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T Imanaka

Publications and source records attributed to T Imanaka.

At least 109 records · Page 6Linked to original sources
PubMed1998

The O6-methylguanine-DNA methyltransferase from the hyperthermophilic archaeon Pyrococcus sp. KOD1: a thermostable repair enzyme.

The enzyme O6-methylguanine-DNA methyltransferase (MGMT) is the most common form of cellular defense against the biological effects of O6-methylguanine (O6-MeG) in DNA. Based on PCR amplification using primers derived from conserved amino acid sequences of MGMTs from 11 species, we isolated the DNA region coding for MGMT from the hyperthermophilic archaeon Pyrococcus sp. KOD1. The MGMT gene from KOD1 (mgtk) comprises 522 nucleotides, encoding 174 amino acid residues; its product shows considerable similarity to the corresponding mammalian, yeast and bacterial enzymes, especially around putative methyl acceptor sites. Phylogenetic analysis of MGMTs showed that archaeal MGMTs were grouped with their bacterial counterparts. The location of the MGMT gene on the KOD1 chromosome was also determined. The cloned KOD1 MGMT gene was overexpressed using the T7 RNA polymerase expression system, and the recombinant protein was purified by ammonium sulfate fractionation, heat treatment, ion-exchange chromatography and gel filtration chromatography. The purified recombinant protein was assayed for its enzyme activity by monitoring transfer of [3H]methyl groups from the substrate DNA to the MGMT protein; the activity was found to be stable at 90 degrees C for at least 30 min. When the mgtk gene was placed under the control of the lac promoter and expressed in the methyltransferase-deficient Escherichia coli strain KT233 (delta ada, delta ogt) cells, a MGMT was produced. The enzyme was functional in vivo and complemented the mutant phenotype, making the cells resistant to the cytotoxic properties of the alkylating agent N-methyl-N'-nitro-N-nitrosoguanidine.

Amino Acid Sequence↗
PubMed1998

Archaeon Pyrococcus kodakaraensis KOD1: application and evolution.

Archaea is the third domain which is phylogenetically differentiated from the other two domains, bacteria and eucarya. Hyperthermophile within the archaea domain has evolved most slowly retaining many ancestral features of higher eukaryotes. Pyrococcus kodakaraensis KOD1, which grows at 95 degrees C optimally, is a newly isolated hyperthermophilc archaeon. The KOD1 strain possesses a circular genome, whose size is estimated to be approximately 2,036 kb. KOD1 enzymes involved in the genetic information processing system, such as DNA polymerase, Rec protein, aspartyl tRNA synthetase and molecular chaperonin, share features of eukaryotic enzymes. Rapid and accurate PCR method by KOD1 DNA polymerase and enzyme stabilization system by KOD1 chaperonin are also introduced in this article.

DNA, Archaeal↗
PubMed1998

Thermostable glycerol kinase from a hyperthermophilic archaeon: gene cloning and characterization of the recombinant enzyme.

The Pk-glpK gene, which encodes glycerol kinase (GK) from a hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1, was cloned and expressed in Escherichia coli. The amino acid sequence of this enzyme (Pk-GK) deduced from the nucleotide sequence showed 57% identity with that of E. coli GK and 47% identity with that of human GK. Pk-GK, which has a molecular weight of 55902 (497 amino acid residues), was purified from E. coli and characterized. Despite the high sequence similarity, Pk-GK and E. coli GK are greatly divergent in structure and function from each other. Unlike E. coli GK, which exists as a tetramer, Pk-GK exists as a dimer. The preferred divalent cation for Pk-GK is Co2+, instead of Mg2+. The optimum pH and temperature for Pk-GK activity are 8.0 and 80 degrees C, respectively. Pk-GK can utilize other nucleoside triphosphates than ATP as a phosphoryl donor. It is fairly resistant to an allosteric inhibitor of E. coli GK, fructose-1,6-bisphosphate. Determination of the kinetic parameters indicates that the Km value of the enzyme is 15.4 microM for ATP and 111 microM for glycerol and its kcat value is 940 s(-1). The enzyme was shown to be fairly resistant to irreversible heat inactivation and still retained 50% of its enzymatic activity even after heating at 100 degrees C for 30 min. Construction of a model for the three-dimensional structure of the enzyme suggests that the formation of extensive ion-pair networks is responsible for the high stability of this enzyme.

