Biomedical subjects

O Adachi

Publications and source records attributed to O Adachi.

At least 37 records · Page 2Linked to original sources

PubMed1999

Spectroscopic characterization of carbon monoxide complexes generated for copper/topa quinone-containing amine oxidases.

Carbon monoxide complexes have been generated for copper/topa quinone (TPQ)-containing amine oxidases from Arthrobactor globiformis (AGAO) and Aspergillus niger (AO-I) and characterized by various spectroscopic measurements. Addition of CO to AGAO anaerobically reduced with its substrate 2-phenylethylamine led to a slight increase of absorption bands at 440 and 470 nm derived from the semiquinone form (TPQ(sq)) of the TPQ cofactor, concomitantly giving rise to new CO-related absorption bands at 334 and 434 nm. The intensity of the TPQ(sq) radical EPR signal at g = 2.004 also increased in the presence of CO, while its hyperfine coupling structure was affected insignificantly. FT-IR measurements revealed C-O stretching bands (nu(CO)) at 2063 and 2079 cm(-1) for the CO complex of the substrate-reduced AGAO (at 2085 cm(-1) for AO-I), which shifted nearly 100 cm(-1) to lower frequencies upon using (13)C(18)O. Collectively, these results suggest that CO is bound to the Cu(I) ion in the Cu(I)/TPQ(sq) species formed in the reductive half-reaction of amine oxidation, thereby shifting the Cu(II)/aminoresorcinol right arrow over left arrow Cu(I)/semiquinone equilibrium toward the latter. When AGAO was reduced with dithionite, an intermediary form of the enzyme with Cu(II) reduced to Cu(I) but TPQ still in the oxidized state (TPQ(ox)) was produced. Dithionite reduction of AGAO in the presence of CO resulted in the immediate formation of FT-IR bands at 2064 and 2083 cm(-1), which were assigned to the nu(CO) bands of the CO bound to the TPQ(ox) enzyme. The intense 2083 cm(-1) band was then displaced by a new band at 2077 cm(-1), corresponding to the formation of the fully reduced topa. Significant variation of these nu(CO) frequencies indicates that vibrational properties of CO bound to copper amine oxidases are sensitively influenced by the coordination structure of the Cu(I) ion, which may be modulated by the chemical and redox states of the TPQ cofactor.

Amine Oxidase (Copper-Containing)↗

PubMed1999

Electron transfer from quinohemoprotein alcohol dehydrogenase to blue copper protein azurin in the alcohol oxidase respiratory chain of Pseudomonas putida HK5.

A blue copper protein was purified together with a type II quinohemoprotein alcohol dehydrogenase (ADH IIB) from the soluble fraction of Pseudomonas putida HK5 grown on n-butanol. The purified blue copper protein was shown to be azurin, on the basis of several properties such as its absorption maximum (623 nm), its low molecular mass (17 500 Da), its acidic nature (pI of 4.1), its relatively high redox potential (306 mV), the presence of an intramolecular disulfide bond, and N-terminal amino acid sequence homology with respect to azurins from other sources, especially from P. putida NCIB 9869 and Pseudomonas fluorescens. Direct electron transfer from ADH IIB to azurin was shown to occur at a rate of 48-70 s-1. The apparent Km value of ADH IIB for azurin, determined by steady-state kinetics, was decreased several-fold by increasing the ionic strength. Furthermore, the extent of fluorescence quenching of ADH IIB due to the interaction with azurin was increased by increasing the ionic strength, but the binding constant for binding between ADH IIB and azurin was unchanged. The redox potential of azurin was increased 12 mV by incubation with ADH but not vice versa. Furthermore, the redox potential gap between ADH and azurin was increased from 102 to 126 mV by increasing the ionic strength. It is conceivable that a hydrophobic interaction is involved in the electron transfer between both proteins, and it is also suggested that the electron transfer may occur by a freely reversible on and off binding process but may not be related to the global binding process of both proteins. Thus, the results presented here strongly suggest that azurin works as an electron-transfer mediator in a PQQ-dependent alcohol oxidase respiratory chain in P. putida HK5.

