Daijiro Ohmori

Purification and kinetic characterization of recombinant alternative oxidase from Trypanosoma brucei brucei

The trypanosome alternative oxidase (TAO) functions in the African trypanosomes as a cytochrome-independent terminal oxidase, which is essential for their survival in the mammalian host and as it does not exist in the mammalian host is considered to be a promising drug target for the treatment of trypanosomiasis. In the present study, recombinant TAO (rTAO) overexpressed in a haem-deficient Escherichia coli strain has been solubilized from E. coli membranes and purified to homogeneity in a stable and highly active form. Analysis of bound iron detected by inductively coupled plasma-mass spectrometer (ICP-MS) reveals a stoichiometry of two bound iron atoms per monomer of rTAO. Confirmation that the rTAO was indeed a diiron protein was obtained by EPR analysis which revealed a signal, in the reduced forms of rTAO, with a g-value of 15. The kinetics of ubiquiol-1 oxidation by purified rTAO showed typical Michaelis-Menten kinetics (K(m) of 338microM and V(max) of 601micromol/min/mg), whereas ubiquinol-2 oxidation showed unusual substrate inhibition. The specific inhibitor, ascofuranone, inhibited the enzyme in a mixed-type inhibition manner with respect to ubiquinol-1.

Induction of Oogenesis in Mosquitoes (Diptera: Culicidae) by Infusion of the Hemocoel with Amino Acids

Abstract As done previously with adult females of Culex pipiens pallens Coquillett, a mixture of 17 amino acids was infused into the hemocoel of females of seven anautogenous and one autogenous mosquito species belonging to three genera. In Culex. p. quinquefasciatus Say, Cx. tritaeniorhynchus Giles, Cx. kyotoensis Yamaguti & LaCasse, Aedes albopictus (Skuse), Armigeres subalbatus (Coquillett), and Cx. p. molestus Forskal, which previously had laid autogenously matured first batch of eggs, ovarian development was stimulated and frequently continued to maturity. In most mosquitoes, the number of mature follicles nearly doubled when the period of infusion was extended from 24 to 48 h. Therefore, the two previously indicated roles of amino acids, one to initiate ovarian development and the other to regulate the number of maturing oocytes, were confirmed in these species. In Cx. halifaxii Theobald and Ae. japonicus (Theobald), however, the frequency of activation and maturation of ovaries was low compared with the other species, indicating that those species may require some factors other than an increase in amino acids for normal ovarian development after a blood meal.

Glyceraldehyde-3-phosphate dehydrogenase interacts with phosphorylated Akt resulting from increased blood glucose in rat cardiac muscle

MINT‐7891324, MINT‐7891304, MINT‐7891314: GAPDH (uniprotkb:P04797) physically interacts (MI:0915) with Akt (uniprotkb:P47196) by anti bait coimmunoprecipitation (MI:0006)

Isolation and characterization of a ferredoxin from Mycobacterium smegmatis takeo

Abstract A soluble ferredoxin was isolated in a crystalline form from Mycobacterium smegmatis Takeo. (This species has been identified as Mycobacterium avium strain Takeo in some previous papers. See Kusunose, M., et al. (1976) Arch. Microbiol., 108, 65–73, for the rationale for this new name.) The molecular weight was calculated to be 12035 from the amino acid sequence (Hase, T., et al. (1979) FEBS Lett., 103, 224–228). It contained 5.7 mol non-heme iron, 5.9 mol acid-labile sulfur and 8 mol cysteine residues per 12 035 g. The ferredoxin exhibited absorption maxima at 280 and 406 nm with shoulders around 330 and 450 nm, and the absorbance ratio ( A 405 A 280 ) was 0.61. The absorbance in the visible region was partially reduced by the addition of sodium dithionite, but was not affected by the addition of potassium ferricyanide. The two midpoint oxidation-reduction potentials at pH 7.0 were determined potentiometrically to be −15 and −435 mV. The EPR spectra in the isolated stateexhibited an almost isotropic signal at g 2.00, while in the dithionite-reduced state the signal was at g 2.015. Though its physiological role in the cell is unknown, the ferredoxin served as an electron mediator for cytochrome c reduction by NADPH in the presence of spinach ferredoxin-NADP+ reductase. From the data described above, M. smegmatis ferredoxin would seem to be a two FeS cluster containing ferredoxin, and at least one of the two FeS clusters is thought to be of the 3Fe3S type.

Continuous-wave and Pulsed EPR Characterization of the [2Fe-2S](Cys)3(His)1 Cluster in Rat MitoNEET

CW EPR spectra of reduced [2Fe-2S](Cys)(3)(His)(1) clusters of mammalian mitoNEET soluble domain appear to produce features resulting from the interaction of the electron spins of the two adjacent clusters, which can be explained by employing the local spin model. This model favors the reduction of the outermost iron with His87 and Cys83 ligands, which is supported by orientation-selected hyperfine sublevel correlation (HYSCORE) characterization of the uniformly (15)N-labeled mitoNEET showing one strongly coupled nitrogen from the His87 N(delta) ligand with hyperfine coupling (15)a = 8 MHz. The (14)N and (15)N HYSCORE spectra also exhibit at least two different cross-peaks located near diagonal in the (++) quadrant, with frequencies approximately 2.8 and 2.4 MHz (N2), and the other approximately 4.0 and 3.5 MHz (N1), but did not show any of the larger splitting approximately 1.1-1.4 MHz previously seen with Rieske proteins. Further analysis with partially (15)N(3)-His-labeled protein indicates that His87 N(epsilon) cross-peaks produce resolved features (N2) in the (14)N spectrum but contribute much less than weakly coupled peptide nitrogen species to the (++) quadrant in the (15)N spectrum. It is suggested that these quantitative data may be used in future functional and theoretical studies on the mammalian mitoNEET [2Fe-2S] cluster system.