Adenosine Triphosphate↗
PubMed1998

Residual 60Co activity in steel samples exposed to the Hiroshima atomic-bomb neutrons.

Residual 60Co radioactivity produced by the neutrons from the Hiroshima atomic bomb has been measured by means of gamma-ray spectrometry for seven steel samples located up to 1,800 m slant range and one control sample obtained at 4.5 km from the hypocenter. After removing the main constituent of iron ions from the steel sample by the solvent extraction method, nickel and cobalt were chemically separated by the ion exchange process. Gamma-ray measurements were performed with a low background well-type germanium detector. Derived specific activities 60Co:Co were compared with previous measurements and with the calculation based on the neutron fluence of current dosimetry system DS86. It has been shown that a systematic discrepancy exists between the measured and calculated activity. The calculated-to-measured ratios for 60Co are consistent with those of 152Eu and 36Cl activity.

Chlorine↗
PubMed1998

Gene cloning and characterization of recombinant RNase HII from a hyperthermophilic archaeon.

We have cloned the gene encoding RNase HII (RNase HIIPk) from the hyperthermophilic archaeon Pyrococcus kodakaraensis KOD1 by screening of a library for clones that suppressed the temperature-sensitive growth phenotype of an rnh mutant strain of Escherichia coli. This gene was expressed in an rnh mutant strain of E. coli, the recombinant enzyme was purified, and its biochemical properties were compared with those of E. coli RNases HI and HII. RNase HIIPk is composed of 228 amino acid residues (molecular weight, 25,799) and acts as a monomer. Its amino acid sequence showed little similarity to those of enzymes that are members of the RNase HI family of proteins but showed 40, 31, and 25% identities to those of Methanococcus jannaschii, Saccharomyces cerevisiae, and E. coli RNase HII proteins, respectively. The enzymatic activity was determined at 30 degreesC and pH 8.0 by use of an M13 DNA-RNA hybrid as a substrate. Under these conditions, the most preferred metal ions were Co2+ for RNase HIIPk, Mn2+ for E. coli RNase HII, and Mg2+ for E. coli RNase HI. The specific activity of RNase HIIPk determined in the presence of the most preferred metal ion was 6. 8-fold higher than that of E. coli RNase HII and 4.5-fold lower than that of E. coli RNase HI. Like E. coli RNase HI, RNase HIIPk and E. coli RNase HII cleave the RNA strand of an RNA-DNA hybrid endonucleolytically at the P-O3' bond. In addition, these enzymes cleave oligomeric substrates in a similar manner. These results suggest that RNase HIIPk and E. coli RNases HI and HII are structurally and functionally related to one another.

Amino Acid Sequence↗
PubMed1998

Characterization of two Chinese hamster ovary cell lines expressing the COOH-terminal domains of sterol regulatory element-binding protein (SREBP)-1.

Sterol regulatory element-binding proteins (SREBPs) regulate transcription of genes encoding enzymes in the cholesterol biosynthetic pathway and the LDL receptor. These proteins are synthesized as membrane-bound precursors and processed to generate the NH2-terminal domains, mature transcription factors. We established two Chinese hamster ovary (CHO) cell lines, CHO-421 expressing the truncated hamster SREBP-1 (amino acids 421-1133) with two transmenbrane domains and CHO-557 expressing another truncated SREBP-1 (amino acids 557-1133) without any transmembrane domains, to investigate the fate of the COOH terminus after cleavage of the NH2-terminal mature SREBP. The cell fractionation experiments revealed that the two proteins, regardless of the absence of transmembrane domains in the SREBP (557-1133), similarly localized in the nuclear envelope and the microsomal membrane fractions, suggesting that these proteins appear to be tightly bound to a membrane protein(s) localizing on the nuclear and endoplasmic reticulum (ER) membranes. Although we predicted that overexpression of the COOH-terminal domains, which were thought to be involved in the regulation of SREBP processing, would result in disruption of the SREBP-dependent transcriptional regulation of several genes, the mRNA levels for 3-hydroxy-3-methylglutaryl coenzyme A (HMG CoA) synthase in these two cell lines were regulated in a sterol-dependent manner. Pulse-chase experiments revealed that the SREBP (421-1133) was relatively rapidly degraded (t1/2 = 4-6 hr) and that the intracellular cholesterol level did not affect the half-life time. The degradation of the SREBP (421-1133) was not suppressed by the treatment of a calpain inhibitor, N-acetyl-leucyl-leucyl-norleucynal (ALLN), which blocks the proteolysis of some proteins within or near the ER. In CHO-557 cells the SREBP (557-1133) was much more rapidly degraded (t1/2 = 1-2 hr), suggesting that the cytosolic COOH-terminal domain is accessible to the enzymatic attacks from the cytoplasm. Taken together, overexpression of the COOH-terminal domains does not affect the regulation of SREBP processing and the domains are rapidly turned over by the cytosolic proteolytic process distinct from the ALLN-sensitive ER degradative pathway.