Alcohol Dehydrogenase↗

PubMed1999

Limb and skin abnormalities in mice lacking IKKalpha.

The gene encoding inhibitor of kappa B (IkappaB) kinase alpha (IKKalpha; also called IKK1) was disrupted by gene targeting. IKKalpha-deficient mice died perinatally. In IKKalpha-deficient fetuses, limb outgrowth was severely impaired despite unaffected skeletal development. The epidermal cells in IKKalpha-deficient fetuses were highly proliferative with dysregulated epidermal differentiation. In the basal layer, degradation of IkappaB and nuclear localization of nuclear factor kappa B (NF-kappaB) were not observed. Thus, IKKalpha is essential for NF-kappaB activation in the limb and skin during embryogenesis. In contrast, there was no impairment of NF-kappaB activation induced by either interleukin-1 or tumor necrosis factor-alpha in IKKalpha-deficient embryonic fibroblasts and thymocytes, indicating that IKKalpha is not essential for cytokine-induced activation of NF-kappaB.

Animals↗

PubMed1999

The quinohemoprotein alcohol dehydrogenase of Gluconobacter suboxydans has ubiquinol oxidation activity at a site different from the ubiquinone reduction site.

Alcohol dehydrogenase (ADH) of acetic acid bacteria functions as the primary dehydrogenase of the ethanol oxidase respiratory chain, where it donates electrons to ubiquinone. In addition to the reduction of ubiquinone, ADHs of Gluconobacter suboxydans and Acetobacter aceti were shown to have a novel function in the oxidation of ubiquinol. The oxidation activity of ubiquinol was detected as an ubiquinol:ferricyanide oxidoreductase activity, which can be monitored by selected wavelength pairs at 273 and 298 nm with a dual-wavelength spectrophotometer. The ubiquinol oxidation activity of G. suboxydans ADH was shown to be two times higher in 'inactive ADH', whose ubiquinone reductase activity is 10 times lower, than with normal 'active' ADH. No activity could be detected in the isolated subunit II or subunit I/III complex, but activity was detectable in the reconstituted ADH complex. Inactive and active ADHs exhibited a 2-3-fold difference in their affinity to ubiquinol despite having the same affinity to ubiquinone. Furthermore, the ubiquinol oxidation site in ADH could be distinguished from the ubiquinone reduction site by differences in their sensitivity to ubiquinone-related inhibitors and by their substrate specificity with several ubiquinone analogues. Thus, the results strongly suggest that the reactions occur at different sites. Furthermore, in situ reconstitution experiments showed that ADH is able to accept electrons from ubiquinol present in Escherichia coli membranes, suggesting the ubiquinol oxidation activity of ADH has a physiological function. Thus, ADH of acetic acid bacteria, which has ubiquinone reduction activity, was shown to have a novel ubiquinol oxidation activity, of which the physiological function in the respiratory chain of the organism is also discussed.

Acetobacter↗

PubMed1999

Unresponsiveness of MyD88-deficient mice to endotoxin.

MyD88 is a general adaptor protein that plays an important role in the Toll/IL-1 receptor family signalings. Recently, Toll-like receptors 2 and 4 (TLR2 and TLR4) have been suggested to be the signaling receptors for lipopolysaccharide (LPS). In this study, we demonstrate that MyD88 knockout mice lack the ability to respond to LPS as measured by shock response, B cell proliferative response, and secretion of cytokines by macrophages and embryonic fibroblasts. However, activation of neither NF-kappaB nor the mitogen-activated protein (MAP) kinase family is abolished in MyD88 knockout mice. These findings demonstrate that signaling via MyD88 is essential for LPS response, but the inability of MyD88 knockout mice to induce LPS-dependent gene expression cannot simply be attributed to lack of the activation of MAP kinases and NF-kappaB.

Adaptor Proteins, Signal Transducing↗

PubMed1999

Crystallization and preliminary diffraction studies of two quinoprotein alcohol dehydrogenases (ADHs): a soluble monomeric ADH from Pseudomonas putida HK5 (ADH-IIB) and a heterotrimeric membrane-bound ADH from Gluconobacter suboxydans (ADH-GS).