Mosquito (Culex pipiens pallens) egg development induced by infusion of amino acids into the hemocoel

Abstract A mixture of amino acids was infused into the hemocoel of female Culex pipiens pallens mosquitoes at a very slow rate. We attempted to simulate the natural concentration increase of hemolymph amino acids, in order to determine whether amino acids derived from a blood meal trigger mosquito oogenesis. Infusion of a 7.5% (w/v) amino acid mixture for 14–16 h at a flow rate of 0.083 μl/h (2.0 μl/24h) activated oocytes to develop to intermediary stages (stages IIb-IIIa). The same infusion continued for more than 20 h resulted in egg maturation in most of the treated females. The number of mature eggs was not significantly different among the 14–24 h infusions but showed a proportional increase between the 24 and 48 h infusions. The hemolymph amino acid concentrations of these infused females did not greatly differ from the levels observed in blood-fed females, suggesting that the oogenic stimulatory effect of the infused amino acids was close to the natural process. This indicated that amino acids resulting from blood meal digestion are not only utilized for yolk protein synthesis but also have the potential to initiate and promote mosquito oogenesis. In addition, oocyte growth caused by infusion without any distention of organs or body wall clearly indicates that stretching is not an essential trigger for this event.

Proton magnetic resonance studies of 7Fe ferredoxins Conversion of a 4Fe core to a 3Fe core with ferricyanide

The redox couples in 7Fe ferredoxins (Fd) treated with ferricyanide were monitored by 1H‐NMR. An excess amount of ferricyanide was found to effect conversion of one of the two redox centers, the 4Fe core, to a 3Fe core in the ferredoxins extracted from Thermus thermophilus, Mycobacterium smegmatis and Pseudomonas ovalis. On long term incubation in air, the converted 3Fe core showed even further change in NMR spectra.

Two Tandemly Arranged Ferredoxin Genes in the Hydrogenobacter thermophilus Genome: Comparative Characterization of the Recombinant [4Fe–4S] Ferredoxins

A thermophilic, obligately chemolithoautotrophic hydrogen-oxidizing bacterium, Hydrogenobacter thermophilus TK-6, assimilates carbon dioxide via the reductive tricarboxylic acid cycle. A gene encoding a ferredoxin involved in this cycle as an electron donor (HtFd1) was cloned and sequenced. Interestingly, another ferredoxin gene (encoding HtFd2) was found in tandem with the HtFd1 gene. These two ferredoxin genes overlapped by four bp, and transcriptional analysis revealed that they are co-transcribed as an operon. The deduced amino acid sequences of HtFd1 and HtFd2 were 42.9% identical and each contained four cysteine residues that serve as probable ligands to an iron-sulfur cluster. Spectroscopic analyses of the purified recombinant ferredoxins heterologously expressed in Escherichia coli indicated that each ferredoxin contains a single [4Fe–4S]2+⁄1+ cluster.

Novel Zinc-containing Ferredoxin Family in Thermoacidophilic Archaea

The dicluster-type ferredoxins from the thermoacidophilic archaea such as Thermoplasma acidophilum and Sulfolobus sp. are known to contain an unusually long extension of unknown function in the N-terminal region. Recent x-ray structural analysis of the Sulfolobus ferredoxin has revealed the presence of a novel zinc center, which is coordinated by three histidine ligand residues in the N-terminal region and one aspartate in the ferredoxin core domain. We report here the quantitative metal analyses together with electron paramagnetic resonance and resonance Raman spectra of T. acidophilum ferredoxin, demonstrating the presence of a novel zinc center in addition to one [3Fe-4S] and one [4Fe-4S] cluster (Fe/Zn = 6.8 mol/mol). A phylogenetic tree constructed for several archaeal monocluster and dicluster type ferredoxins suggests that the zinc-containing ferredoxins of T. acidophilum and Sulfolobus sp. form an independent subgroup, which is more distantly related to the ferredoxins from the hyperthermophiles than those from the methanogenic archaea, indicating the existence of a novel group of ferredoxins, namely, a “zinc-containing ferredoxin family” in the thermoacidophilic archaea. Inspection of the N-terminal extension regions of the archaeal zinc-containing ferredoxins suggested strict conservation of three histidine and one aspartate residues as possible ligands to the novel zinc center.

Amino acid sequence of iron-superoxide dismutase from Pseudomonas ovalis

The amino acid sequence of iron‐superoxide dismutase from Pseudomonas ovalis was deduced by the analyses of peptides derived from limited hydrolysis of the aminoethylated or pyridylethylated apoprotein with trypsin, Staphylococcus aureus V8 protease, and diluted acid hydrolysis. The polypeptide chain contains 195 amino acid residues and has a calculated M r of 21 421. the sequence is highly homologous (65% identity) to the recently published sequence of the iron‐superoxide dismutase from Photobacterium leiognathi. It is also homologous to the known sequences of the manganese‐superoxide dismutase by sharing 33–53% identical residues. Alignment of the superoxide dismutase sequences and the available structural information from X‐ray crystallography suggest that the ligands to the iron in the P. ovalis superoxide dismutase are His‐26, His‐74, Asp‐156 and His‐160, which align with the ligands to the manganese in the Thermus thermophilus manganese‐superoxide dismutase. The sequence information of the P. ovalis dismutase will facilitate refinement of the X‐ray crystallographic data that are now available at 2.9 Å resolution.