Animals↗
PubMed1997

Effect of heat treatment on proper oligomeric structure formation of thermostable glutamate dehydrogenase from a hyperthermophilic archaeon.

Natural glutamate dehydrogenase (Pk-GDH) was purified from hyperthermophilic archaeon Pyrococcus sp. KOD1 to homogeneity and its activity and structure were compared with those of recombinant enzyme, which was expressed in Escherichia coli. Determination of the molecular weight of these enzymes by SDS-PAGE and gel filtration revealed that the natural enzyme was purified only as a hexameric form, whereas the recombinant enzyme was purified as both monomeric and hexameric forms. Determination of the enzymatic activities indicated that only the enzyme in a hexameric form is active. Moreover, it is noted that the specific activity of the hexameric form of the recombinant enzyme is much lower than that of the natural enzyme and that circular dichroism spectra of these enzymes are distinctly different from each other. These results suggest that the structure of the hexameric form of the recombinant enzyme with low specific activity (Type I) is different from that of the natural enzyme with high specific activity (Type II). Upon heat treatment (80 degrees C, 15 min), the Type I structure was effectively converted to Type II structure and the specific activity of the enzyme was increased by 2.6-fold. Likewise, upon heat treatment (70 degrees C for 15 min), the inactive monomeric form of the recombinant enzyme was at least partially associated with the hexameric form. These results indicate that high temperature plays an important role for proper folding and oligomerization of Pk-GDH.

Archaeal Proteins↗
PubMed1997

Structural characterization of mouse monoclonal antibody 13-1 against a porphyrin derivative: identification of a disulfide bond in CDR-H3 of Mab 13-1.

The amino acid sequence of a mouse monoclonal antibody Mab13-1, a catalytic antibody against TCPP (meso-tetrakis(4-carboxyphenyl)porphyrin), was confirmed by mass spectrometric (MS) peptide mapping. The amino-terminal sequence of the heavy chain was established by MS/MS analysis of the isolated N-terminal peptide. The presence of a unique disulfide bond between Cys93H and Cys102H was identified by MS peptide mapping and sequence analysis of an S-S containing peptide. Positions of other disulfide bonds were identified to be conserved. The non-conserved disulfide bridge was found to be resistant as other intra-chain disulfide bonds against reduction under non-denaturing condition, and to be buried inside the molecule. This extra disulfide bond is expected to support antigen-binding by restricting the flexibility of CDR-H3 loop, and it might be favorable for the recognition of a plane antigen, a porphyrin derivative.

Amino Acid Sequence↗
PubMed1997

Studies of high thermostability and peroxidase activity of recombinant antibody L chain-porphyrin Fe(III) complex.

A complex of an independent L chain from anti-mesotetrakis (4-carboxyphenyl) porphyrin (TCPP) monoclonal antibody 13-1 and TCPP Fe(III) was designated as L-zyme and shown to exhibit high peroxidase activity and high optimal reaction temperature (90 degrees C). Heat denaturation study and circular dichroism (CD) spectra analysis suggested that refolded structure of 13-1 L chain exhibited significantly reduced inactivation rate after heat treatment. The secondary structure of 13-1 L chain changed slightly by the encapsulation of TCPP Fe(III) and the complex was found to be less thermostable than the L chain alone. Furthermore, by characterization of truncated forms of the L chain, it was revealed that the hydrophobic region (115-146) and hydrophilic region (147-189) in CL are important for thermostability and activity, respectively. Tertiary structure of L-zyme was predicted by AbM. Comparison of residues of L-zyme with those in the active centre of known structure of the peroxidase from Arthromyces ramosus (ARP) indicated that His38(CDR1), His94(CDR3), Arg96(CDR3) of L-zyme are important residues for peroxidase activity. Moreover, the steric arrangements of these residues in both L-zyme and ARP are similar, respectively. Distance between proximal His and distal His in L-zyme is 9.09 A, whereas that of ARP is 7.8 A.