Crystals of a soluble monomeric quinocytochrome alcohol dehydrogenase (ADH-IIB) and of a trimeric membrane-associated quinocytochrome alcohol dehydrogenase (ADH-GS) have been obtained. The ADH-IIB crystals are triclinic, with one monomer in the unit cell, and were obtained in the presence of PEG 8000, sodium citrate, HEPES buffer and 2-propanol. X-ray data were collected at 110 K to 1. 9 A resolution (R(merge) = 6.4%) and the orientation of a methanol dehydrogenase search molecule (from Methylophilus methylotrophus W3A1) was obtained by molecular replacement. Preliminary refinement of this model (10.0-3.0 A resolution, R = 0.37, R(free) = 0.40) led to tentative identification of the two highest peaks in a native anomalous difference Fourier map as the Fe atom of the heme and a calcium ion interacting with the PQQ prosthetic group. The ADH-GS crystals are tetragonal, displaying six similar lattices, both primitive and centered, and were grown by the sitting-drop method after replacement of Triton X-100 by dodecylmaltoside or octaethylene glycol monododecyl ether in the presence of ammonium sulfate and sodium acetate buffer, with and without PEG 3500 and calcium ion. The best diffraction is obtained at 110 K where the resolution extends to about 4 A in the a and b directions and about 3 A in the c direction.

Alcohol Oxidoreductases↗

PubMed1999

Purification and characterization of methylamine oxidase induced in Aspergillus niger AKU 3302.

Crude extract of Aspergillus niger AKU 3302 mycelia incubated with methylamine showed a single amine oxidase activity band in a developed polyacrylamide gel that weakly cross-reacted with the antibody against a copper/topa quinone-containing amine oxidase (AO-II) from the same strain induced by n-butylamine. Since the organism cannot grow on methylamine and the already known quinoprotein amine oxidases of the organism cannot catalyze oxidation of methylamine, the organism was forced to produce another enzyme that could oxidize methylamine when the mycelia were incubated with methylamine. The enzyme was separated and purified from the already known two quinoprotein amine oxidases formed in the same mycelia. The purified enzyme showed a sharp symmetric sedimentation peak in analytical ultracentrifugation showing S20,w0 of 6.5s. The molecular mass of 133 kDa estimated by gel chromatography and 66.6 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The purified enzyme was pink in color with an absorption maximum at 494 nm. The enzyme readily oxidized methylamine, n-hexylamine, and n-butylamine, but not benzylamine, histamine, or tyramine, favorite substrates for the already known two quinoprotein amine oxidases. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topa quinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme-p-nitrophenylhydrazone (469 nm at neutral to 577 nm at alkaline pH) supported the identity of the cofactor with topaquinone. Nothern blot analysis indicated that the methylamine oxidase encoding gene is largely different from the already known amine oxidase in the organism.

Amino Acid Sequence↗

PubMed1998

Mutant isolation of the Escherichia coli quinoprotein glucose dehydrogenase and analysis of crucial residues Asp-730 and His-775 for its function.

Several mutants of quinoprotein glucose dehydrogenase (GDH) in Escherichia coli were obtained and characterized. Of these, significant mutants were further characterized by kinetic analysis after purification or by site-directed mutagenesis to introduce different amino acid substitutions. H775R and H775A showed a pronounced reduction of affinity for a prosthetic group, pyrroloquinoline quinone (PQQ), suggesting that His-775 may directly interact with PQQ. D730N and D730A showed low glucose oxidase activity without influence on the affinity for PQQ, Mg2+, or substrate, but D730R showed reduced affinity for PQQ. The spectrum of tryptophan fluorescence revealed that the local structure surrounding PQQ was not changed by D730N mutation. Based on these data, we assume that Asp-730 may occur close to PQQ and function as a proton (and also electron) donor to PQQ or acceptor from PQQH2. Substitutions of Gly-689, that are located at the end of a unique segment of GDH among homologous quinoprotein dehydrogenases, directed reduction of the affinity for PQQ or GDH activity. Therefore, the unique segment and Asp-730 may play a specific role for GDH, which might be related to the intramolecular electron transfer from PQQ to ubiquinone.

