mainly the second and third ester bonds from the free hydroxy terminus at different frequencies, depending upon the chain length of the substrates.

Carboxylic Ester Hydrolases↗
PubMed1985

Cloning of the Escherichia coli gene for the stringent starvation protein.

In order to clone the Escherichia coli gene for the stringent starvation protein (SSP), we determined its N-terminal sequence as well as the sequence of two peptide fragments obtained by cyanogen bromide cleavage of the protein. We then chemically synthesized four sets of oligodeoxyribonucleotide mixtures that represented possible codon combinations for parts of these amino acid sequences. The synthetic oligonucleotides were labelled with 32P at their 5'-termini and used as hybridization probes to detect DNA fragments containing the complementary sequences. Genomic Southern hybridization of E. coli chromosomal DNA gave up to ten DNA fragments hybridizing with each probe but only a few hybridized with two or more of the probes. The latter fragments were cloned in pBR322. By determining partial base sequences with a rapid method and examining proteins encoded by the DNA fragments, we were able to show that we had isolated a clone containing the complete SSP structural gene.

Amino Acid Sequence↗
PubMed1985

Effects of histamine-2 receptor blockade on lidocaine kinetics.

The hypothesis that the H2-receptor blockers cimetidine and ranitidine have different effects on the disposition of lidocaine, a microsomally metabolized drug dependent on hepatic blood flow for elimination, was tested. Six normal men received lidocaine infusions (2 mg/kg over 10 minutes) and lidocaine levels were determined by HPLC. Lidocaine kinetics were studied in the untreated state (O) and in a double-blind, double-dummy design after 2 days of placebo (P), cimetidine (C, 300 mg every 6 hours by mouth), or ranitidine (R, 160 mg every 12 hours by mouth). Model-independent kinetics were estimated by the statistical moment theory. The steady-state volume of distribution was lower after cimetidine (means +/- SD: O, 156 +/- 39 L; P, 156 +/- 48 L; C, 123 +/- 20 L; and R, 174 +/- 38 L). A trend toward decreased lidocaine clearance after cimetidine was also noted (O, 1011 +/- 140 ml/min; P, 1087 +/- 227 ml/min; C, 886 +/- 214 ml/min; and R, 1143 +/- 225 ml/min). Elimination rate constants were of the same order in all four treatments. Only higher levels of alpha 1-acid glycoprotein appeared to limit the lidocaine steady-state volume of distribution. Cimetidine and ranitidine have distinctly different effects on lidocaine kinetics in normal subjects. The absence of ranitidine effects on the disposition of lidocaine, a high-extraction, high-clearance drug, suggests that H2-receptor blockade may not decrease hepatic blood flow, and that cimetidine impairs drug elimination only by inhibition of hepatic microsomal enzymes. Such interactions are not likely to occur with ranitidine.

Administration, Oral↗
PubMed1985

Efficient production of a human tumour necrosis factor in Escherichia coli.

To examine the effect of altering the nucleotide sequence of the Shine-Dalgarno (SD) region and the spacer sequence between the SD sequence and the AUG translation start signal, several plasmids were constructed which directed the synthesis of mature human tumour necrosis factor (TNF) under the control of the E. coli trp leader promoter. We found that the presence of the SD sequence, AAGGAGGT, which is complementary to the 3' end of 16S rRNA, gave the higher translational efficiency, and also, the presence of the spacer sequence which consists of only A and T residues raised the production of TNF 2-3 fold. The levels of expression of TNF were elevated over 20-fold by the alteration of the SD and spacer sequences and amounted to 20% of the total cellular proteins or approx. 5 X 10(6) molecules of TNF per cell.

Base Sequence↗
PubMed1985

A silica-based reversed-phase column for single-stranded oligodeoxyribonucleotides.

A novel, silica-based, reversed-phase column targeted to the separation of single-stranded oligodeoxyribonucleotides has been developed by grafting monochlorooctadecylsilane onto silica which is a base material of reversed-phase columns (TSK 120A and 120T), in which polychlorosilane is used as a grafting reagent. By applying a shallow gradient elution of an aqueous acetonitrile solution containing 0.1 M ammonium acetate, chromatography with this column produced well-resolved peaks for large samples such as those having bases up to 26.

Chromatography, High Pressure Liquid↗
PubMed1985

[Physical problems as to standardization and optimization of radiation therapy].

In order to systemize and standardize radiation therapy it is emergent and important to organize radiation therapy in wide regions, which precedes physical, biological and clinical problems. By the way in this paper practical problems, of dose distributions and/or present state of a few radiotherapists--this controls accuracy of radiotherapy--were reported: dose distribution of connection of various field, irregular or uneven field, unhomogeneous tissue and target volume in boost therapy.

Humans↗
PubMed1984

Molecular cloning and the complete nucleotide sequence of cDNA to mRNA for S-100 protein of rat brain.

The complete nucleotide sequence of mRNA for beta-subunit of rat brain S-100 protein was determined from recombinant cDNA clones. The sequence was composed of 1488 bp which included the 276 bp of the complete coding region, the 120 bp of the 5'-noncoding region and the 1092 bp of the 3'-noncoding region containing two polyadenylation signals. In addition, the poly(A) tail was also found. The amino acid sequence deduced from the nucleotide sequence was homologous to the amino acid sequence of bovine S-100 beta subunit except 4 residues showing species differences. From the viewpoint of evolutionary implications, the homology between the nucleotide sequence of S-100 and those of rat intestinal Ca-binding protein (ICaBP) and calmodulin (CaM) was examined. A dot-blot hybridization of poly(A) RNA from the developing rat brains using a labeled cDNA showed a rapid increase in S-100 mRNA at 10-20 postnatal days. The presence of S-100 mRNA in C-6 glioma cells is also described.

Age Factors↗
PubMed1984

Purification and characterization of acetoacetyl-CoA synthetase from rat liver.

Acetoacetyl-CoA synthetase (acetoacetate:CoA ligase) was purified to electrophoretic homogeneity from a rat liver supernatant by ammonium sulfate fractionation and successive chromatographies on DEAE-Sepharose, Blue-Sepharose, Red-Sepharose, CoA-Sepharose, Ultrogel AcA-44, and DEAE-Sepharose once again. The purified enzyme had a specific activity of 2.3 mumol acetoacetyl-CoA formed per min per mg protein, which constituted a 960-fold purification compared to the crude extract, with a 7.4% yield. The enzyme absolutely required ATP, CoA, a monovalent cation (K+, Rb+, Cs+ or NH4+) and a divalent cation (Mg2+, Mn2+ or Ni2+) for its activity, yielding acetoacetyl-CoA, AMP and PPi in equimolar amounts. The molecular weight of the enzyme was approximately 80 000 as determined by Ultrogel AcA-34 gel filtration, and 71 000 by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. The enzyme was active only on acetoacetate and to a lesser extent on L-(+)-3-hydroxybutyrate, and the Km values for acetoacetate, L-(+)-3-hydroxybutyrate, ATP and CoA were 8 microM, 75 microM, 60 microM and 10 microM, respectively.

Acetyl Coenzyme A↗