Animals↗
PubMed1997

Indolepyruvate ferredoxin oxidoreductase from Pyrococcus sp. KOD1 possesses a mosaic structure showing features of various oxidoreductases.

Indolepyruvate ferredoxin oxidoreductase (IOR) catalyzes the oxidative decarboxylation of arylpyruvates. Gene cloning and sequencing analysis of the IOR gene from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was performed. Two genes, iorA and iorB. encoding alpha and beta subunits of IOR were found to be tandemly arranged, which suggests that gene expression is translationally coupled. Sequence analysis showed the C-terminal region of the alpha subunit to have a typical ferredoxin-type [4Fe-4S] cluster motif (CXXCXXCXXCXXXCP), which is similar to that present in the delta subunits of other oxidoreductases such as pyruvate ferredoxin oxidoreductase (POR) and 2-ketoisovalerate ferredoxin oxidoreductase (VOR). We suggest that the alpha subunit of KOD1-IOR has a mosaic structure composed of features characteristic of the alpha, beta and delta subunits from POR and VOR. KOD1-IOR was overproduced in anaerobically incubated Escherichia coli cells and the crude enzyme was extracted under anaerobic conditions. The optimal temperature for activity of recombinant IOR was 70 degrees C and the half-life of this enzyme in the presence of air was 15 min at 25 degrees C.

Amino Acid Sequence↗
PubMed1997

Enzymatic assay for chloride ion with chloride-dependent sarcosine oxidase created by site-directed mutagenesis.

Serum chloride ion is routinely assayed in clinical laboratories. We have developed a new enzymatic assay for determining the chloride ion concentration. The method involves the use of a mutant sarcosine oxidase, which was created as desired by site-directed mutagenesis and showed chloride-dependent activity. The enzyme which is reactivated by the chloride ion forms hydrogen peroxide from sarcosine. The production of hydrogen peroxide is measured by the 4-amino-antipyrine peroxidase system. The increase of the reaction rate was proportional to the chloride ion concentration. A lag time of the time course was not observed, and the reaction rate for a blank was not detected. Therefore, a rate assay could be adopted. A standard curve of the assay reagent was linear up to 180 mM chloride ion of the sample. The specificity for the bromide ion was 43% of that of the chloride ion, although it was 0% for other ion species. When serum samples were used, within-day coefficient variations (CVs) and day-to-day CVs were below 1.5%. A good correlation with the comparison assay was observed by using 160 samples of normal and abnormal patient sera. This method can easily and reliably be used for the accurate determination of chloride ion concentration in serum or other samples.

Amino Acid Sequence↗
PubMed1997

Characterization of a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcus sp. KOD1.

The Pk-rec gene, encoding a RecA/RAD51 homologue from the hyperthermophilic archaeon Pyrococcussp. KOD1, was expressed in Escherichia coli. The recombinant Pk-REC was purified to homogeneity and was shown to be in a dimeric form. A striking property of the purified recombinant Pk-REC was the unusual DNase activity on both single- and double-stranded DNAs along with the ATPase activity. The reaction product of this DNase activity was mononucleotides. The optimum temperature and pH for the DNase activity were 60 degrees C and 8-8.5, respectively. In addition, the metal ion requirement for DNase activity was different from that for the ATPase activity. The protein exhibited no DNase activity in the presence of Zn2+ion, which was one of the most preferable divalent cations for ATPase activity. Another unique characteristic of the recombinant protein was that the reaction product of ATPase activity was AMP instead of ADP.Pk-REC may represent a common prototype of the RecA family proteins with high RecA-like activity.

Adenosine Triphosphatases↗
PubMed1997

Gene cloning, sequencing and enzymatic properties of glutamate synthase from the hyperthermophilic archaeon Pyrococcus sp. KOD1.