Amino Acid Sequence↗

PubMed1998

Intramolecular electron transport in quinoprotein alcohol dehydrogenase of Acetobacter methanolicus: a redox-titration study

Quinohemoprotein-cytochrome c complex alcohol dehydrogenase (ADH) of acetic acid bacteria consists of three subunits, of which subunit I contains pyrroloquinoline quinone (PQQ) and heme c, and subunit II contains three heme c components. The PQQ and heme c components are believed to be involved in the intramolecular electron transfer from ethanol to ubiquinone. To study the intramolecular electron transfer in ADH of Acetobacter methanolicus, the redox potentials of heme c components were determined with ADH complex and the isolated subunits I and II of A. methanolicus, as well as hybrid ADH consisting of the subunit I/III complex of Gluconobacter suboxydans ADH and subunit II of A. methanolicus ADH. The redox potentials of hemes c in ADH complex were -130, 49, 188, and 188 mV at pH 7.0 and 24, 187, 190, and 255 mV at pH 4.5. In hybrid ADH, one of these heme c components was largely changed in the redox potential. Reduced ADH was fully oxidized with potassium ferricyanide, while ubiquinone oxidized the enzyme partially. The results indicate that electrons extracted from ethanol at PQQ site are transferred to ubiquinone via heme c in subunit I and two of the three hemes c in subunit II. Copyright 1998 Elsevier Science B.V.

Journal Article↗

PubMed1998

Defective NK cell activity and Th1 response in IL-18-deficient mice.

IL-18 is a cytokine that is secreted from activated macrophages and induces IFNgamma production. To investigate the in vivo role of IL-18, we generated IL-18-deficient mice. In Propionibacterium acnes (P. acnes)-primed IL-18-deficient mice, LPS-induced IFNgamma production was markedly reduced, despite normal IL-12 induction. Natural killer cell activity was significantly impaired. Th1 cell response after injection of P. acnes or Mycobacterium bovis (bacillus Calmette-Guerin [BCG]) was significantly reduced. Similar results were observed in IL-12-deficient mice. Interestingly, Th1 response was induced after BCG infection in IL-12-deficient mice. We therefore generated mice lacking both IL-18 and IL-12. In these mice, NK activity and Th1 response were further impaired. This demonstrates the important role of both IL-18 and IL-12 in NK activity, as well as in in vivo Th1 response.

Animals↗

PubMed1998

Targeted disruption of the MyD88 gene results in loss of IL-1- and IL-18-mediated function.

MyD88, originally isolated as a myeloid differentiation primary response gene, is shown to act as an adaptor in interleukin-1 (IL-1) signaling by interacting with both the IL-1 receptor complex and IL-1 receptor-associated kinase (IRAK). Mice generated by gene targeting to lack MyD88 have defects in T cell proliferation as well as induction of acute phase proteins and cytokines in response to IL-1. Increases in interferon-gamma production and natural killer cell activity in response to IL-18 are abrogated. In vivo Th1 response is also impaired. Furthermore, IL-18-induced activation of NF-kappaB and c-Jun N-terminal kinase (JNK) is blocked in MyD88-/- Th1-developing cells. Taken together, these results demonstrate that MyD88 is a critical component in the signaling cascade that is mediated by IL-1 receptor as well as IL-18 receptor.

Adaptor Proteins, Signal Transducing↗

PubMed1997

Resonance Raman, infrared, and EPR investigation on the binuclear site structure of the heme-copper ubiquinol oxidases from Acetobacter aceti: effect of the heme peripheral formyl group substitution.