The gltA gene encoding a glutamate synthase (GOGAT) from the hyperthermophilic archaeon Pyrococcus sp. KOD1 was cloned as a 6.6 kb HindIII-BamHI fragment. Sequence analysis indicates that gltA encodes a 481- amino acid protein (53,269 Da). The deduced amino acid sequence of KOD1-GltA includes conserved regions that are found in the small subunits of bacterial GOGAT: two cysteine clusters, an adenylate-binding consensus sequence and an FAD-binding consensus sequence. However, no sequences homologous to the large subunit of bacterial GOGAT were found in the upstream or downstream regions. In order to examine whether GltA alone can act as a functional GOGAT, GltA was overexpressed in Escherichia coli BL21 (DE3) cells using an expression plasmid. GltA was purified to homogeneity and shown to be functional as a homotetramer of approximately 205 kDa, which is equivalent to the molecular weight of the native GOGAT from KOD1, thus indicating that KOD1-GOGAT is the smallest known active GOGAT. GltA is capable of both glutamine-dependent and ammonia-dependent synthesis of glutamate. Synthesis of glutamate by KOD1-GltA required NADPH, indicating that this enzyme is an NADPH-GOGAT (EC 1.4.1.13). The optimum pH for both activities was 6.5. However, GltA exhibited different optimum temperatures for activity depending on the reaction assayed (glutamine-dependent reaction, 80 degrees C; ammonia-dependent reaction, 90 degrees C).

Amino Acid Sequence↗
PubMed1997

Characterization of DNA polymerase from Pyrococcus sp. strain KOD1 and its application to PCR.

The DNA polymerase gene from the archaeon Pyrococcus sp. strain KOD1 (KOD DNA polymerase) contains a long open reading frame of 5,013 bases that encodes 1,671 amino acid residues (GenBank accession no. D29671). Similarity analysis revealed that the DNA polymerase contained a putative 3'-5' exonuclease activity and two in-frame intervening sequences of 1,080 bp (360 amino acids; KOD pol intein-1) and 1,611 bp (537 amino acids; KOD pol intein-2), which are located in the middle of regions conserved among eukaryotic and archaeal alpha-like DNA polymerases. The mature form of the DNA polymerase gene was expressed in Escherichia coli, and the recombinant enzyme was purified and characterized. 3'-5' exonuclease activity was confirmed, and although KOD DNA polymerase's optimum temperature (75 degrees C) and mutation frequency (3.5 x 10(-3)) were similar to those of a DNA polymerase from Pyrococcus furiosus (Pfu DNA polymerase), the KOD DNA polymerase exhibited an extension rate (100 to 130 nucleotides/s) 5 times higher and a processivity (persistence of sequential nucleotide polymerization) 10 to 15 times higher than those of Pfu DNA polymerase. These characteristics enabled the KOD DNA polymerase to perform a more accurate PCR in a shorter reaction time.

Amino Acid Sequence↗
PubMed1997

In vitro stabilization and in vivo solubilization of foreign proteins by the beta subunit of a chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The gene encoding the beta subunit of a molecular chaperonin from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1 (cpkB) was cloned, sequenced, and expressed in Escherichia coli. The cpkB gene is composed of 1,641 nucleotides, encoding a protein (546 amino acids) with a molecular mass of 59,140 Da. The enhancing effect of CpkB on enzyme stability was examined by using Saccharomyces cerevisiae alcohol dehydrogenase (ADH). Purified recombinant CpkB prevents thermal denaturation and enhances thermostability of ADH. CpkB requires ATP for its chaperonin function at a low CpkB concentration; however, CpkB functions without ATP when present in excess. In vivo chaperonin function for the solubilization of insoluble proteins was also studied by coexpressing CpkB and CobQ (cobryic acid synthase), indicating that CpkB is useful for solubilizing the insoluble proteins in vivo. These results suggest that the beta subunit plays a major role in chaperonin activity and is functional without the alpha subunit.

Adenosine Triphosphate↗
PubMed1997

Characterization of recombinant glutamine synthetase from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1.

The glnA gene encoding glutamine synthetase was cloned from the hyperthermophilic archaeon Pyrococcus sp. strain KOD1, and its nucleotide sequence was determined. The glnA gene was expressed in Escherichia coli ME8459 (glnA mutant strain), and the protein was purified to homogeneity and shown to be functional in a dodecameric from (637,000 Da), exhibiting both transferase and synthetase activities. However, kinetic studies indicated that the enzyme possessed low biosynthetic activity, suggesting that the reaction was biased towards glutamate production. The optimum temperature for both activities was 60 degrees C, which was lower than the optimal growth temperature of KOD1. Recombinant KOD1 GlnA exhibited different optimum pHs depending on the reaction employed (pH 7.8 for the synthetase reaction and pH 7.2 for the transferase reaction). Of the various nucleoside triphosphates tested, GTP as well as ATP was involved in the synthetase reaction.

Adenosine Triphosphate↗