Acetobacter aceti produces two different terminal ubiquinol oxidases (cytochromes a1 and o) depending on the culture conditions. Two types of oxidases share a common protein moiety but with different heme components at the binuclear center (heme A for cytochrome a1 and heme O for cytochrome o). We investigated the structure of the binuclear site of the two oxidases using resonance Raman, Fourier transform-infrared (FT-IR), and EPR spectroscopies to clarify the interactions of heme A formyl group with protein moiety. We found that the overall architecture and the electronic configuration at the binuclear center in the oxidized state seem to be well conserved irrespective of the heme peripheral group at position 8, except for the azide-inhibited state. In contrast, we observed great variations in the C-N stretching frequency and cyanide-binding affinity in the CN-reduced state, in addition to multiple C-O stretching bands in the CO-reduced state. Present and previous studies suggest that the conformational flexibility of the binuclear center in the reduced ligand-bound state may be a common feature among the heme-copper oxidase superfamily. In the CN-reduced state, a hydrogen bond network may be formed among the formyl group, water molecule(s), and the surrounding amino acid residue(s). This network may be very important to maintain proper orientations of the distal amino acid residues and/or the CuB1+ ion relative to the cyanide ion bound to the ferrous heme iron and could play a critical role for the high affinity in cyanide binding.

Acetobacter↗

PubMed1997

Characterization of the gntT gene encoding a high-affinity gluconate permease in Escherichia coli.

We characterized the gntT gene encoding a high-affinity gluconate permease of Escherichia coli K-12. Primer extension and lacZ-operon fusion analyses revealed that gntT has one strong and two weak promoters, all of which are regulated positively by cAMP-CRP and negatively by GntR. The weak promoters became constitutive when separated from the upstream region including the strong promoter that overlaps a putative GntR-binding sequence. Gluconate-specific uptake activity was observed with cells harboring the gntT plasmid clone, which was enhanced by the presence of gntK encoding gluconate kinase.

Amino Acid Sequence↗

PubMed1997

Gene organization and transcriptional regulation of the gntRKU operon involved in gluconate uptake and catabolism of Escherichia coli.

We cloned and characterized the gntRKU operon encoding part of the GntI system involved in gluconate uptake and catabolism by Escherichia coli. The operon was shown to encode its repressor, a thermoresistant gluconate kinase, and a low affinity gluconate permease. CAT fusion analysis revealed that the operon has a promoter for gntR and another for gntKU, and that the gntR gene is constitutively expressed, while that of gntKU is regulated positively by the cAMP-CRP complex and negatively by GntR. Read-through transcription from the gntR promoter into gntK was decreased in the presence of GntR, although GntR did not repress its own promoter. In addition, transcriptional attenuation was observed after the gntK gene, so gntU expression is reduced presumably to modulate the production of the low affinity gluconate permease according to the available concentration of gluconate.

Amino Acid Sequence↗

PubMed1997

The fungus Gibberella fujikuroi produces copper/topaquinone-containing amine oxidase when induced by N-butylamine.

Crude extract of Gibberella fujikuroi AKU 3802 mycelium induced with n-butylamine showed a single amine oxidase activity band in a non-denaturing gel that cross-reacted with the antibody against copper/topaquinone-containing amine oxidase from Aspergillus niger. The enzyme was purified by a procedure involving four chromatographic steps. Purified enzyme was pink with an absorption maximum at 490 nm. Molecular mass of 135 kDa estimated by gel chromatography and 70 kDa found by SDS-PAGE confirmed the dimeric structure of the enzyme. The enzyme readily oxidized n-hexylamine, n-butylamine, benzylamine and histamine, but not spermine or spermidine. Inactivation by carbonyl reagents and copper chelators suggested the presence of a copper/topaquinone cofactor. Spectrophotometric titration by p-nitrophenylhydrazine showed one reactive carbonyl group per subunit and redox-cyclic quinone staining confirmed the presence of a quinone cofactor. pH-dependent shift of the absorption spectrum of the enzyme p-nitrophenylhydrazone (465 nm at neutral to 580 nm at alkaline pH) supports the identity of the cofactor, with topaquinone. The N-terminal amino acid sequence of the enzyme showed high similarity to other microbial copper/topaquinone-containing amine oxidases.

Amine Oxidase (Copper-Containing)↗

PubMed1997

Escherichia coli is unable to produce pyrroloquinoline quinone (PQQ).

Many bacteria can synthesize the cofactor pyrroloquinoline quinone (PQQ), a cofactor of several dehydrogenases, including glucose dehydrogenase (GCD). Among the enteric bacteria, Klebsiella pneumoniae has been shown to contain the genes required for PQQ biosynthesis. Escherichia coli and Salmonella typhimurium were thought to be unable to synthesize PQQ but it has been reported that strain EF260, a derivative of E. coli FB8, can synthesize PQQ after mutation and can oxidize glucose to gluconate via the GCD/PQQ pathway (F. Biville, E. Turlin & F. Gasser, 1991, J Gen Microbiol 137, 1775-1782). We have re-investigated this claim and conclude that it is most likely erroneous. (i) Strain EF260, isolated originally by Biville and coworkers, was unable to synthesize a holo-enzyme GCD unless PQQ was supplied to the growth medium. No GCD activity could be detected in membrane fractions. (ii) The amount of PQQ detected in the growth medium of EF260 was very low and not very different from that found in a medium with its parent strain or in a medium containing no cells. (iii) EF260 cells were unable to produce gluconate from glucose via the PQQ/GCD pathway. (iv) Introduction of a gcd::Cm deletion in EF260, eliminating GCD, did not affect glucose metabolism. This suggested a pathway for glucose metabolism other than the PQQ/GCD pathway. (v) Glucose uptake and metabolism in EF260 involved a low-affinity transport system of unknown identity, followed most likely by phosphorylation via glucokinase. It is concluded that E. coli cannot synthesize PQQ and that it lacks genes required for PQQ biosynthesis.

Apoenzymes↗

PubMed1996

Purification and characterization of the Escherichia coli thermoresistant glucokinase encoded by the gntK gene.

A thermoresistant gluconokinase encoded by the gntK gene of Escherichia coli K-12 was purified and characterized. The Km values of the purified enzyme for gluconate and ATP are 42 microM and 123 microM, respectively, and the activity was not altered by the presence of pyruvate. The enzyme was shown to function as a dimer with two identical subunits of 18.4 kDa. These characteristics appear to be distinct from those of the gluconokinase reported by E.I. Vivas, A. Liendo, K. Dawidowicz, and T. Istúriz (1994) J. Basic. Microbiol. 16, 117-122.

Dimerization↗

PubMed1996

Two amine oxidases from Aspergillus niger AKU 3302 contain topa quinone as the cofactor: unusual cofactor link to the glutamyl residue occurs only at one of the enzymes.

Amine oxidases (EC 1.4.3.6) from Aspergillus niger, AO-I (2 x 75 kDa) and AO-II (80 kDa), were examined to determine the cofactor structure. Inactivated with p-nitrophenylhydrazine, they showed absorption and fluorescence spectra similar to those published for other copper amine oxidases and to topa hydantoin p-nitrophenylhydrazone. After digestion by thermolysin and pronase, cofactor peptides were purified by HPLC and sequenced. For thermolytic peptides, a typical topa consensus sequence, Asn-X-Glu-Tyr, was obtained for AO-II, although in case of AO-I it overlapped with Val-Val-Ile-Glu-Pro-Tyr-Gly. For pronase peptides of AO-I, only the latter sequence was obtained. NMR and mass spectroscopy confirmed the residue X as topa p-nitrophenylhydrazone in AO-II and revealed the presence of a residue Z attached to the Glu in the peptide Val-Val-Ile-Glu(Z)-Pro of AO-I. This residue was separated from the peptide by hydrolysis and identified as a product derived from topa quinone. The data, together with amino-acid sequence of AO-I, confer strong evidence for topa quinone as the cofactor, bound in the typical consensus sequence. Raman spectra of the p-nitrophenylhydrazone derivative of AO-I and its pronase peptide showed essentially the same peaks matching to a model compound for topa p-nitrophenylhydrazone. However, there may exist an unusual ester link between the topa-404 and Glu-145 in the native enzyme.

Amine Oxidase (Copper-Containing)↗